Skip to main content
PMC home page
eLife logoLink to eLife
. 2023 Mar 8;12:e85739. doi: 10.7554/eLife.85739

Kindlin-1 regulates IL-6 secretion and modulates the immune environment in breast cancer models

Emily R Webb 1,✉, Georgia L Dodd 1, Michaela Noskova 1, Esme Bullock 1, Morwenna Muir 1, Margaret C Frame 1, Alan Serrels 1, Valerie G Brunton 1,✉
Editors: Tatyana Chtanova2, Tadatsugu Taniguchi3
PMCID: PMC10023156  PMID: 36883731

Abstract

The adhesion protein Kindlin-1 is over-expressed in breast cancer where it is associated with metastasis-free survival; however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in mouse models of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells led to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs. Similar changes in T cell populations were seen following depletion of Kindlin-1 in the polyomavirus middle T antigen (PyV MT)-driven mouse model of spontaneous mammary tumorigenesis. There was a significant increase in IL-6 secretion from Met-1 cells when Kindlin-1 was depleted and conditioned media from Kindlin-1-depleted cells led to a decrease in the ability of Tregs to suppress the proliferation of CD8+ T cells, which was dependent on IL-6. In addition, deletion of tumor-derived IL-6 in the Kindlin-1-depleted tumors reversed the reduction of tumor-infiltrating Tregs. Overall, these data identify a novel function for Kindlin-1 in regulation of anti-tumor immunity, and that Kindlin-1 dependent cytokine secretion can impact the tumor immune environment.

Research organism: Mouse

Introduction

Kindlin-1 is a four-point-one, ezrin, radixin, moesin (FERM) domain-containing adaptor protein that localises to focal adhesions, where it plays an important role in controlling integrin activation via binding to integrin β subunits (Rognoni et al., 2016). Loss-of-function mutations in the gene encoding Kindlin-1, FERMT1, leads to Kindler Syndrome, a rare autosomal recessive genodermatosis that causes skin atrophy, blistering, photosensitivity, hyper or hypo-pigmentation, increased light sensitivity and an enhanced risk of developing aggressive squamous cell carcinoma (Guerrero-Aspizua et al., 2019; Lai-Cheong et al., 2009). However, Kindlin-1’s role in cancer is complex as it can also have a tumor-promoting role (Rognoni et al., 2016; Zhan and Zhang, 2018).

In breast cancer, Kindlin-1 expression is higher in tumor versus normal breast tissue and its expression is associated with metastasis-free survival (Azorin et al., 2018; Sin et al., 2011). Consistent with its recognised role in regulating integrin-extracellular matrix interactions, Kindlin-1 controls both breast cancer cell adhesion, migration and invasion (Azorin et al., 2018; Sarvi et al., 2018). Kindlin-1 also regulates TGFβ signaling and epithelial to mesenchymal transition (EMT) in breast cancer (Sin et al., 2011). We have previously shown in the polyomavirus middle T antigen (PyV MT)-driven mouse model of mammary tumorigenesis, that loss of Kindlin-1 significantly delays tumor onset and reduces the incidence of lung metastasis (Sarvi et al., 2018). Mechanistically, Kindlin-1 stimulates metastatic growth in this model via integrin-dependent adhesion of circulating tumor cells to endothelial cells in the metastatic niche (Sarvi et al., 2018).

Kindlin-1 has also been shown to regulate inflammation in the skin of Kindler syndrome patients, where a number of pro-inflammatory cytokines are upregulated (Heinemann et al., 2011; Maier et al., 2016), and increased expression of genes associated with cytokine signaling have been reported (Chacón-Solano et al., 2019). Progressive fibrosis of the dermis that follows inflammation in Kindler Syndrome is consistent with enhanced cytokine signaling, and the resulting ‘activation’ of fibroblasts leads to enhanced extracellular matrix deposition (Heinemann et al., 2011; Chacón-Solano et al., 2019). Although inflammatory cytokines can play an important role in tumor progression, it is not known whether, and if so how, Kindlin-1 regulation of inflammatory cytokines in the tumor microenvironment influences tumor growth. Here we report that Kindlin-1 promotes an immunosuppressive and pro-tumorigenic microenvironment in a mouse model of breast cancer. Specifically, genetic deletion of the gene encoding Kindlin-1 leads to a reduction in tumor infiltrating Tregs and impairment of their immune-suppressive activities, and the generation of an immunological memory response. This implicates Kindlin-1 in a previously unrecognised function of immune-modulation in the breast cancer microenvironment in vivo.

Results

Loss of Kindlin-1 leads to tumor clearance and immunological memory

We used a syngeneic model in which Fermt1 had been deleted in the Met-1 murine breast cancer cell line (Kin1-NULL) and to which either wild type Kindlin1 (Kin1-WT), or a mutant that is unable to bind β-integrin (Kin1-AA) were reintroduced (Sarvi et al., 2018). Tumor growth was monitored following subcutaneous injection of cells into both CD-1 nude immune-compromised and FVB (syngeneic) mice. Loss of Kindlin-1 led to reduced tumor growth in CD-1 nude mice (Figure 1A and B) with significant differences in tumor size noted from day 10 onwards. A similar reduced tumor growth rate in CD1 nude mice was seen following injection of human MDA-MB-231 cells in which Kindlin-1 was depleted using shRNA (Figure 1—figure supplement 1). In both cell models loss of Kindlin-1 had no effect on in vitro cell proliferation (Figure 1—figure supplement 1), while immunohistochemical analysis of Ki67 and phospho-histone H3 in tumors showed there was no effect on proliferation in vivo (Figure 1—figure supplement 2). In contrast when Met-1 cells were injected into immune competent FVB mice, although Kindlin-1 loss led to a similar delay in tumor growth at day 10, there was complete tumor regression by day 19 (Figure 1C and D). Thus, Kindlin-1 is required for tumor growth of Met-1 cells in mice with a functional immune system, similar to what we reported previously for FAK-deficiency in a mouse model of squamous cell carcinoma (SCC) (Serrels et al., 2015). Growth of the Kin1-AA mutant-expressing tumors was indistinguishable from Kin1-WT tumors in both CD1 nude and FVB mice (Figure 1A and C respectively), implying that integrin dependent functions of Kindlin-1 are not important for the growth of Met-1 tumors.

Figure 1. Loss of Kindlin-1 leads to tumor clearance and immunological memory.

(A, C) Met-1 Kin1-WT, Kin1-NULL or Kin1-AA tumors were established via subcutaneous injection into flanks of CD1 nude mice (A) or FVB mice (C). Tumor growth was monitored and recorded until day 30, with average tumor growth shown. (B, D) Tumor size at day 10 post injection shown in CD1 nude mice (B) and FVB mice (D). (E) Left flank of FVB mice was injected with Met-1 Kin1-WT or Kin1-NULL cells. At day 35, when no tumor was present, Kin1-NULL injected mice were rechallenged with either Kin1-WT or Kin1-NULL Met-1 cells on the right flank. Naïve FVB mice were also injected concurrently. Tumor growth and survival (F) were monitored throughout. Combined data from three independent experiments (A–D). Example of two independent experiments (E–F). n=5–16 per group. Unpaired t-test (A–D) or Log Rank (F) with * =< 0.05, ** =< 0.01, *** =< 0.001. Analysis of human cell line MDA-MB-231 is shown in Figure 1—figure supplement 1, with proliferation analysis of tumours shown in Figure 1—figure supplement 2.

Figure 1.

Figure 1—figure supplement 1. Loss of Kindlin-1 leads to reduction of tumor growth in human breast cancer model.

Figure 1—figure supplement 1.

(A) Representative western blot demonstrating Kindlin-1 protein knockdown after addition of doxycycline to MDA-MB-231 cells expressing inducible non-targeting (NT) or FERMT1 shRNA. (B) Quantification of A normalised to loading control, n=3 per group, error bars = SEM. (C) Western blot demonstrating Kindlin-1 deletion in Met-1 cells. (D) Loss of Kindlin-1 has no effect on in vitro proliferation of MDA-MB-231 (NT shRNA) and FERMT1 shRNA (K1a), or Met-1 cells, n=3 per group, error bars = SEM. (E) MDA-MB-231 cells carrying doxycycline inducible FERMT1 shRNA were subcutaneously injected into CD1 nude mice, after which doxycycline treatment was commenced for half the group. Tumor growth was monitored and recorded until day 25, with average tumor growth shown. n = 8 mice per group, error bars = SD. Unpaired t-test with * =< 0.05.
Figure 1—figure supplement 1—source data 1. Raw western blot images for Figure 1—figure supplement 1A, C.
Figure 1—figure supplement 2. Loss of Kindlin-1 does not alter proliferation rate of Met-1 cells and tumors.

Figure 1—figure supplement 2.

(A) Nanostring PanCancer panel of cell cycle genes in Met-1 cells. n=3 per group. (B, C) Immunohistochemical analysis of Ki67 (B) and phospho-histone H3 (C) in Met-1 tumors, with representative images and quantification shown. n=3 per group, error bars = SD (BC).
Figure 1—figure supplement 2—source data 1. Nanostring PanCancer panel analysis of Met-1 Kin1-WT and NULL cells in vitro.
Open in a new tab

To further investigate whether an immune response was generated in mice with Kin1-NULL tumors, a re-challenge experiment was conducted. Following regression of Kin1-NULL tumors, mice were re-challenged with either Kin1-WT or Kin1-NULL cells on day 35 (Figure 1E and F). Neither Kin1-WT nor Kin1-NULL cells grew palpable tumors in mice which had been pre-challenged with Kin1-NULL cells, while injection of Kin1-WT or Kin1-NULL cells into naive mice on the same day showed normal tumor growth and survival (Figure 1E and F). These data suggest that deletion of Kindlin-1 promotes effective immunosurveillance, resulting in tumor regression and lasting immunological memory. Furthermore, the inability of Kin1-WT cells to give rise to tumors in mice previously harbouring a Kin1-NULL tumor suggests that Kindlin-1 does not regulate key antigens permitting T-cell tumor recognition and effective immunosurveillance.

Loss of Kindlin-1 modulates tumor associated myeloid populations

To understand how loss of Kindlin-1 promotes immunosurveillance, flow cytometry was used to profile the immune landscape of Kin1-WT and Kin1-NULL tumors at 10 days post tumor challenge. The percentages of major myeloid subsets were quantified within the tumors (Figure 2A, Figure 2—figure supplement 1). Of note there were significantly reduced CD45+ cells within Kin1-NULL tumors, alongside a reduction in both monocytes and macrophages (Figure 2A). However, no significant differences were observed in expression of the phenotype markers MHC II, CD206 and SIRPα, between Kin1-WT and Kin1-NULL tumor-associated macrophages (Figure 2—figure supplement 2A), suggesting that there is no change in the ‘polarisation’ status of these cells. Although there was no difference in total dendritic cell (DC) percentages, analysis of DC subsets demonstrated a significant increase in conventional type I DCs (cDC1) within Kin1-NULL tumors compared to Kin1-WT (Figure 2B and C). cDC1s are efficient at cross presentation, essential for CD8+ responses and have been demonstrated to be important for anti-tumor immune responses (Ferris et al., 2020; Laoui et al., 2016). Furthermore, we observed increased expression of the T-cell co-stimulatory molecule CD80 on DCs in Kin1-NULL tumors (Figure 2D, Figure 2—figure supplement 2B), and increased expression of the T cell inhibitory PD-1 receptor ligand PD-L1 (Figure 2E, Figure 2—figure supplement 2B). Additionally, analysis of bulk tumor RNA demonstrated an increase in antigen presentation (H2-Q2 and H2-Eb1) and antigen transport (Tap2) related genes within Kin1-NULL tumors (Figure 2F). An increase in Ifng was also seen in the Kin1-NULL tumors (Figure 2G), consistent with increased expression of IFNγ-inducible PD-L1, and MHC/Antigen processing genes (Zhou, 2009; Garcia-Diaz et al., 2017). These data suggest that loss of Kindlin-1 may result in increased cross-presentation of tumor antigen by DCs, promoting T-cell activation and anti-tumor immunity. Despite an increase in PD-L1 protein expression noted on cDC1 cells, overall PD-L1 gene (Cd274) expression was found to be decreased on CD45+ cells isolated from both tumors and draining lymph nodes (dLN) of Kin1-NULL tumors, compared to Kin1-WT tumors, although this did not reach significance in the tumors (Figure 2H). Analysis of a publicly available human breast cancer data set (METABRIC), demonstrated a small but significant correlation between FERMT1 (Kindlin-1) and CD274 (PD-L1) gene expression (Figure 2—figure supplement 2C). Together these data show that loss of Kindlin-1 can lead to modulation of PD-L1 expression on tumor infiltrating immune cells, suggesting that the PD-1/L1 pathway may contribute to the anti-tumor immune response.

Figure 2. Loss of Kindlin-1 reduces tumor associated macrophages and increases cDC1 dendritic cells.

(A) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Major myeloid populations were quantified as a percentage of live (total) cells. Gating demonstrated in Figure 2—figure supplement 1. (B) Raw FACS plots demonstrating gating of cDC1 and cDC2 cells, and quantified (C) as a percentage of total DCs (CD11c+ MHC II+). (D) Quantification of CD80 expression on total DCs by flow cytometry. (E) Quantification of PD-L1 expression on cDC1 cells. (F) As in A but bulk tumors were harvested for RNA expression analysis using Nanostring PanCancer Immune panel. Differentially expressed genes related to the gene sets ‘MHC’ and ‘Antigen (Ag) presentation’ are shown. (G) Expression of Ifng using Nanostring PanCancer Immune panel comparing Met-1 Kin1-WT and Kin1-NULL cells. (H) Expression of Cd274 (PD-L1) on isolated CD45+ cells using Nanostring Immune Exhaustion panel comparing draining lymph nodes (dLN) and tumors from Met-1 Kin1-WT and Kin1-NULL tumor bearing mice. Example of two independent experiments (A–E), n=3–5 per group, error bars = SD. For F, fold change cut off = 1.2, FDR =< 0.05. Unpaired t-test with * =< 0.05, ** =< 0.01, *** =< 0.001. Further macrophage and dendritic cell profiling shown in Figure 2—figure supplement 2.

Figure 2.

Figure 2—figure supplement 1. Flow cytometry gating examples of myeloid populations.

Figure 2—figure supplement 1.

Example of tumor myeloid cell gating shown. First debris is removed by All cells gate, followed by singlets and live cells. CD45+ cells are selected for downstream identification of Macrophages (F4/80+), Neutrophils (CD11b+ F4/80- Ly6Ghi Ly6cint) and Monocytes (CD11b+ F4/80- Ly6Glo Ly6C+). Total dendritic cells (DCs) were defined as CD11c+ MHC II+ with further downstream gating of cDC1 (CLEC9a+ SIRPα-) and cDC2 (CLEC9a- SIRPα+). Expression of CD103 was then assessed on cDC1s and cDC2s.
Figure 2—figure supplement 2. Macrophage and dendritic cell profiling in Met-1 tumors and PD-L1- Kindlin-1 correlation in human breast cancer dataset.

Figure 2—figure supplement 2.

(A) Quantification of MHC II and CD206 (left), MHC II and SIRPα (middle), and SIRPα and CD206 (right) as a percentage of total macrophages infiltrating MET-1 tumors. Data representative of two independent experiments. n=5 per group, error bars = SD. (B) Representative histograms of PD-L1 and CD80 expression on cDC1 cells in Met-1 Kin1-WT and NULL tumors. (C) Correlation analysis of FERMT1 (Kindlin-1) and CD274 (PD-L1) using METABRIC human breast cancer data set. n=1904,, r=0.1375 (95% confidence interval 0.09–0.18), p<0.0001.
Open in a new tab

Loss of Kindlin-1 reduces Treg infiltration and memory phenotype

The modulation of PD-L1 in Kin1-NULL tumors, and generation of immunological memory, suggested that the tumor clearance may be tied to a T cell directed anti-tumor immune response. To that end, further immunophenotyping of Kin1-WT and Kin1-NULL tumors was conducted with focus on T cell subsets (Figure 3—figure supplement 1). Tumors were taken at day 10 post tumor cell implantation. We found a significant reduction of total CD3+ cells in Kin1-NULL tumors (Figure 3—figure supplement 2A), which was driven by a decrease in CD4+ T cells. Within the CD4+ cell compartment, a significant decrease of regulatory T (Treg) cells as a percentage of total cells was evident in Kin1-NULL tumors compared to Kin1-WT tumors (Figure 3A). The reduction of Tregs was also observed when analysing cells as a percentage of total CD3+ cells, with a significant increase in non-Treg CD4+ cells as a proportion of total CD3+ (Figure 3—figure supplement 2B). As Treg cells are widely reported to control anti-tumor T cell responses (Hatzioannou et al., 2021), these data suggest that Kindlin-1 loss results in fewer infiltrating suppressive T cells within the tumor microenvironment. Furthermore, the reduction in Tregs was mirrored in the tumor draining lymph nodes of Kin1-NULL tumors at day 10, but not observed systemically in the spleen (Figure 3—figure supplement 2C). This suggests that the reduction in Tregs is due to changes in the local tumor environment.

Figure 3. Loss of Kindlin-1 reduces tumor infiltrating Treg cells.

(A) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Gating of major T cell populations was conducted and quantified as percentage of total (alive) cells. Gating provided and further population analysis in Figure 3—figure supplements 1 and 2. (B) Quantification of effector (CD62L- CD44+), memory (CD62L+ CD44+) and naive (CD62L+ CD44-) populations as a percentage of corresponding T cell subset. (C) Representative example of gating resting Tregs (CD62L+) and activated Tregs (CD62L-) in tumors, with quantification on the right. (D, E) Quantification of PD-1 and TIM-3 expression on T cell subset effector (or CD62L-) populations (D) and memory (or CD62L+) populations (E). Example of two independent experiments (A–E). n=3–5 per group, error bars = SD. Unpaired t-test with * =< 0.05, ** =< 0.01, *** =< 0.001. Similar analysis of MMTV-PyV tumors provided in Figure 3—figure supplement 3.

Figure 3.

Figure 3—figure supplement 1. Flow cytometry gating examples of T cell populations.

Figure 3—figure supplement 1.

Example of tumor T cell cell gating shown. First debris is removed by All cells gate, followed by singlets, live cells and lymphocytes. γδT cells (CD3+ γδTCR+) and T cells (CD3+ γδTCR-) were gated. T cells were then further subdivided into CD8+ and CD4+ T cells. From the CD4+ gate Tregs (FoxP3+) and non-Treg CD4s (FoxP3-) were gated.
Figure 3—figure supplement 2. Loss of Kindlin-1 reduces tumor infiltrating Treg cells.

Figure 3—figure supplement 2.

(A) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Gating of CD3+ cells was conducted with subsequent gating and quantification of major T cell populations (B) as percentage of total CD3+ cells. (C) As in A but quantification of T cell subset in spleen (left) and draining (inguinal) lymph nodes (right). Example of two independent experiments (A–C). n=3–5 per group. Unpaired t-test with *=<0.05, ** =< 0.01, *** =< 0.001.
Figure 3—figure supplement 3. Immune modulation in MMTV-PyV MMTV-Kin-1wt/wt and MMTV-Kin-1fl/fl spontaneous tumor model.

Figure 3—figure supplement 3.

(A) Spontaneous MT-Kin-1wt/wt or MT-Kin-1fl/fl tumors were harvested once 1 tumour reached 10 mm for immunophenotyping by flow cytometry. Quantification of cDC1 cells as a percentage of total DCs (CD11c+ MHC II+). (B) Quantification of PD-L1 expression on non-immune (CD45-) and myeloid subsets from MT-Kin-1wt/wt or MT-Kin-1fl/fl tumours, as a percentage of the corresponding cell population. (C) As in A but quantification of major T cell subsets as a % of live (total) cells and, (D) % of CD3+ cells. n=5–6 mice per group, error bars = SD. Unpaired t-test with *=<0.05, ** =< 0.01.
Open in a new tab

To further elucidate whether any T cell phenotypic changes occurred upon loss of Kindlin-1 in tumors, analysis of memory markers (Figure 3B and C) and activation/exhaustion markers (Figure 3D and E) was conducted. We found no significant changes in memory marker expression on non-Treg CD4+ cells; however, an increase in naïve (CD62L+ CD44-) CD8+ cells was observed (Figure 3B). Interestingly, a significant increase in CD62L+ Treg cells was evident within Kin1-NULL tumors when compared to Kin1-WT tumors at day 10 (Figure 3C). A corresponding reduction in CD62L- Treg proportions were also noted. CD62L+ Treg cells have been categorised into resting Tregs, with reduced proliferative capacity, whereas CD62L- populations are reported to be activated Tregs with increased suppressive capacity, and accumulation of these cells into tumors drives CD8+ T cell suppression (Luo et al., 2016; Ren et al., 2019; Huehn et al., 2004). The switch in the prominence of these two subsets in the Kin1-NULL tumors suggests that more resting Tregs are present compared to Kin1-WT tumors. We found minimal changes in activation (PD-1+ TIM-3-) and exhaustion (PD-1+ TIM-3+) markers on effector (or CD62L- Tregs) (Figure 3D) non-Treg T cells. Of note, a reduction of double positive (exhaustion) cells within CD4mem and TregCD62L+ populations was seen in Kin1-NULL tumors, alongside a corresponding increase in double negative (PD-1- TIM-3-) cells (Figure 3E). These data suggest that loss of Kindlin-1 in Met-1 tumors is primarily modulating CD4+ T cell phenotypes, specifically that of Tregs.

Loss of Kindlin-1 also causes immune changes in a spontaneous breast cancer model

To investigate whether similar immune modulation is evident when Kindlin-1 is depleted in a spontaneous mammary tumor model, immunophenotyping of tumors from the MMTV-PyV MT mouse model was carried out. MMTV-PyV MT mice were crossed with mice in which exons 4 and 5 of the Fermt1 gene were flanked with LoxP1 recombination sites, and in which Cre recombinase was expressed in the mammary epithelium under transcriptional control of the mouse mammary tumor virus (MMTV; Sarvi et al., 2018). Tumors that formed were collected from MMTV-PyV MMTV-Kin-1wt/wt (MT-Kin-1wt/wt) and MMTV-PyV MMTV-Kin-1fl/fl (MT-Kin-1fl/fl) mice and immune cell populations analyzed. We observed an increase of cDC1 cells in tumors from the MT-Kin-1fl/fl mice when compared to tumors in the MT-Kin-1wt/wt mice (Figure 3—figure supplement 3A), similar to the Met-1 cell-line-derived tumors already described (Figure 2B, C). Furthermore, there was a reduction of PD-L1 expression on CD45+ cells in MT-Kin-1fl/fl tumors (Figure 3—figure supplement 3B), as well as reduced PD-L1 on several myeloid subsets, demonstrating modulation of the PD-L1 pathway in Kindlin-1-deficient tumors also in this spontaneous breast cancer model.

Analysis of T cell subsets demonstrated significant reduction of T cells in MT-Kin-1fl/fl tumors as a percentage of total cells when compared to MT-Kin-1wt/wt (Figure 3—figure supplement 3C), although there was large variability between animals. This may be due to the asynchronous growth characteristics of the spontaneous MMTV model, with some of the mammary tumors enveloping nearby lymph nodes as they progress. Of note, total CD3 infiltration of these tumors was lower than in the Met-1 model (Figure 3—figure supplement 2A). However, when analyzed as a percentage of total CD3+ cells, MT-Kin-1fl/fl tumors had significantly fewer Treg cells with an increase in percentage of non-Treg CD4+ cells (Figure 3—figure supplement 3D), that was similar to the Met-1 cell-line-derived tumor model (Figure 3—figure supplement 2B). These data demonstrate immune modulation upon the loss of Kindlin-1 tumor expression in distinct models of breast cancer, with consistent changes in Treg and cDC1 cells resulting from Kindlin deficiency. However, in the MT-Kin-1fl/fl mice we do not see tumor regression, which most likely reflects the incomplete loss of Kindlin-1 in the mammary tumor cells in this model (Sarvi et al., 2018).

Kindlin-1 knock-out cells modulate Treg phenotype and function

As Tregs, which drive suppression of effector T cell function, were observed to be reduced in number in Kin1-NULL tumors, assessment of immunosuppressive pathways was conducted. Analysis of RNA from CD45+ tumor infiltrating cells isolated from Kin1-WT and Kin1-NULL tumors was carried out which showed a reduction of various T cell inhibitory checkpoint pathway related genes, including Cd274, Vsir and Havcr2 in Kin1-NULL tumors (Figure 4A). As many of these suppressive receptor pathways are known to be utilised by Treg cells for effector cell suppression, we next addressed whether Kindlin-1 deficiency leads to widespread disruption of Treg phenotype and function. Detailed Treg profiling from the Met-1 cell line derived tumors at day 10 by flow cytometry, was carried out (Figure 4B–D and Figure 4—figure supplement 1). Of note, downregulation of TNF superfamily co-stimulatory receptors GITR, 4-1BB and OX40 was observed in Kin1-NULL infiltrating Tregs (Figure 4B, Figure 4—figure supplement 1A, D), which are critical for Treg development and promoting their proliferation (Willoughby et al., 2017; Alissafi et al., 2019). Expression of the inhibitory receptors LAG-3, CTLA-4, TIGIT and PD-1 were also downregulated on the surface of Tregs in Kin1-NULL tumors (Figure 4C, Figure 4—figure supplement 1B, D). Although ligation of these receptors in CD8+ and non-Treg CD4+ cells inhibits effector function, these receptors are crucial for Treg differentiation and immunosuppressive activity (Alissafi et al., 2019). There was significant downregulation of both CD73 and CD39 expression on Tregs from Kin1-NULL tumors (Figure 4D, Figure 4—figure supplement 1C, D). The CD39/CD73 pathway is a major modulator of Treg activity via metabolism of ATP to create extracellular adenosine, in turn inhibiting effector T cell function (Antonioli et al., 2013; Allard et al., 2017). This suggests that loss of Kindlin-1 may cause metabolic changes in Treg cells, resulting in impairment of their suppressive capacity. Despite these phenotypic changes, there was no overt modulation of Treg proliferation seen (Figure 4E). Taken together, downregulation of these phenotypic markers suggests that Tregs from Kin-1 NULL tumors could be less immunosuppressive, and therefore allow development of a sufficient anti-tumor immune response, leading to tumor clearance. To that end, analysis of activation markers on CD8+ T cells was assessed. Although, we have previously shown little modulation of CD8+ cell number and expression of PD-1 and TIM-3 (Figure 2), this expanded panel allowed for assessment of CD8 activation in greater depth. There was an increase in expression of OX40, CD83 and CD29 on CD8+ T cells infiltrating Kin1-NULL tumors compared to those in Kin1-WT tumors (Figure 4F). These receptors are associated with activated T cells and an increase in cytotoxic potential (Willoughby et al., 2017; Hirano, 2006; Nicolet et al., 2020).To confirm this, analysis of CD107a expression (degranulation marker) and Granzyme B expression (cytotoxic granule) demonstrated a marked and significant increase in expression of these two markers on CD8+ effector T cells in Kin1-NULL tumors (Figure 4F and G). Together, these data suggest that loss of Kindlin-1 causes a reduction in Treg suppressive function, which can enhance the activation of CD8+ cytotoxic T cells, leading to reduced tumor growth.

Figure 4. Loss of Kindlin-1 modulates Treg phenotype and function.

(A) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for RNA analysis of isolated CD45+ cells using Nanostring Immune Exhaustion panel. Shown is log2 normalised expression of known T cell inhibitory receptors and pathways. n = 3 per group. (B) As in A but tumors harvested for immunophenotyping by flow cytometry. Analysis of expression of markers were assessed on gated CD4+ FoxP3+ T cells (Tregs). Quantification of expression as geo mean fluorescent intensity (geoMFI) shown for TNF superfamily members (B), known inhibitory receptors (C), metabolism related receptors (D) and proliferation marker (E). Histograms and percentage expression is shown in Figure 4—figure supplement 1. (F) As in A with quantification of activation associated receptors on tumor infiltrating CD8+T cells. (G) Expression of markers of degranulation (CD107a) and cytotoxicity (Granzyme B) in tumor infiltrating CD8+ T cells. Example contour plots (left) and quantification of double positive cells (right). n=4–5 per group, error bars = SD. Unpaired t-test with * =< 0.05, ** =< 0.01, *** =< 0.001, **** =< 0.0001.

Figure 4.

Figure 4—figure supplement 1. Loss of Kindlin-1 modulates Treg phenotype markers.

Figure 4—figure supplement 1.

(A–D) Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Analysis of expression of markers was assessed on Tregs (CD4+ FoxP3+). (A–C) Example histograms of marker expression. (D) Expression shown as a percentage of Treg population. n=4–5 per group.
Open in a new tab

Loss of Kindlin-1 leads to altered cytokine secretion and regulates Treg differentiation

In the skin Kindlin-1 controls signaling through the TGF-β pathway, a key regulator of the immune environment (Rognoni et al., 2016). Analysis of Tgfb ligands showed a significant increase in Tgfb2 but not Tgfb1 or Tgfb3 in the Kin1-NULL cells (Figure 5—figure supplement 1A). The increase in Tgb2 was not seen in analysis of bulk tumors (Figure 5—figure supplement 1B). Furthermore, there was no difference in phospho-SMAD3 between the Kin1-WT and Kin1-NULL tumors indicating that the TGF-β-SMAD signaling pathway is not altered (Figure 5—figure supplement 1C). We then carried out a forward phase protein array of 64 cytokines from conditioned media collected from Kin1-WT and Kin1-NULL Met-1 cells. Decreases in CXCL11, 12 and IL-12 and significant increases in CXCL13, IL-1RA, and IL-6 were detected in conditioned media from Kin1-NULL cells compared to Kin1-WT and Kin1-AA cells (Figure 5A, Figure 5—figure supplement 2A). Although secretion of CXCL13, a chemokine involved in B cell migration (Kazanietz et al., 2019), was greatly increased, there was a trend towards decreased B cells within Kin-1 NULL tumors (Figure 5—figure supplement 3). We therefore focussed on IL-6 as previous studies have demonstrated the importance of IL-6 in influencing the differentiation of naïve CD4+ T cells into Tregs (Kimura and Kishimoto, 2010) and Treg suppressive function in various settings (Guo et al., 2019; Yang et al., 2008; Ye et al., 2020; Garg et al., 2019). The increase in secretion of IL-6 protein in conditioned media from Kin1-NULL cells was confirmed by ELISA (Figure 5B), while bulk tumor RNA analysis of Kin1-WT and Kin1-NULL tumors showed changes in IL-6-related genes, with an increase in Il6, Il6ra, and Il6st found in Kin1-NULL tumors (Figure 5C). However, analysis of Il6 in Kin1-WT and Kin1-NULL cells in vitro showed no difference in expression indicating that Kindlin-1 is not regulating transcription of Il6 in the tumor cells themselves (Figure 5D). Interestingly expression of Cxcl13 was also not altered between the Kin1-WT and Kin1-NULL cells (Figure 5—figure supplement 2B). Overall, this suggests that the altered cytokine profile secreted by Kin1-NULL cells is able to modulate signaling within local immune microenvironments.

Figure 5. Loss of Kindlin-1 leads to altered cytokine secretion.

(A) Met-1 Kin1-WT or Kin1-NULL cells were cultured for 48 hr before conditioned media (CM) was harvested for analysis by forward phase protein array. Proteins detected above background are shown as fold change over Kin1-WT. Individual data points and Met-1 Kin1-AA are shown in Figure 5—figure supplement 2A. (B) Quantification of IL-6 in Met-1 conditioned media via ELISA. (C) Bulk tumor RNA analysis of Met-1 Kin1-WT or Kin1-NULL tumors at day 10. Log2 normalised expression of IL-6-related genes are shown. Expression of Cxcl13 genes is shown in Figure 5—figure supplement 2B. (D) As in C for Met-1 Kin1-WT or Kin1-NULL cells in vitro. n = 3-6 per group, error bars = SD. Unpaired t-test with *=<0.05, ** =< 0.01, *** =< 0.001, **** =< 0.0001. Expression of TGFβ signaling genes is shown in Figure 5—figure supplement 1, with quantification of B cells shown in Figure 5—figure supplement 3.

Figure 5—source data 1. Forward Phase Protein Array of Met-1 cells (raw values).
Figure 5—source data 2. Nanostring PanCancer Immune panel analysis of Met-1 Kin1-WT and NULL cells in vitro.

Figure 5.

Figure 5—figure supplement 1. Analysis of TGFβ signaling in Met-1 Kin1-WT, NULL tumors.

Figure 5—figure supplement 1.

(A) RNA analysis of TGFβ signalling related genes in Met-1 Kin1-WT and NULL cells in vitro, using Nanostring PanCancer immune profiling panel. (B) Met-1 Kin1-WT, NULL and AA tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for bulk tumour RNA analysis of TGFβ signalling related genes using Nanostring PanCancer immune profiling panel. (C) Immunohistochemistry analysis of phosphorylated SMAD3 in FFPE sections of Met-1 Kin-1 WT, NULL and AA tumours. n=3 biological replicates (A), n=4 mice per group (B), n=3-4 mice per group, error bars = SD (C). Unpaired t-test with * =< 0.05 and ** =< 0.01.
Figure 5—figure supplement 2. Quantification of CXCL13 and IL-6 in Met-1 Kin1-WT, Kin1-NULL and Kin1-AA cells.

Figure 5—figure supplement 2.

(A) Met-1 Kin1-WT, Kin1-NULL, and Kin1-AA cells were cultured for 48 hr before conditioned media (CM) was harvested for analysis by forward phase protein array. Data for CXCL13 and IL-6 from Figure 5 are presented here with individual data points and Met-1 Kin-1-WT, -NULL and -AA shown as fold change to WT. (B) RNA analysis of Met-1 Kin1-WT and Kin1-NULL cells in vitro. Log2 normalised expression of Cxcl13 and its receptor Cxcr5 are shown. n=3 biological replicates, error bars = SD. Unpaired t-test with * =< 0.05, ** =< 0.01, *** =< 0.001.
Figure 5—figure supplement 3. Quantification of B cells in Met-1 Kin1-WT and Kin1-NULL tumors.

Figure 5—figure supplement 3.

Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Gating of CD3- cells was conducted with subsequent gating of B220+B cells as a percentage of total cells. n=3 per group. Unpaired t-test.
Open in a new tab

Previous studies have demonstrated the importance of IL-6 in influencing the differentiation of naïve CD4+ T cells into either Tregs or Th17 cells when in the presence of TGFβ (Kimura and Kishimoto, 2010). We therefore addressed whether tumor cell conditioned media could influence the differentiation of naïve CD4+ T cells in vitro. Conditioned media from Kin1-NULL cells resulted in a reduction of differentiation of CD4+ T cells into FoxP3+ Tregs compared to that seen following incubation with conditioned media from Kin1-WT cells (Figure 6A and B). We then generated Kin1-NULL cells in which Il6 was deleted using CRISPR-Cas9. This resulted in a complete block of IL-6 secretion (Figure 6C), and conditioned media from these cells was not able to reduce the differentiation of the CD4+ T cells into FoxP3+ Tregs (Figure 6D). Furthermore, blocking CXCL13 did not affect Treg differentiation (Figure 6—figure supplement 1), suggesting that IL-6 is the main component in the conditioned media driving this change. The decrease in CD4+FoxP3+ cells following treatment with conditioned media from Kin1-NULL cells was accompanied by an increase in CD107a and TNFα expression by CD4+ FoxP3- cells, suggesting an increase in their activation and function (Figure 6E and F). Furthermore, a corresponding increase in CD4+RoRγT+ Th17 cells (Figure 6G) was demonstrated, implying that the conditioned media from Kin1-NULL cells is diverting naïve CD4+ differentiation towards a more Th17 cell than Treg cell phenotype. When we looked at RNA expression in CD45+ cells isolated from Kin1-NULL and Kin1-WT tumors, we found a significant increase in Th17 associated gene, Rorc (Ruan et al., 2011), in Kin1-NULL tumors compared to Kin1-WT (Figure 6H) supporting a role for Kindlin-1 in modulating CD4+ T cell differentiation.

Figure 6. Loss of Kindlin-1 leads to modulation of Treg differentiation and function.

(A–B) Met-1 Kin1-WT or Kin1-NULL cells were cultured for 48 hr before conditioned media (CM) was harvested. Naïve CD4+ T cells were isolated from FVB mice spleens and stimulated in the presence of either Met-1 Kin1-WT or Kin1-NULL CM. At day 5 T cells were harvested for analysis of Treg differentiation by expression of FoxP3 (A). Example of gating is shown together with, (B) quantification as percentage of CD4+ cells. (C) Genetic knockout of IL-6 was performed in Met-1 Kin1-NULL cells (Kin1-NULL IL-6 NULL), with a no crRNA control (Kin1-NULL IL-6 CTRL). IL-6 knockout was assessed by ELISA of CM. (D) Naive CD4+ differentiation assay was performed as in A, using CM from Met-1 Kin1-NULL IL-6 NULL and CTRL cells. Analysis of Treg differentiation was conducted. Same is shown for CXCL13 blocking in Figure 6—figure supplement 1. (E, F) As in A with expression of degranulation marker CD107a (E) and functional cytokine TNFα (F) production in FoxP3- CD4 T cells. (G) As in A with quantification of RoRγT expression as a percentage of CD4+ FoxP3- CD44+ cells. (H) Rorc gene expression in isolated CD45+ cells from either Met-1 Kin1-WT or Kin1-NULL tumors as shown as Log2 normalised expression. (I) CD8+ CD4- CD25- and CD8- CD4+ CD25hi (Treg) cells were sorted from FVB spleens. CD8+ cells were labelled with CellTrace Violet and co-cultured with Tregs under stimulation at a ratio of 1:8 (Treg:CD8), in the presence of conditioned media +/-anti-IL-6 blocking antibody. At day 5, cells were harvested and analysis of proliferation of CD8 cells was conducted. Example histogram of CellTrace Violet staining with (J) quantification of CD8 proliferation shown. n=3–7 per group, error bars = SD. Unpaired t-test with * =< 0.05 and ** =< 0.01.

Figure 6.

Figure 6—figure supplement 1. Blocking CXCL13 does not alter Treg differentiation in vitro.

Figure 6—figure supplement 1.

Met-1 Kin1-NULL cells were cultured for 48 hr before conditioned media (CM) was harvested. Naive CD4+ T cells were isolated from FVB mice spleens and stimulated in the presence of Kin1-NULL CM +/– anti-CXCL13 blocking antibody. At day 5, T cells were harvested for analysis of Treg differentiation by expression of FoxP3 as a percentage of CD4+ cells. n=5 per group. Unpaired t-test.
Open in a new tab

To establish whether proteins secreted by Kin1-NULL cells can impair the ability of Tregs to suppress the proliferation of CD8+ T cells, a Treg suppression assay was conducted with addition of conditioned media from either Kin1-WT or Kin1-NULL cells. Conditioned media from Kin1-NULL cells lead to a decrease in Treg suppressive capacity, shown as an increase in percentage of divided CD8+ T cells compared to incubation with Kin1-WT conditioned media (Figure 6I and J). Treatment with an antibody that blocks the function of IL-6 reduced the ability of conditioned media from Kin1-NULL to increase division of CD8+ T cells. Thus, loss of Kindlin1 in the Met-1 cells leads to increased secretion of IL-6, which in turn reduces the ability of Tregs to suppress CD8+ T cell proliferation.

To address whether the increased secretion of IL-6 in the Kin1-NULL cells could alter the effects on T cell populations in vivo immunophenotyping was carried out 10 days post tumor cell implantation in Kin1-NULL tumors and Kin-NULL tumors in which IL-6 had also been deleted. There was a significant increase in the number of CD4+ T cells and Tregs in the IL-6-depleted tumors indicating that the tumor cell derived increase in IL-6 secretion following Kindlin-1 loss can drive changes in Tregs in vivo (Figure 7A). Furthermore, deletion of IL-6 in Kin1-NULL tumors resulted in a significant decrease in CD107a+ Granzyme B+ CD8+ T cells, suggesting their effector functions are impaired (Figure 7B). However, this was not sufficient to prevent clearance of the Kin1-NULL tumors (Figure 7C and D). In addition, treatment with an IL-6 blocking antibody was not able to prevent clearance of the Kin1-NULL tumors (Figure 7E).

Figure 7. Loss of tumor-derived IL-6 drives changes in Treg numbers and function but is not sufficient to reverse clearance of Kin1-NULL tumors.

(A) Met-1 Kin1-NULL IL-6-CTRL or Kin1-NULL IL-6-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Gating of CD4+ T cell populations was conducted and quantified as percentage of total (alive) cells. (B) As in A with quantification of expression of markers of degranulation (CD107a) and cytotoxicity (Granzyme B) in tumor infiltrating CD8+ T cells. Example contour plots (left) and quantification of double positive cells (right). (C) Weights of tumors from mice shown in A-B. (D) Met-1 Kin1-NULL IL-6-CTRL or Kin1-NULL IL-6-NULL tumors were established via subcutaneous injection in FVB mice. Tumor size was recorded. (E) Met-1 Kin1-NULL tumors were established via subcutaneous injection in FVB mice, with 20 μg anti-IL-6 neutralising antibody administered on Day –1, 0, 4, 8, 12, and 16 post tumor cell injection. Tumor size was recorded. (F) Met-1 tumors were established in mice pre-treated with anti-CD25 to deplete Tregs. Tumor growth for individual mice (left and middle) and averages (right) are shown. Depletion demonstrated in Figure 7—figure supplement 1 (G) Tumor size at Day 17 from F. n=3–7 per group, error bars = SD. Unpaired t-test with * =< 0.05, ** =< 0.01, *** =< 0.001, **** =< 0.0001.

Figure 7.

Figure 7—figure supplement 1. Depletion of Tregs with anti-CD25 antibody treatment.

Figure 7—figure supplement 1.

(A) After 3 day pre-treatment with anti-CD25 or isotype control antibody, Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice. Antibody treatment was continued weekly for 21 days at which point tissue was harvested to assess Treg-specific deletion. Representative plots showing depletion of Treg cells in tumour (top), spleen (middle) and draining lymph node (dLN – bottom). (B) Quantification of tumour data for both FoxP3+ (Treg) and FoxP3- (non-Treg CD4+) cells. (C) Example histogram demonstrating CD25 expression on Treg cells. Representative example of two experiments. n=3 per group, error bars = SD. Unpaired t-test * =< 0.05, ** = <0.01.
Open in a new tab

To demonstrate whether Tregs can control the anti-tumor immune response in Kin1-WT tumors, mice were depleted of Treg cells using an anti-CD25 antibody administered before tumor implantation. Although expression of CD25 is not specific to Tregs, it is a cell surface protein that has been widely used to deplete Treg cells (Hayes et al., 2020; Clemente-Casares et al., 2016; Göschl et al., 2018), and anti-CD25 antibody treatment resulted in depletion of CD4+ FoxP3+, but not CD4+ FoxP3- T cells, demonstrating deletion of Tregs, due to their high and constitutive expression of CD25 (Peng et al., 2023; Figure 7—figure supplement 1). Depletion of CD25+ cells resulted in a reduction of tumor growth compared to controls (Figure 7F), demonstrating a similar growth pattern as seen in Kin-1 NULL tumors (Figure 1C). There was a significant reduction in tumor size at Day 17 (Figure 7G) in Kin1-WT tumors with depleted Tregs. Overall, these data demonstrate the importance of Treg-mediated immune suppression in Kin1-WT tumors, leading to an increase in tumor growth. Although the Kindlin-1-dependent regulation of IL-6 secretion from the Met-1 tumor cells was able to able to regulate Treg infiltration and the function of the cytotoxic T cells in vivo this was not sufficient to induce tumor clearance.

Discussion

Previous studies have identified an important pro-tumorigenic role for Kindlin-1 in breast cancer, where it promotes cell migration, adhesion and EMT, and is associated with increased pulmonary metastasis and lung metastasis-free survival (Azorin et al., 2018; Sin et al., 2011; Sarvi et al., 2018). Here we show that Kindlin-1 can also regulate breast cancer progression by modulating the anti-tumor immune response through regulation of the immune composition of the tumor microenvironment.

Kindlin-1 is part of a family of proteins consisting of Kindlin-1,–2, and –3. They bind β integrin subunits and are required for integrin activation (Rognoni et al., 2016) with most studies reporting their integrin-dependent roles in cellular phenotypes, such as cell adhesion, migration, and invasion. However, integrin-independent functions of Kindlin-1 have also been reported, where it can initiate downstream signaling independently of integrin adhesions (Rognoni et al., 2014; Patel et al., 2013). Here, we used a mutant of Kindlin-1 (Kin1-AA) that cannot bind β integrins, which we have previously shown leads to reduced levels of activated β1 integrin and integrin-dependent adhesion in Met-1 cells (Sarvi et al., 2018). We show that the ability of Kindlin-1 to bind integrins is not required for the growth of Met-1 tumors, or their immune clearance in immunocompetent animals. Furthermore, the secretion of both IL-6 and CXCL13 was not altered by the ability of Kindlin-1 to bind β1 integrins, supporting a role for secreted cytokines in driving the integrin-independent immune changes (Figure 5—figure supplement 2). Redundancy between Kindlin-1 and Kindlin-2 has been reported in relation to integrin-dependent functions, where they have overlapping roles (Azorin et al., 2018; He et al., 2011). However, in the Met-1 cell line model we used here, we saw no change in Kindlin-2 expression following depletion of Kindlin-1 (Figure 1—figure supplement 1), implying that an increase in Kindlin-2 is not driving the effects on immune cell populations and anti-tumor immunity. Interestingly, Kindlin-2 has been reported to control the recruitment of immunosuppressive (F4/80+, CD206+) macrophages in orthotopic breast cancer models; Kindlin-2 in the tumor cells is required for the secretion of colony-stimulating factor-1 that acts as a chemoattractant for the macrophages (Sossey-Alaoui et al., 2017). Although we observed a reduction in macrophages in the Met-1 tumors lacking Kindlin-1, analysis of phenotypic markers (MHC II, SIRPα, and CD206), did not suggest any changes in their ‘polarisation’.

Our previous work has demonstrated the importance of the focal adhesion protein FAK in regulating anti-tumor immunity (Serrels et al., 2015; Canel et al., 2020; Serrels et al., 2017), which is driven in part by transcriptional regulation of cytokine production. Here we show that Kindlin-1 can also alter cytokine production leading to changes in the immune environment. However, for IL-6 (and CXCL13) this is not controlled at the level of transcription. Although Il6 is primarily regulated via transcription the secretion of IL-6 is also regulated by other processes including via trafficking through the endocytic pathway (Revelo et al., 2019). Further studies are required to establish how Kindlin regulates IL-6 secretion, although Kindlin-dependent regulation of integrin trafficking has been reported (Margadant et al., 2012). Of note is the observation that IL-6 secretion is also increased from keratinocytes from Kindler syndrome patients that lack Kindlin-1 (Maier et al., 2016; Qu et al., 2012), although its function is not known.

Although IL-6 has well-known pro-tumorigenic roles, a number of studies have demonstrated the importance of IL-6 in reducing Treg suppressive function in various settings (Guo et al., 2019; Yang et al., 2008; Ye et al., 2020; Garg et al., 2019), and also in the differentiation of naive CD4+ T cells into Tregs (Kimura and Kishimoto, 2010). Here, we show that the Kindlin-1-dependent regulation of IL-6 secretion controls the differentiation of CD4+ T cells into FoxP3+ Tregs and the ability of Tregs to suppress CD8+ T cell proliferation in vitro, while also regulating Treg numbers in the tumor microenvironment. We also saw an IL-6 dependent reduction in Granzyme B production, and degranulation of cytotoxic T cells. As Granzyme B is an important cytotoxic molecule secreted alongside Perforin in granules by activated CD8+ T cells in order to induce apoptosis of target cells (Voskoboinik et al., 2015), these data imply that Kindlin-1 can impair the ability of T cells to mediate tumor cell killing. However, the inability of IL-6 blockade to impact on the clearance of Kin1-NULL tumors means that other mechanisms of immune regulation are also involved. Indeed, in addition to widespread downregulation of numerous functional markers on Tregs in Kindlin-1-depleted tumors that are required for their activation and suppressive activity, an increase in cDC1 cell numbers was also observed in Kindlin-1-depleted tumors. These cells are a subset of dendritic cells that express CD103 and are capable of efficient cross-presentation of antigens to both CD4 and CD8 T cells. They have also been implicated in generation of T-cell-driven anti-tumor immune responses (Ferris et al., 2020; Laoui et al., 2016; Noubade et al., 2019). Thus, loss of Kindlin-1 impacts several immune cell types that are known to contribute to anti-tumor immunity and future studies will explore how other factors act in concert with IL-6 to drive anti-tumor immunity in response to Kindlin-1 depletion.

In summary, we provide novel mechanistic insight into how Kindlin-1-expressing tumors can evade immune destruction. As Kindlin-1 is upregulated in breast cancer and linked to survival, targeting Kindlin-1-dependent pathways linked to immune phenotypes may provide a novel strategy to increase the efficacy of immunotherapies in breast cancer, particularly methods relating to reinvigorating the anti-tumor T cell response.

Materials and methods

Cell lines

Met-1 cells were originally acquired from B. Qian (University of Edinburgh) and have been described previously (Borowsky, 2005). Generation of Kin1-WT and Kin1-NULL cells was detailed previously (Sarvi et al., 2018). Genetic knockout of IL-6 in Met-1 Kin1-NULL cells was conducted using CRISPR-Cas9. Briefly, IL-6 crRNAs (IDT predesigned: Mm.Cas9.IL6.1.AA, Mm.Cas9.IL6.1.AB) were annealed to Alt-R CRISPR-Cas9 tracrRNA (IDT 1072533) at 95 °C. Alt-R S.p. Cas9 Nuclease V3 (IDT 1081059) was added to form the Cas9-RNP complex alongside Alt-R Cas9 Electroporation enhancer (IDT 1075916), and transfected into Met-1 Kin-1 NULL cells using nucleofection with Amaxa SE Cell Line 4D-Nucleofector X Kit S (Lonza V4XC-1032) to generate a pool of Met-1 Kin-1 NULL IL-6 NULL cells. A pool of Met-1 Kin-1 NULL IL-6 CTRL cells was created by the same method minus IL-6 crRNAs. Knockout of IL-6 was confirmed by ELISA of conditioned media (as detailed below). Cells were cultured in DMEM high glucose with 10% FBS and hygromycin for selection. Cells were split using TrypLE (Gibco) expression upon 70% confluency. Cells were mycoplasma tested every month and were used within 3 months of recovery from frozen. Cell viability over 7 days was monitored in 96-well plates using alamarBlue (Thermo Fisher): fluorescence emission was read at 590 nm following a 3 hr incubation.

Mice

All experiments were carried out in compliance with UK Home Office regulations. Met-1 Kin1-WT, Kin1-NULL, Kin1-NULL IL-6 NULL and Kin1-NULL IL-6 CTRL cells (1x106) were injected subcutaneously into both flanks of 8- to 12-week-old female FVB/N mice and tumor growth measured twice weekly using calipers. For tumor growth rechallenge experiments, 1x106 cells were injected into FVB/N mice as above. Following tumor regression, mice were housed for 35 days prior to rechallenge with 1x106 cells and tumor growth measured. At the time of rechallenge, age matched mice that had not previously been challenged with tumor cells (naive), were injected with the same cells and tumor growth measured as above. For CD25+ cell depletion anti-mouse CD25 depleting antibody (BioXCell, via 2BScientific BP0012) and isotype control (BioXCell, via 2BScientific BP0290) were dosed at 250 μg on days −5,–4, and –3 before 1x106 cells were injected as above on day 0. Antibodies were dosed weekly from day 0 until day 21 at which point the mice were culled and tissue taken for depletion assessment. For anti-IL-6 blocking, anti-IL-6 antibody (Biolegend, 504513) was dosed at 20 μg on days –1, 0, 4, 8, 12, and 16 (as previously detailed Benevides et al., 2015) post 1x106 tumor cell implantation as detailed above.

Generation of MMTV-PyV MMTV-Kin-1wt/wt and MMTV-PyV MMTV-Kin-1fl/fl have been described previously (Sarvi et al., 2018). Female mice were monitored weekly for tumor formation by palpation. Sample sizes were determined by tumor growth patterns observed in previous experiments (for example, Sarvi et al., 2018). Mice were randomised to groups upon tumor implantation. Technicians conducting tumor measurements were blinded. Mice were excluded if culls occurred due to non-experimental reasons, and noted in figure legends where applicable. Each mouse was considered an experimental unit.

Tissue dissociation

After harvesting, tumors were minced and digested with Liberase TL (Roche) and DNase. Tumors were then passed through a 100 μm strainer to achieve a single cell suspension. Spleen and lymph nodes were minced and passed directly through a 100 μm strainer. All tissues underwent red blood cell lysis using RBC lysis buffer (Biolegend).

Flow cytometry

Following tumor dissociation cells were stained with ZombieUV viability dye (Biolegend), before being resuspended in PBS + 1% BSA. Approximately 1x106 cells were aliquoted in 5 ml tubes and incubated with Fc Blocking antibody (Biolegend). Antibodies used for immune profiling are detailed in Supplementary file 1, and master mixes were prepared before incubation with cells. After washing, cells were fixed and permeabilised overnight using FoxP3/transcription factor staining buffer set following manufacturer’s instructions (eBiosceince, ThermoFischer), before staining with intracellular antibody master mixes. Finally, cells were washed before being acquired on BD LSR Fortessa (BD Biosciences). Gating and analysis of flow cytometry data was conducted using FlowJo (Version 10.8, Tree Star). Gating of major populations are demonstrated in Figure 2—figure supplement 1 and Figure 3—figure supplement 1. For immunophenotyping experiments, sample size was chosen to ensure biological differences could be determined, with minimum mice numbers to allow for collection and processing in one day. Samples were excluded if intracellular staining was unsuccessful.

IL-6 ELISA

Met-1 Kin1-WT and Kin1-NULL cells were seeded at 1.5x106 cells per 10 cm3 dish. Media was removed after 24 hr and 5 ml of fresh media was added. After 48 hr media was harvested, centrifuged and passed through a 0.22 μm filter to remove cell debris. Media was stored at –80 °C until use for maximum of 1 month. If stated, media was concentrated 2 X using 3 kDa cut off centrifuge tubes (ThermoFisher) immediately before use. ELISA was conducted using the Mouse IL-6 ELISA Max Delux kit (Biolegend), following the manufacturer’s instructions. Plates were read at 450 nm using Tecan Spark 20 M plate reader. A reference wavelength reading at 570 nm was subtracted from 450 nm values. Quantification of IL-6 concentration was calculated by extrapolating values using a standard curve of known concentrations.

Forward phase protein array

Conditioned media was prepared as detailed above. Cells were lysed in RIPA buffer and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher) following the manufacturer’s instructions. The cytokine assay was carried out in microarray format using validated capture/detection antibody pairs (R&D Systems). For each sample, a selected panel of 64 capture antibodies was printed as four-replicate sub-array sets on a single nitrocellulose-coated glass slide (Supernova Grace Biolabs). Each sample was incubated overnight with a 64 sub-array slide. After washing and blocking each sub-array was incubated with the appropriate biotin-labelled detection antibody. A final incubation with fluorescently labelled streptavidin followed by slide scanning using an InnoScan710IR scanner (Innopsys) generated array images. Images were analyzed and signals quantified using Mapix software (Innopsys). Only proteins which were determined to be above background binding were further analyzed. All values were normalised to protein concentration, and calculated as fold change over the mean of Kin1-WT values.

Naïve CD4+ differentiation assay

Spleens and lymph nodes (inguinal, axillary, brachial, and mesenteric) were harvested from FVB/N mice, minced and passed through a 70 μm strainer. After washing, cells were incubated in RBC lysis buffer to remove red blood cells and resuspended in PBS + 0.5% BSA + EDTA. 1x108 cells were used to isolate by negative selection naive (CD44-) CD4+ T cells using Mouse naive CD4+ cell isolation kit (Miltenyi Biotech) following manufactures instructions. 96-well plates were coated overnight in anti-CD3 antibody (7.5 μg/ml; cat# 100340), and anti-CD28 (2 μg/ml; cat#), TGFβ (1 ng/ml; cat#763102) and IL-2 (5 ng/ml, cat# 575402; All Biolegend) were added along with T cell media (RPMI, 10% FBS, 1% L-Glutamine, 0.5% Penicillin-Streptomycin). For CXCL13 blocking, anti-CXCL13 antibody (Biolegend; cat# 934503) was added. 5x104 cells per well were added, and stimulated for 5 days in the presence of either Kin1-WT, Kin1-NULL, Kin1-NULL IL-6 NULL or Kin1-NULL IL-6 CTRL conditioned media (30%). Cells were harvested from plates, added to 5 ml FACS tubes and analyzed by flow cytometry as detailed above.

Treg suppression assay

Spleens were harvested from FVB/N mice, minced and passed through 70 μm strainer. After washing cells were resuspended in PBS + 1% BSA and incubated with anti-CD3 (PerCP-Cy5.5; Cat# 100218), anti-CD4 (Brilliant Violet 711; Cat# 100447), anti-CD8 (Brilliant Violet 510; Cat# 100100752) and anti-CD25 (PE; Cat# 102008) antibodies. Cells were then sorted using FACS Aria system (BD Biosciences) for CD3+ CD8+ CD4- CD25- (effector CD8+ cells) and CD3+ CD8- CD4+ CD25hi (Treg cells). Effector CD8+ cells were labelled with CellTrace Violet dye (Thermo Fischer) following manufacturer’s instructions. 96-well plates were coated in anti-CD3 antibody (1 μg/ml Biolegend, cat #100340), and splenocytes from CD-1 nude mice were added as antigen presenting cells (APCs). Isolated Tregs and CellTrace violet labelled CD8 cells were co-cultured in T cell media at 1:8 ratio for 5 days in the presence of either Kin1-WT or Kin1-NULL conditioned media (50% concentrated 2 X), and with or without anti-IL-6 antibody (20 μg/ml, Biolegend Cat#504512). Cells were then harvested from plates, added to 5 ml FACS tubes and analyzed by flow cytometry as detailed above.

Nanostring analysis

For in vitro analysis: Cells were plated and harvested as detailed in ‘IL-6 ELISA’ section. For bulk RNA analysis: tumors were harvested at day 10 post tumor implantation as described above. Tumors were snap frozen in liquid nitrogen and then disrupted and homogenised using RLT buffer (Qiagen). For CD45+ cell analysis: After harvesting, tumors were processed into a single cell suspension as detailed above in ‘Tissue dissociation’. Cells were incubated with CD45+ MACS beads (Miltenyi Biotec) and isolated by positive selection using LS columns (Miltenyi Biotec). For all of the above RNA was extracted using RNAeasy kit (Qiagen). A total of 100 μg of RNA was processed using either the mouse Nanostring PanCancer Immune Profiling panel (in vitro cells and bulk tumor) or mouse Nanostring Immune Exhaustion Panel (Isolated CD45+ cells), following manufacturer’s instructions. Hybridization was performed for 18 hr at 65 °C and samples processed using the Nanostring prep station set on high sensitivity. Images were analyzed at maximum (555 fields of view). All data was analyzed by ROSALIND (https://rosalind.bio/), with a HyperScale architecture developed by ROSALIND, Inc (San Diego, CA).

Immunohistochemistry

Formalin-fixed tumor samples from Day 10 post tumor were deparaffinised and antigen retrieval performed with Citrate buffer. After peroxidase inhibitor incubation and blocking, sections were incubated with either anti-Ki67 (Cell Signaling Technology 12202), anti-pHistone H3 (Cell Signaling Technology 9701 S) or anti-pSMAD3 (Thermo Scientific PA5-110155) antibodies overnight at 4 °C. After washing, sections were incubated with EnVision System HRP Labelled Polymer Anti-Rabbit (Dako k4003), DAB chromogen (Agilent Technologies K346811-2), and counterstained with Mayer’s hematoxylin. Finally, sections were dehydrated, xylene washed as mounted.

Western blot

Cell lysates were resolved by gel electrophoresis, transferred to nitrocellulose, and probed with either anti–Kindlin-1 (1:1000; Abcam ab68041), anti–Kindlin-2 (1:1000; Sigma K3269) or anti-GAPDH (1:1000; Cell Signaling Technology 5174 S) antibodies, followed by goat anti-rabbit HRP secondary (1:5000 Cell Signaling Technology 7074 S). Membranes were imaged using an BioRad Chemi doc with Clarity Western ECL Substrate (Bio-Rad 1705061).

Human data

Pearson correlation of expression of FERMT1 and CD274 in all breast cancers the METABRIC microarray dataset (expression log intensity levels), n=1904,, r=0.1375 (95% confidence interval 0.09–0.18). Data was downloaded from cbioportal. Pearson correlation and two-tailed t-test were carried out in GraphPad Prism 9.3.0.

Statistical analysis

Statistical analyzes and graphs were produced and performed using a combination of GraphPad Prism version 9.3.0 (GraphPad) and Excel 2016 (Microsoft Corporation). Statistical methods used as detailed in figure legends. Comparisons were considered significantly different when p-value < 0.05. All data are biological replicates unless otherwise stated in figure legends.

Acknowledgements

We thank the Host and Tumor Profiling Unit at the University of Edinburgh Cancer Research UK Centre for help with the Nanostring and forward phase protein arrays. We also thank Marina Jodrell for assistance with performing ELISA assays.

This work was funded by Cancer Research UK grants C157/A24837 and C157/A29279.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Emily R Webb, Email: Emily.webb@ed.ac.uk.

Valerie G Brunton, Email: v.brunton@ed.ac.uk.

Tatyana Chtanova, Garvan Institute of Medical Research, Australia.

Tadatsugu Taniguchi, University of Tokyo, Japan.

Funding Information

This paper was supported by the following grants:

  • Cancer Research UK C157/A24837 to Emily R Webb.

  • Cancer Research UK C157/A29279 to Emily R Webb, Georgia L Dodd, Michaela Noskova, Esme Bullock, Morwenna Muir, Margaret C Frame, Alan Serrels, Valerie G Brunton.

Additional information

Competing interests

No competing interests declared.

Reviewing editor, eLife.

Author contributions

Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing.

Investigation.

Investigation, Methodology.

Formal analysis.

Investigation.

Funding acquisition, Writing - review and editing.

Conceptualization, Writing - review and editing.

Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing.

Ethics

All experiments were carried out in compliance with UK Home Office regulations underproject licence PP7510272. All animal procedures were approved by the University of Edinburgh Animal Welfare & Ethical Review Body (AWERB) approval PL05-21, and in accordance with the principles of the 3Rs. Every effort was made to minimise suffering.

Additional files

Supplementary file 1. List of antibodies used for immunophenotyping.
elife-85739-supp1.xlsx (10.1KB, xlsx)
MDAR checklist
Source data 1. NanoString PanCancer Immune panel analysis of bulk Met-1 tumors at day 10 (normalised expression), related to Figure 2 and Figure 5.
elife-85739-data1.csv (101.9KB, csv)
Source data 2. Nanostring immune exhaustion panel of isolated CD45+ cells from Met-1 tumors at Day 10, related to Figure 2, Figure 4 and Figure 6.
elife-85739-data2.csv (93.6KB, csv)

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1, 2, 4, 5 and 6.

The following previously published dataset was used:

Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, Speed D, Lynch AG, Samarajiwa S, Yuan Y, Gräf S, Ha G, Haffari G, Bashashati A, Russell R, McKinney S, Langerød A, Green A, Provenzano E, Wishart G, Pinder S, Watson P, Markowetz F, Murphy L, Ellis I, Purushotham A, Børresen-Dale AL, Brenton JD, Tavaré S, Caldas C, Aparicio S, METABRIC Group 2012. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. European Genome-Phenome Archive. EGAS00000000083

References

  1. Alissafi T, Hatzioannou A, Legaki AI, Varveri A, Verginis P. Balancing cancer immunotherapy and immune-related adverse events: the emerging role of regulatory T cells. Journal of Autoimmunity. 2019;104:102310. doi: 10.1016/j.jaut.2019.102310. [DOI] [PubMed] [Google Scholar]
  2. Allard B, Longhi MS, Robson SC, Stagg J. The ectonucleotidases CD39 and CD73: novel checkpoint inhibitor targets. Immunological Reviews. 2017;276:121–144. doi: 10.1111/imr.12528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Antonioli L, Pacher P, Vizi ES, Haskó G. CD39 and CD73 in immunity and inflammation. Trends in Molecular Medicine. 2013;19:355–367. doi: 10.1016/j.molmed.2013.03.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Azorin P, Bonin F, Moukachar A, Ponceau A, Vacher S, Bièche I, Marangoni E, Fuhrmann L, Vincent-Salomon A, Lidereau R, Driouch K. Distinct expression profiles and functions of kindlins in breast cancer. Journal of Experimental & Clinical Cancer Research. 2018;37:281. doi: 10.1186/s13046-018-0955-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benevides L, da Fonseca DM, Donate PB, Tiezzi DG, De Carvalho DD, de Andrade JM, Martins GA, Silva JS. IL17 promotes mammary tumor progression by changing the behavior of tumor cells and eliciting tumorigenic neutrophils recruitment. Cancer Research. 2015;75:3788–3799. doi: 10.1158/0008-5472.CAN-15-0054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borowsky AD. Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior. Clinical & Experimental Metastasis. 2005;22:47–59. doi: 10.1007/s10585-005-2908-5. [DOI] [PubMed] [Google Scholar]
  7. Canel M, Taggart D, Sims AH, Lonergan DW, Waizenegger IC, Serrels A. T-cell co-stimulation in combination with targeting FAK drives enhanced anti-tumor immunity. eLife. 2020;9:e48092. doi: 10.7554/eLife.48092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chacón-Solano E, León C, Díaz F, García-García F, García M, Escámez MJ, Guerrero-Aspizua S, Conti CJ, Mencía Á, Martínez-Santamaría L, Llames S, Pévida M, Carbonell-Caballero J, Puig-Butillé JA, Maseda R, Puig S, de Lucas R, Baselga E, Larcher F, Dopazo J, Del Río M. Fibroblast activation and abnormal extracellular matrix remodelling as common hallmarks in three cancer-prone genodermatoses. The British Journal of Dermatology. 2019;181:512–522. doi: 10.1111/bjd.17698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clemente-Casares X, Blanco J, Ambalavanan P, Yamanouchi J, Singha S, Fandos C, Tsai S, Wang J, Garabatos N, Izquierdo C, Agrawal S, Keough MB, Yong VW, James E, Moore A, Yang Y, Stratmann T, Serra P, Santamaria P. Expanding antigen-specific regulatory networks to treat autoimmunity. Nature. 2016;530:434–440. doi: 10.1038/nature16962. [DOI] [PubMed] [Google Scholar]
  10. Ferris ST, Durai V, Wu R, Theisen DJ, Ward JP, Bern MD, Davidson JT, Bagadia P, Liu T, Briseño CG, Li L, Gillanders WE, Wu GF, Yokoyama WM, Murphy TL, Schreiber RD, Murphy KM. Cdc1 prime and are licensed by CD4+ T cells to induce anti-tumour immunity. Nature. 2020;584:624–629. doi: 10.1038/s41586-020-2611-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garcia-Diaz A, Shin DS, Moreno BH, Saco J, Escuin-Ordinas H, Rodriguez GA, Zaretsky JM, Sun L, Hugo W, Wang X, Parisi G, Saus CP, Torrejon DY, Graeber TG, Comin-Anduix B, Hu-Lieskovan S, Damoiseaux R, Lo RS, Ribas A. Interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression. Cell Reports. 2017;19:1189–1201. doi: 10.1016/j.celrep.2017.04.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Garg G, Muschaweckh A, Moreno H, Vasanthakumar A, Floess S, Lepennetier G, Oellinger R, Zhan Y, Regen T, Hiltensperger M, Peter C, Aly L, Knier B, Palam LR, Kapur R, Kaplan MH, Waisman A, Rad R, Schotta G, Huehn J, Kallies A, Korn T. Blimp1 prevents methylation of foxp3 and loss of regulatory T cell identity at sites of inflammation. Cell Reports. 2019;26:1854–1868. doi: 10.1016/j.celrep.2019.01.070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Göschl L, Preglej T, Hamminger P, Bonelli M, Andersen L, Boucheron N, Gülich AF, Müller L, Saferding V, Mufazalov IA, Hirahara K, Seiser C, Matthias P, Penz T, Schuster M, Bock C, Waisman A, Steiner G, Ellmeier W. A T cell-specific deletion of HDAC1 protects against experimental autoimmune encephalomyelitis. Journal of Autoimmunity. 2018;86:51–61. doi: 10.1016/j.jaut.2017.09.008. [DOI] [PubMed] [Google Scholar]
  14. Guerrero-Aspizua S, Conti CJ, Escamez MJ, Castiglia D, Zambruno G, Youssefian L, Vahidnezhad H, Requena L, Itin P, Tadini G, Yordanova I, Martin L, Uitto J, Has C, Del Rio M. Assessment of the risk and characterization of non-melanoma skin cancer in Kindler syndrome: study of a series of 91 patients. Orphanet Journal of Rare Diseases. 2019;14:183. doi: 10.1186/s13023-019-1158-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guo H, Xun L, Zhang R, Hu F, Luan J, Lao K, Wang X, Gou X. Stability and inhibitory function of treg cells under inflammatory conditions in vitro. Experimental and Therapeutic Medicine. 2019;18:2443–2450. doi: 10.3892/etm.2019.7873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hatzioannou A, Boumpas A, Papadopoulou M, Papafragkos I, Varveri A, Alissafi T, Verginis P. Regulatory T cells in autoimmunity and cancer: a duplicitous lifestyle. Frontiers in Immunology. 2021;12:731947. doi: 10.3389/fimmu.2021.731947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hayes ET, Hagan CE, Khoryati L, Gavin MA, Campbell DJ. Regulatory T cells maintain selective access to IL-2 and immune homeostasis despite substantially reduced CD25 function. Journal of Immunology. 2020;205:2667–2678. doi: 10.4049/jimmunol.1901520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. He Y, Esser P, Heinemann A, Bruckner-Tuderman L, Has C. Kindlin-1 and -2 have overlapping functions in epithelial cells implications for phenotype modification. The American Journal of Pathology. 2011;178:975–982. doi: 10.1016/j.ajpath.2010.11.053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heinemann A, He Y, Zimina E, Boerries M, Busch H, Chmel N, Kurz T, Bruckner-Tuderman L, Has C. Induction of phenotype modifying cytokines by FERMT1 mutations. Human Mutation. 2011;32:397–406. doi: 10.1002/humu.21449. [DOI] [PubMed] [Google Scholar]
  20. Hirano N. Engagement of CD83 ligand induces prolonged expansion of CD8 +T cells and preferential enrichment for antigen specificity. Blood. 2006;107:1528–1536. doi: 10.1182/blood-2005-05-2073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huehn J, Siegmund K, Lehmann JCU, Siewert C, Haubold U, Feuerer M, Debes GF, Lauber J, Frey O, Przybylski GK, Niesner U, de la Rosa M, Schmidt CA, Bräuer R, Buer J, Scheffold A, Hamann A. Developmental stage, phenotype, and migration distinguish naive- and effector/memory-like CD4+ regulatory T cells. The Journal of Experimental Medicine. 2004;199:303–313. doi: 10.1084/jem.20031562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kazanietz MG, Durando M, Cooke M. CXCL13 and its receptor CXCR5 in cancer: inflammation, immune response, and beyond. Frontiers in Endocrinology. 2019;10:471. doi: 10.3389/fendo.2019.00471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kimura A, Kishimoto T. IL-6: regulator of treg/th17 balance. European Journal of Immunology. 2010;40:1830–1835. doi: 10.1002/eji.201040391. [DOI] [PubMed] [Google Scholar]
  24. Lai-Cheong JE, Tanaka A, Hawche G, Emanuel P, Maari C, Taskesen M, Akdeniz S, Liu L, McGrath JA. Kindler syndrome: a focal adhesion genodermatosis. The British Journal of Dermatology. 2009;160:233–242. doi: 10.1111/j.1365-2133.2008.08976.x. [DOI] [PubMed] [Google Scholar]
  25. Laoui D, Keirsse J, Morias Y, Van Overmeire E, Geeraerts X, Elkrim Y, Kiss M, Bolli E, Lahmar Q, Sichien D, Serneels J, Scott CL, Boon L, De Baetselier P, Mazzone M, Guilliams M, Van Ginderachter JA. The tumour microenvironment harbours ontogenically distinct dendritic cell populations with opposing effects on tumour immunity. Nature Communications. 2016;7:13720. doi: 10.1038/ncomms13720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Luo CT, Liao W, Dadi S, Toure A, Li MO. Graded FoxO1 activity in Treg cells differentiates tumour immunity from spontaneous autoimmunity. Nature. 2016;529:532–536. doi: 10.1038/nature16486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maier K, He Y, Wölfle U, Esser PR, Brummer T, Schempp C, Bruckner-Tuderman L, Has C. UV-B-induced cutaneous inflammation and prospects for antioxidant treatment in Kindler syndrome. Human Molecular Genetics. 2016;25:5339–5352. doi: 10.1093/hmg/ddw350. [DOI] [PubMed] [Google Scholar]
  28. Margadant C, Kreft M, de Groot DJ, Norman JC, Sonnenberg A. Distinct roles of talin and kindlin in regulating integrin α5β1 function and trafficking. Current Biology: CB. 2012;22:1554–1563. doi: 10.1016/j.cub.2012.06.060. [DOI] [PubMed] [Google Scholar]
  29. Nicolet BP, Guislain A, van Alphen FPJ, Gomez-Eerland R, Schumacher TNM, van den Biggelaar M, Wolkers MC. CD29 identifies IFN-γ-producing human CD8+ T cells with an increased cytotoxic potential. PNAS. 2020;117:6686–6696. doi: 10.1073/pnas.1913940117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Noubade R, Majri-Morrison S, Tarbell KV. Beyond cdc1: emerging roles of dc crosstalk in cancer immunity. Frontiers in Immunology. 2019;10:1014. doi: 10.3389/fimmu.2019.01014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Patel H, Zich J, Serrels B, Rickman C, Hardwick KG, Frame MC, Brunton VG. Kindlin-1 regulates mitotic spindle formation by interacting with integrins and plk-1. Nature Communications. 2013;4:2056. doi: 10.1038/ncomms3056. [DOI] [PubMed] [Google Scholar]
  32. Peng Y, Tao Y, Zhang Y, Wang J, Yang J, Wang Y. CD25: A potential tumor therapeutic target. International Journal of Cancer. 2023;152:1290–1303. doi: 10.1002/ijc.34281. [DOI] [PubMed] [Google Scholar]
  33. Qu H, Wen T, Pesch M, Aumailley M. Partial loss of epithelial phenotype in kindlin-1-deficient keratinocytes. The American Journal of Pathology. 2012;180:1581–1592. doi: 10.1016/j.ajpath.2012.01.005. [DOI] [PubMed] [Google Scholar]
  34. Ren J, Han L, Tang J, Liu Y, Deng X, Liu Q, Hao P, Feng X, Li B, Hu H, Wang H. Foxp1 is critical for the maintenance of regulatory T-cell homeostasis and suppressive function. PLOS Biology. 2019;17:e3000270. doi: 10.1371/journal.pbio.3000270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Revelo NH, Ter Beest M, van den Bogaart G. Membrane trafficking as an active regulator of constitutively secreted cytokines. Journal of Cell Science. 2019;133:e81. doi: 10.1242/jcs.234781. [DOI] [PubMed] [Google Scholar]
  36. Rognoni E, Widmaier M, Jakobson M, Ruppert R, Ussar S, Katsougkri D, Böttcher RT, Lai-Cheong JE, Rifkin DB, McGrath JA, Fässler R. Kindlin-1 controls wnt and TGF-β availability to regulate cutaneous stem cell proliferation. Nature Medicine. 2014;20:350–359. doi: 10.1038/nm.3490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rognoni E, Ruppert R, Fässler R. The kindlin family: functions, signaling properties and implications for human disease. Journal of Cell Science. 2016;129:17–27. doi: 10.1242/jcs.161190. [DOI] [PubMed] [Google Scholar]
  38. Ruan Q, Kameswaran V, Zhang Y, Zheng S, Sun J, Wang J, DeVirgiliis J, Liou H-C, Beg AA, Chen YH. The th17 immune response is controlled by the rel-RORγ-RORγ T transcriptional axis. The Journal of Experimental Medicine. 2011;208:2321–2333. doi: 10.1084/jem.20110462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sarvi S, Patel H, Li J, Dodd GL, Creedon H, Muir M, Ward J, Dawson JC, Lee M, Culley J, Salter DM, Sims AH, Byron A, Brunton VG. Kindlin-1 promotes pulmonary breast cancer metastasis. Cancer Research. 2018;78:1484–1496. doi: 10.1158/0008-5472.CAN-17-1518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Serrels A, Lund T, Serrels B, Byron A, McPherson RC, von Kriegsheim A, Gómez-Cuadrado L, Canel M, Muir M, Ring JE, Maniati E, Sims AH, Pachter JA, Brunton VG, Gilbert N, Anderton SM, Nibbs RJB, Frame MC. Nuclear FAK controls chemokine transcription, Tregs, and evasion of anti-tumor immunity. Cell. 2015;163:160–173. doi: 10.1016/j.cell.2015.09.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Serrels B, McGivern N, Canel M, Byron A, Johnson SC, McSorley HJ, Quinn N, Taggart D, Von Kreigsheim A, Anderton SM, Serrels A, Frame MC. IL-33 and ST2 mediate FAK-dependent antitumor immune evasion through transcriptional networks. Science Signaling. 2017;10:eaan8355. doi: 10.1126/scisignal.aan8355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sin S, Bonin F, Petit V, Meseure D, Lallemand F, Bièche I, Bellahcène A, Castronovo V, de Wever O, Gespach C, Lidereau R, Driouch K. Role of the focal adhesion protein kindlin-1 in breast cancer growth and lung metastasis. Journal of the National Cancer Institute. 2011;103:1323–1337. doi: 10.1093/jnci/djr290. [DOI] [PubMed] [Google Scholar]
  43. Sossey-Alaoui K, Pluskota E, Bialkowska K, Szpak D, Parker Y, Morrison CD, Lindner DJ, Schiemann WP, Plow EF. Kindlin-2 regulates the growth of breast cancer tumors by activating CSF-1-mediated macrophage infiltration. Cancer Research. 2017;77:5129–5141. doi: 10.1158/0008-5472.CAN-16-2337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Voskoboinik I, Whisstock JC, Trapani JA. Perforin and granzymes: function, dysfunction and human pathology. Nature Reviews. Immunology. 2015;15:388–400. doi: 10.1038/nri3839. [DOI] [PubMed] [Google Scholar]
  45. Willoughby J, Griffiths J, Tews I, Cragg MS. OX40: structure and function - what questions remain? Molecular Immunology. 2017;83:13–22. doi: 10.1016/j.molimm.2017.01.006. [DOI] [PubMed] [Google Scholar]
  46. Yang XO, Nurieva R, Martinez GJ, Kang HS, Chung Y, Pappu BP, Shah B, Chang SH, Schluns KS, Watowich SS, Feng X-H, Jetten AM, Dong C. Molecular antagonism and plasticity of regulatory and inflammatory T cell programs. Immunity. 2008;29:44–56. doi: 10.1016/j.immuni.2008.05.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ye M, Joosse ME, Liu L, Sun Y, Dong Y, Cai C, Song Z, Zhang J, Brant SR, Lazarev M, Li X. Deletion of IL-6 exacerbates colitis and induces systemic inflammation in IL-10-deficient mice. Journal of Crohn’s & Colitis. 2020;14:831–840. doi: 10.1093/ecco-jcc/jjz176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhan J, Zhang H. Kindlins: roles in development and cancer progression. The International Journal of Biochemistry & Cell Biology. 2018;98:93–103. doi: 10.1016/j.biocel.2018.03.008. [DOI] [PubMed] [Google Scholar]
  49. Zhou F. Molecular mechanisms of IFN-gamma to up-regulate MHC class I antigen processing and presentation. International Reviews of Immunology. 2009;28:239–260. doi: 10.1080/08830180902978120. [DOI] [PubMed] [Google Scholar]

Editor's evaluation

Tatyana Chtanova 1

This study provides compelling evidence that deletion of Kindlin-1 impairs breast cancer growth by reducing the recruitment of tumor Tregs and decreasing their immunosuppressive activity. This leads to an increase in cytotoxic T-cell activity and tumor growth suppression. Collectively, the findings in this study identify an important novel function for Kindlin-1 in regulating anti-tumor immunity.

Decision letter

Editor: Tatyana Chtanova1

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "Kindlin-1 controls anti-tumor immunity by regulating T-regs in breast cancer mouse models" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.

We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered further for publication by eLife in its present form.

Comments to the Authors:

Specifically, the reviewers found your study uncovering a novel role for the adhesion protein Kindlin-1 in regulating anti-tumor immunity in breast cancer interesting. But they also concluded that the manuscript lacks in vivo confirmation of the mechanism and that extensive additional data is required to support the central hypothesis.

However, should further revisions allow you to improve the paper by addressing the issues raised by the reviewers, we will be happy to consider the revised paper, which will be considered as a new submission.

Reviewer #1 (Recommendations for the authors):

In this manuscript, the authors have elucidated how Kindlin-1 loss impairs breast cancer growth. They have shown that genetic deletion of Kindlin-1 reduces the recruitment of tumor-infiltrating Tregs in the breast tumor microenvironment. It also severely impacted their immune-suppressive abilities, which helped in the generation of immunological memory. Moreover, Kindlin-1 deletion upregulates IL-6 production, which decreases the immune-suppressive activity of Tregs thereby, reduces breast tumor growth. The authors present intriguing observations, and the findings are well supported by the data, however, the data lacks some important mechanistic details. Overall, the study will be of substantial interest to researchers engaged in basic and clinical sciences.

Reviewer #2 (Recommendations for the authors):

Here the authors show that the deletion of Kindlin-1 in tumor causes a delay in its growth and reduces overall tumor burden. This effect was more pronounced in T cell sufficient hosts compared to the T cell deficient ones. Therefore, the authors hypothesized that tumor infiltrating T cells are controlled, at least in part, by Kindlin-1. Although the authors present some phenotypical changes in the DC and T cell compartments, it is still unclear whether these changes account for an altered anti-tumor T cell activity. Furthermore, the authors claim that the effect is possibly driven by increased IL-6 in the TME in the absence of Kindlin-1, however, in vivo data supporting this claim are missing. While the overall effects of the lack of tumor derived Kindlin-1 is interesting, how this regulates T cell activity and which mechanism(s) are responsible remain elusive due to insufficient evidence.

Figure 1 -

To prove that the deletion of Kindlin-1 promotes lasting immunological memory, have the authors also rechallenged Kindlin WT group with Kindlin WT tumor? Does that group have impaired immunological memory?

Figure 2-

Overall, this reviewer doesn't think the claim "the PD-1/L1 pathway is important for Kin1-WT tumors to control the anti-tumor immune response." is substantiated here.

D-E: What is the expression level (MFI) for CD80 and PD-L1 on the DCs? Histograms comparing the expression levels of PD-L1 for DCs from Kindlin-WT vs null need to be demonstrated.

H: PD-L1 expression doesn't seem to decrease in tumor infiltrating CD45+ cells as indicated by the p value (=0.05).

I: This reviewer doesn't understand the relevance of this experiment to the hypothesis tested here. What happens when PD-L1 is blocked in Kindlin-null group?

Also, absolute cell numbers should be added to the supplementary.

Figure 3 -

How does the homeostatic proliferation of Tregs change? Have the authors checked Ki-67 expression?

This figure lacks information on the functionality of effector CD4+ and CD8+ T cells. Is there an effect of reduced Treg infiltration on the ability of CD4 and CD8 cells to produce cytokines, GzmB etc.?

Also, absolute numbers of CD4+, CD8+ and Tregs should be added to the supplementary.

Figure 4-

Kindlin-1 deletion indeed seem to alter Treg phenotype however, the FACS plots for these phenotypical changes should be added to supplementary for better clarity.

Regarding Treg function, the phenotypical changes should not be used as a means to interpret the changes in Treg activity. For instance, a downregulation of GITR and PD-1 may as well have boosting effects on Treg function. Ideally, the gold standard test for Treg function would be performing a suppression assay. However, due to the difficulty of isolating Tregs from tumor, the functionality and proliferation of CD4 and CD8 effector cells (indicated by cytokine, GzmB production and Ki-67+ %) in addition to CD83 etc. are crucial.

Figure 6-

in vitro Treg/Th17 conversion seem to be only marginally affected, if at all, by the proinflammatory milieu created by Kindlin-null cells. However, to what extent such a conversion takes place in the TME is largely unknown. Depleting Tregs by a-CD25 can reduce tumor burden in multiple tumor models and its effect doesn't have to be due presence or absence of Kindlin. One key experiment here could be treating Kindlin WT mice with blocking anti-IL-6 antibody, and comparing it to the Kindlin-null tumor to see whether the proinflammatory environment attributed to the lack of Kindlin can be recapitulated. Also, have the authors thought about the possibility that Kindlin-1 wt TME may have more TGF-b? Can a TGF-b blockade reverse the amelioration of tumor burden in Kindlin-1 null mice?

Reviewer #3 (Recommendations for the authors):

Webb et al. investigated the role of Kindlin-1 in the development of breast cancer using both the Met-1 tumor cell xenograft model and the PyV-MT mice breast tumor model. The authors found that deletion/depletion of kindlin-1 resulted in the suppression of tumor growth, concluding kindlin-1 promotes breast tumor development. Surprisingly, they find that the integrin-binding capacity of kindlin-1 is not required for this function. They analyzed the infiltration of immune cells within the tumor and find that the population of Treg cells was significantly decreased in the kindlin-1 null tumor. Cytokine-profiling revealed that the secretion of IL-6 and several other cytokines were altered. The author used conditioned medium from Kindlin-1 WT and Kindlin-1 null cells to treat the co-culture of Treg and CD8+ T cells isolated from the mice spleen. They found that kindlin-1 deletion increased the proliferation of CD8+ T cells in the co-culture system and this effect can be blocked by addition of IL-6 antibody. The author suggests that the upregulation of IL-6 secretion from the kindlin-1 null tumor cells modulates the T cell differentiation, which lead to decrease of the Treg population and function and therefore the increase of anti-tumor immune response.

Overall, this is an interesting study which focus on the role of kindlin-1 in the anti-tumor immunity. However, in my opinion, some experiments need to be refined and more data is needed to clarify and support the hypothesis.

Specific points

1. The authors showed that deletion of kindlin-1 impaired tumor growth in both breast cancer models. The rest of the manuscript completely focuses on the immunoregulation within the tumor. Kindlin proteins are essential activator of integrin signaling, which can cooperate with growth factor receptors to promote cell proliferation. The proliferation of the tumor cells themselves should be monitored as well (in vitro MTT assay, kindlin-1 WT VS kindlin-1 null cells; phospho-histone H3 staining in the WT and kindlin-1 null tumors) to exclude the possibility that kindlin-1 directly promotes tumor cell proliferation.

2. The authors mentioned that kindlin-1 expression is higher in tumor compared to normal tissues. What happen to the xenograft if kindlin-1 is overexpressed in the met-1 cells? Will it promote the onset of tumorigenesis/ increase the tumor size and also decrease IL-6 secretion? What happen to the tumor growth and cytokine profile if an integrin-binding deficient mutant of kindlin-1 (AA mutant) is overexpressed?

3. In the discussion, although it was mentioned that kindlin-2 expression was not changed when kindlin-1 was deleted, there is not kindlin-2 western blot nor staining provided in the manuscript to support this claim. Regarding the functional redundancy between kindlin-1 and kindlin-2, which was also pointed out by the authors, it is important to include this data in the manuscript.

4. IL-6 and CXCL13 were both upregulated in the secretome of met-1 cells when kindlin-1 was deleted. CXCL13's upregulation was much more pronounced than IL-6 (20 folds VS 5 folds). Although CXCL13 is mainly involved in B cell recruitment, it was also reported to recruit T cells and regulate their functions (Li et al., DOI: 10.1016/j.jhep.2019.09.031; Zhou et al., DOI: 10.1126/science.abd5926). It is risky to exclude the possibility that CXCL13 also plays a role in the increased anti-tumor immunity induced by kindlin-1 deletion. Furthermore, in the supplementary Figure 5, which is the basis of the claim that CXCL13 does not play a role, it seems to me that there is a trend of decrease in the B cell recruitment when kindlin-1 was deleted. The P value is very close to 0.05 and the sample number is only 3. In this case, increasing the N number is essential to justify the claim. I strongly suggest to include CXCL13 antibody in the experiment in Figure 6 E and F.

5. IL-6 can have opposing effects on tumor development depending on the context. Chronic IL-6 signaling has been linked in many mouse tumor models (Fisher et al., DOI: 10.1016/j.smim.2014.01.008; Bent et al., DOI: 10.1038/s41467-021-26407-4). As far as I know, the authors did not directly test the role of IL-6 in the in vivo xenograft or PyV-MT models. Can injection of IL-6 or its antibody modulate the tumor growth and T cell differentiation in these two models? In my opinion, the best way to justify the essential role of IL-6 in the proposed model would be knocking out IL-6 on top of Kindlin-1 deletion in met-1 cells and check if this could abrogate the decreases of Treg population and tumor growth.

6. It is a surprise that re-expression of kindlin-1 AA mutant was able to restore the tumor growth. This is an important observation as it implies that integrin activation by kindlin-1 is not required for the growth of met-1 tumor xenograft. Interestingly, in their previous study (Sarvi et al., Cancer Research, 2018), the authors demonstrated that the integrin-binding capacity of kindlin-1 is required for the lung metastasis of met-1 cells. Combining both studies, it seems that the integrin signaling mediated by kindlin-1 is not required for the tumor growth itself but is indispensable for the tumor metastasis. I suggest the authors perform more control experiments to strengthen this conclusion. For example, 9EG7 staining in the WT or AA kindlin-1 expressing cells or tumor and then quantify the integrin activation. Can the re-expression of AA kindlin-1 mutant in the kindlin-1 null cells restore the cytokine profile?

This study focuses on the effect of kindlin-1 deletion on immunoregulation of myeloid cells within breast tumor. Although this is an interesting observation, the author did not provide any mechanistic explanation about how kindlin-1 regulates cytokine production/secretion. Does this regulation depend on the mechano-transduction mediated by kindlin-1? It is most likely not because the author showed the integrin-binding deficient kindlin-1 also promoted tumor growth. There are already several studies showed that Kindlin-2 can regulate the transcription of Axin2, Sox9 and YAP/TAZ (Yu et al., EMBO reports, 2012; Wu et al., Nat Comm, 2015; Guo et al., J Cell Biol, 2018). The author should at least add a section discussing the potential mechanism by which kindlin-1 regulates IL-6 secretion in the discussion.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Kindlin-1 regulates IL-6 secretion and modulates the immune environment in breast cancer models" for further consideration by eLife. Your revised article has been evaluated by Tadatsugu Taniguchi (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Please address Reviewer#1 recommendations (below) by revising the manuscript text.

Reviewer #1 (Recommendations for the authors):

The resubmitted manuscript clarified some of this reviewer's concerns, however, there are still some remaining.

1. Please adjust the size and legend of Figure 1—figure supplement 1. In the revised version, part of the legend is not visible in the pdf file.

2. Figure 1—figure supplement 1 A, what is the loading control protein? Please make the annotation.

3. Can the authors provide the pictures for Ki-67 and pH3 immunostaining along the quantification in Figure 1 Figure supplement 1G? As this is an important control experiment for the entire manuscript.

4. Figure 7 shows that deletion or blockade of IL-6 was not able to influence tumor growth, although it did increase the Foxp3+ Treg population. On the other hand, the depletion of CD25+ cells reduced tumor growth. Blockade of CD25 could have very complex and profound effects as it is a less specific marker for T reg cells compared to Foxp3. The effects of CD25 blockade might not solely come from the inhibition of Tregs. I suggest directly targeting the Foxp3+ Tregs (e. g. doi: 10.1080/2162402X.2019.1570778; doi: 10.1136/jitc-2021-003892) to confirm whether Treg is responsible for kindlin-1's function in tumor immunity.

Reviewer #2 (Recommendations for the authors):

The authors have addressed (or justified where experiments cannot be performed) all my concerns.

eLife. 2023 Mar 8;12:e85739. doi: 10.7554/eLife.85739.sa2

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #2 (Recommendations for the authors):

Here the authors show that the deletion of Kindlin-1 in tumor causes a delay in its growth and reduces overall tumor burden. This effect was more pronounced in T cell sufficient hosts compared to the T cell deficient ones. Therefore, the authors hypothesized that tumor infiltrating T cells are controlled, at least in part, by Kindlin-1. Although the authors present some phenotypical changes in the DC and T cell compartments, it is still unclear whether these changes account for an altered anti-tumor T cell activity. Furthermore, the authors claim that the effect is possibly driven by increased IL-6 in the TME in the absence of Kindlin-1, however, in vivo data supporting this claim are missing. While the overall effects of the lack of tumor derived Kindlin-1 is interesting, how this regulates T cell activity and which mechanism(s) are responsible remain elusive due to insufficient evidence.

Figure 1 -

To prove that the deletion of Kindlin-1 promotes lasting immunological memory, have the authors also rechallenged Kindlin WT group with Kindlin WT tumor? Does that group have impaired immunological memory?

As the Kin1-WT tumors do not regress it is not possible to do a rechallenge experiment in these mice due to the rapid growth of the initial tumor.

Figure 2-

Overall, this reviewer doesn't think the claim "the PD-1/L1 pathway is important for Kin1-WT tumors to control the anti-tumor immune response." is substantiated here.

We agree with the reviewer that we cannot substantiate this claim and have therefore reworded the text: “These data show that loss of Kindlin-1 leads to modulation of PD-L1 expression on tumor infiltrating immune cells, suggesting that the PD-1/L1 pathway may play a role in controlling the anti-tumor immune response.”

D-E: What is the expression level (MFI) for CD80 and PD-L1 on the DCs? Histograms comparing the expression levels of PD-L1 for DCs from Kindlin-WT vs null need to be demonstrated.

Histograms have now been included (Figure 2—figure supplement 2B).

H: PD-L1 expression doesn't seem to decrease in tumor infiltrating CD45+ cells as indicated by the p value (=0.05).

We have stated in the text that the reduction in PD-L1 in the tumor infiltrating CD45+ cells did not reach significance (p=0.05).

I: This reviewer doesn't understand the relevance of this experiment to the hypothesis tested here. What happens when PD-L1 is blocked in Kindlin-null group?

This experiment was included to demonstrate that the Kin1-WT cells are responsive to inhibition of the PD1/PD-L1 pathway. However, we acknowledge that this does not add to our understanding of how Kindlin-1 may regulate this pathway and have therefore removed Figure 2I. As the Kin1-NULL tumors are cleared it would not be possible to look at the effect of PD-L1 blockade on tumour growth.

Also, absolute cell numbers should be added to the supplementary.

Although cell counts were performed from single cell suspensions of the tissues before staining, this was performed with a cell counter. To accurately collect absolute cell numbers from flow cytometry data, counting beads need to be added to the sample tubes while acquiring the data on the analyser. As this has not been performed, any ‘absolute cell numbers’ would not be accurate, therefore the data has been presented mainly as a percentage of total cells (which is after double and dead cell removal). These data should still accurately represent the proportions of cells within the tumours as it takes into account the amount of total cells within the tumour samples.

Figure 3 -

How does the homeostatic proliferation of Tregs change? Have the authors checked Ki-67 expression?

There is no difference in Ki67 in Tregs between the Kin1-WT and Kin1-NULL tumors. These data have been included in Figure 4E.

This figure lacks information on the functionality of effector CD4+ and CD8+ T cells. Is there an effect of reduced Treg infiltration on the ability of CD4 and CD8 cells to produce cytokines, GzmB etc.?

We see a significant decrease in expression of the degranulation marker CD107a and also granzymeB in CD8+ effector T cells in Kin1-NULL tumors. These data have been included in Figure 4F and G.

Also, absolute numbers of CD4+, CD8+ and Tregs should be added to the supplementary.

See previous comment regarding use of absolute cell numbers in the analysis.

Figure 4-

Kindlin-1 deletion indeed seem to alter Treg phenotype however, the FACS plots for these phenotypical changes should be added to supplementary for better clarity.

We have now included the FACS plots as Figure 4—figure supplement 1.

Regarding Treg function, the phenotypical changes should not be used as a means to interpret the changes in Treg activity. For instance, a downregulation of GITR and PD-1 may as well have boosting effects on Treg function. Ideally, the gold standard test for Treg function would be performing a suppression assay. However, due to the difficulty of isolating Tregs from tumor, the functionality and proliferation of CD4 and CD8 effector cells (indicated by cytokine, GzmB production and Ki-67+ %) in addition to CD83 etc. are crucial.

As detailed above these data have now been included in Figure 4.

Figure 6-

in vitro Treg/Th17 conversion seem to be only marginally affected, if at all, by the proinflammatory milieu created by Kindlin-null cells. However, to what extent such a conversion takes place in the TME is largely unknown. Depleting Tregs by a-CD25 can reduce tumor burden in multiple tumor models and its effect doesn't have to be due presence or absence of Kindlin. One key experiment here could be treating Kindlin WT mice with blocking anti-IL-6 antibody, and comparing it to the Kindlin-null tumor to see whether the proinflammatory environment attributed to the lack of Kindlin can be recapitulated.

We agree that this is an important question. However, as the Kin1-WT cells do not express or secrete IL-6 we took an alternative approach to determine the role of tumor-derived IL6 in the Kin1-NULL tumors. As antibody treatment in vivo would impact on IL6 secreted from both tumour and stromal cells we first generated Kin1-NULL Met-1 cells in which we genetically deleted IL6 using CRISPR Cas-9. This resulted in a significant reduction in secretion of IL6 (Figure 6C) in Kin1-NULL cells. Subsequent immune profiling of tumors collected 10 days following tumor cell implantation showed that deletion of IL6 in Kin1-NULL tumors led to an increased number of CD4+ T cells and also an increase in the number of Tregs (Figure 7A). Although depletion of IL6 in the Kin1-NULL tumours was able to increase the number of Tregs cells to levels equivalent to those seen in the Kin1-WT tumors, and reduce the number of activated (CD107a+ GranzymeB+) cytotoxic T cells (Figure 7B), there was no effect on tumor clearance (Figure 7C, D). We then carried out experiments with an IL-6 neutralizing antibody in mice implanted with the Kin1-NULL cells. There was no effect tumor clearance in mice treated with the IL6 neutralizing antibody (Figure 7E). Together these data show that although the increased secretion of IL6 following loss of Kindlin-1 can reverse the effects on Treg infiltration and also reduce the number of activated cytotoxic T cells, it is not sufficient to prevent tumor clearance indicating that other mechanisms are involved in the anti-tumor immunity. As discussed in the text the data we show support a role for Kindlin-1 in several potential methods of immune cell regulation. It is likely therefore that in the context of the complex tumor environment that the IL6 mediated effects on the suppressive activity of Tregs on CD8+ T cells (demonstrated in vitro) is not sufficient on its own to prevent Kin1-NULL tumor clearance. However, the data presented does provide the first description of how Kindlin-1 regulates the tumor immune environment which IL6 contributes to. Such a detailed study provides a significant advance in our understanding of how this key adhesion protein impacts on tumor progression and we therefore believe it warrants publication. Future studies will explore how other pathways and factors may act in concert with IL6 to drive anti-tumor immunity.

Also, have the authors thought about the possibility that Kindlin-1 wt TME may have more TGF-b? Can a TGF-b blockade reverse the amelioration of tumor burden in Kindlin-1 null mice?

There was no difference in Tgfb1 and Tgfb3 in Kin1-NULL cells while there was a reduction in Tgfb2. However, this difference was not seen upon ex vivo analysis of Kin1-NULL and Kin1-WT tumors. Furthermore, immunohistochemical analysis of phospho-SMAD3 in Kin1-NULL and Kin1-WT tumors showed that there was no difference in pathway activation. These data have now been included as (Figure 5—figure supplement 1). We therefore do not think that TGF-b signalling is playing an important role in tumor growth.

Reviewer #3 (Recommendations for the authors):

Webb et al. investigated the role of Kindlin-1 in the development of breast cancer using both the Met-1 tumor cell xenograft model and the PyV-MT mice breast tumor model. The authors found that deletion/depletion of kindlin-1 resulted in the suppression of tumor growth, concluding kindlin-1 promotes breast tumor development. Surprisingly, they find that the integrin-binding capacity of kindlin-1 is not required for this function. They analyzed the infiltration of immune cells within the tumor and find that the population of Treg cells was significantly decreased in the kindlin-1 null tumor. Cytokine-profiling revealed that the secretion of IL-6 and several other cytokines were altered. The author used conditioned medium from Kindlin-1 WT and Kindlin-1 null cells to treat the co-culture of Treg and CD8+ T cells isolated from the mice spleen. They found that kindlin-1 deletion increased the proliferation of CD8+ T cells in the co-culture system and this effect can be blocked by addition of IL-6 antibody. The author suggests that the upregulation of IL-6 secretion from the kindlin-1 null tumor cells modulates the T cell differentiation, which lead to decrease of the Treg population and function and therefore the increase of anti-tumor immune response.

Overall, this is an interesting study which focus on the role of kindlin-1 in the anti-tumor immunity. However, in my opinion, some experiments need to be refined and more data is needed to clarify and support the hypothesis.

Specific points

1. The authors showed that deletion of kindlin-1 impaired tumor growth in both breast cancer models. The rest of the manuscript completely focuses on the immunoregulation within the tumor. Kindlin proteins are essential activator of integrin signaling, which can cooperate with growth factor receptors to promote cell proliferation. The proliferation of the tumor cells themselves should be monitored as well (in vitro MTT assay, kindlin-1 WT VS kindlin-1 null cells; phospho-histone H3 staining in the WT and kindlin-1 null tumors) to exclude the possibility that kindlin-1 directly promotes tumor cell proliferation.

There was no effect of Kindlin-loss on the in vitro proliferation of the Met-1 or MDA-MB-231 cells as assessed by alamarBlue assay. Furthermore, analysis of the Nanostring PanCancer gene panel showed no difference in cell cycle gene expression in the Met-1 model. Immunohistochemical analysis of Ki67 and phospho-histone H3 was unaltered in the tumors collected from the CD1 nude mice demonstrating that Kindlin-1 does not directly promote tumor cell proliferation. These data have been included in Figure 1—figure supplement 1.

2. The authors mentioned that kindlin-1 expression is higher in tumor compared to normal tissues. What happen to the xenograft if kindlin-1 is overexpressed in the met-1 cells? Will it promote the onset of tumorigenesis/ increase the tumor size and also decrease IL-6 secretion? What happen to the tumor growth and cytokine profile if an integrin-binding deficient mutant of kindlin-1 (AA mutant) is overexpressed?

Reports in other breast cancer cell lines have shown that overexpression of Kindlin-1 can enhance tumour growth (doi: 10.1002/cac2.12338; doi: 10.1093/jnci/djr290). In this study we have focused on the role of Kindlin-1 in immune regulation. With respect to IL6 secretion, the Kin1-WT and parental Met-1 cells do not express IL6 so we would not be able to able to address whether overexpression further reduces IL6 secretion.

The Kin1-AA cells secrete very low levels of IL6 and CXCL13 similar to that seen in the Kin1-WT cells. These data are now included in Figure 5—figure supplement 2A.

3. In the discussion, although it was mentioned that kindlin-2 expression was not changed when kindlin-1 was deleted, there is not kindlin-2 western blot nor staining provided in the manuscript to support this claim. Regarding the functional redundancy between kindlin-1 and kindlin-2, which was also pointed out by the authors, it is important to include this data in the manuscript.

These data are now included in Figure 1—figure supplement 1.

4. IL-6 and CXCL13 were both upregulated in the secretome of met-1 cells when kindlin-1 was deleted. CXCL13's upregulation was much more pronounced than IL-6 (20 folds VS 5 folds). Although CXCL13 is mainly involved in B cell recruitment, it was also reported to recruit T cells and regulate their functions (Li et al., DOI: 10.1016/j.jhep.2019.09.031; Zhou et al., DOI: 10.1126/science.abd5926). It is risky to exclude the possibility that CXCL13 also plays a role in the increased anti-tumor immunity induced by kindlin-1 deletion. Furthermore, in the supplementary Figure 5, which is the basis of the claim that CXCL13 does not play a role, it seems to me that there is a trend of decrease in the B cell recruitment when kindlin-1 was deleted. The P value is very close to 0.05 and the sample number is only 3. In this case, increasing the N number is essential to justify the claim. I strongly suggest to include CXCL13 antibody in the experiment in Figure 6 E and F.

We agree that we cannot exclude the possibility that CXCL13 plays a role in the increased anti-tumor immunity induced by Kindlin-1 deletion and have reworded the text to make this point. We present new data showing that the CXCL13 blocking antibody has no effect on Treg differentiation (Figure 6—figure supplement 1).

5. IL-6 can have opposing effects on tumor development depending on the context. Chronic IL-6 signaling has been linked in many mouse tumor models (Fisher et al., DOI: 10.1016/j.smim.2014.01.008; Bent et al., DOI: 10.1038/s41467-021-26407-4). As far as I know, the authors did not directly test the role of IL-6 in the in vivo xenograft or PyV-MT models. Can injection of IL-6 or its antibody modulate the tumor growth and T cell differentiation in these two models? In my opinion, the best way to justify the essential role of IL-6 in the proposed model would be knocking out IL-6 on top of Kindlin-1 deletion in met-1 cells and check if this could abrogate the decreases of Treg population and tumor growth.

We agree that this is an important question. As antibody treatment in vivo would impact on IL6 secreted from both tumour and stromal cells we first generated Kin1-NULL Met-1 cells in which we genetically deleted IL6 using CRISPR Cas-9. This resulted in a significant reduction in secretion of IL6 (Figure 6C) in Kin1-NULL cells. Subsequent immune profiling of tumors collected 10 days following tumor cell implantation showed that deletion of IL6 in Kin1-NULL tumors led to an increased number of CD4+ T cells and also an increase in the number of Tregs (Figure 7A). Although depletion of IL6 in the Kin1-NULL tumours was able to increase the number of Tregs cells to levels equivalent to those seen in the Kin1-WT tumors, and reduce the number of activated (CD107a+ GranzymeB+) cytotoxic T cells (Figure 7B), there was no effect on tumor clearance (Figure 7C, D). We then carried out experiments with an IL-6 neutralizing antibody in mice implanted with the Kin1-NULL cells. There was no effect tumor clearance in mice treated with the IL6 neutralizing antibody (Figure 7E). Together these data show that although the increased secretion of IL6 following loss of Kindlin-1 can reverse the effects on Treg infiltration and also reduce the number of activated cytotoxic T cells, it is not sufficient to prevent tumor clearance indicating that other mechanisms are involved in the anti-tumor immunity. As discussed in the text the data we show support a role for Kindlin-1 in several potential methods of immune cell regulation. It is likely therefore that in the context of the complex tumor environment that the IL6 mediated effects on the suppressive activity of Tregs on CD8+ T cells (demonstrated in vitro) is not sufficient on its own to prevent Kin1-NULL tumor clearance. However, the data presented does provide the first description of how Kindlin-1 regulates the tumor immune environment which IL6 contributes to. Such a detailed study provides a significant advance in our understanding of how this key adhesion protein impacts on tumor progression and we therefore believe it warrants publication. Future studies will explore how other pathways and factors may act in concert with IL6 to drive anti-tumor immunity.

6. It is a surprise that re-expression of kindlin-1 AA mutant was able to restore the tumor growth. This is an important observation as it implies that integrin activation by kindlin-1 is not required for the growth of met-1 tumor xenograft. Interestingly, in their previous study (Sarvi et al., Cancer Research, 2018), the authors demonstrated that the integrin-binding capacity of kindlin-1 is required for the lung metastasis of met-1 cells. Combining both studies, it seems that the integrin signaling mediated by kindlin-1 is not required for the tumor growth itself but is indispensable for the tumor metastasis. I suggest the authors perform more control experiments to strengthen this conclusion. For example, 9EG7 staining in the WT or AA kindlin-1 expressing cells or tumor and then quantify the integrin activation. Can the re-expression of AA kindlin-1 mutant in the kindlin-1 null cells restore the cytokine profile?

We have previously shown in Sarvi et al. Cancer Res (DOI: 10.1158/0008-5472.CAN-17-1518) (Supplementary Figure S4) that integrin activation (as measured by flow cytometry using the 9EG7 antibody) is reduced in the Kin1-AA cells. As discussed above IL6 and CXCL13 secretion is not restored in the Kin1-AA cells (Figure 5—figure supplement 2).

This study focuses on the effect of kindlin-1 deletion on immunoregulation of myeloid cells within breast tumor. Although this is an interesting observation, the author did not provide any mechanistic explanation about how kindlin-1 regulates cytokine production/secretion. Does this regulation depend on the mechano-transduction mediated by kindlin-1? It is most likely not because the author showed the integrin-binding deficient kindlin-1 also promoted tumor growth. There are already several studies showed that Kindlin-2 can regulate the transcription of Axin2, Sox9 and YAP/TAZ (Yu et al., EMBO reports, 2012; Wu et al., Nat Comm, 2015; Guo et al., J Cell Biol, 2018). The author should at least add a section discussing the potential mechanism by which kindlin-1 regulates IL-6 secretion in the discussion.

We now present evidence that indicate a transcription independent mechanism of IL-6 regulation of IL-6 by Kindlin-1. Analysis of data from the Nanostring PanImmune panel show that there is no difference in expression of Il6 between the Kin1-WT and Kin1-NULL cells (Figure 5D). Although IL-6 is regulated primarily via transcription, the regulation of IL-6 secretion is controlled by additional mechanisms including regulation of trafficking through the exocytic pathway (doi:10.1242/jcs.234781). Further work is required to establish how Kindlin-1 is regulating secretion of IL-6 but of note Kindlin-dependent regulation of integrin trafficking to the plasma membrane has been reported (doi: 10.1016/j.cub.2012.06.060). This has been discussed in the text.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #1 (Recommendations for the authors):

The resubmitted manuscript clarified some of this reviewer's concerns, however, there are still some remaining.

1. Please adjust the size and legend of Figure 1—figure supplement 1. In the revised version, part of the legend is not visible in the pdf file.

We thank the reviewer for spotting this mistake. In order to solve this, and include the representative images requested in point 3, Figure 1—figure supplement 1 has now been broken into 2 separate Figure supplements. Therefore, the legend has been adjusted on Figure 1—figure supplement 1 and the new Figure 1—figure supplement 2, and should both be readable. The corresponding text in the manuscript has been changed and highlighted to reflect this.

2. Figure 1—figure supplement 1 A, what is the loading control protein? Please make the annotation.

This error has now been corrected with the loading control labelled as β-actin, in Figure 1—figure supplement 1.

3. Can the authors provide the pictures for Ki-67 and pH3 immunostaining along the quantification in Figure 1 Figure supplement 1G? As this is an important control experiment for the entire manuscript.

Representative images of both Ki67 staining and pH3 staining have now been added to the newly created Figure 1—figure supplement 2 (see point 1 above), alongside the corresponding quantification.

4. Figure 7 shows that deletion or blockade of IL-6 was not able to influence tumor growth, although it did increase the Foxp3+ Treg population. On the other hand, the depletion of CD25+ cells reduced tumor growth. Blockade of CD25 could have very complex and profound effects as it is a less specific marker for T reg cells compared to Foxp3. The effects of CD25 blockade might not solely come from the inhibition of Tregs. I suggest directly targeting the Foxp3+ Tregs (e. g. doi: 10.1080/2162402X.2019.1570778; doi: 10.1136/jitc-2021-003892) to confirm whether Treg is responsible for kindlin-1's function in tumor immunity.

We agree with the reviewer that CD25 is not a specific Treg cell marker, and that depletion by anti-CD25 antibody may have effects on other cell functions. However, in mice it is constitutively and highly expressed on Treg cells, compared to other immune cells. Furthermore, depletion of Treg cells by anti-CD25 antibodies has been widely used throughout the literature, and therefore could be regarded as a standard method of analysing effects of Treg depletion in vivo. Incidentally, antibodies which target CD25 are currently being optimised and tested in clinical trials, with the main mechanism of action being targeted Treg depletion. This therefore, is why we chose to use this method of depletion. Although, the suggestion of targeting of FoxP3 as proposed by the reviewer, does sound very promising, these involve use of new approaches for which the materials are not readily available. These will however be useful in confirming the effect we see here in future investigations of the role Tregs play in this model.

To ensure that the anti-CD25 antibody was depleting the cells of interest (Tregs), we analysed tumours of mice after treatment, which demonstrated a depletion of CD4+ FoxP3+ (Treg cells), with no significant decrease in CD4+ FoxP3- (non-Treg CD4s) cells (Figure 7—figure supplement 1). We believe these data provide support, that in this model, the anti-CD25 antibody was specifically depleting Treg cells. However, to clarify that CD25 is not an exclusive marker present on T cells, we have added a statement in the text (p19).

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, Speed D, Lynch AG, Samarajiwa S, Yuan Y, Gräf S, Ha G, Haffari G, Bashashati A, Russell R, McKinney S, Langerød A, Green A, Provenzano E, Wishart G, Pinder S, Watson P, Markowetz F, Murphy L, Ellis I, Purushotham A, Børresen-Dale AL, Brenton JD, Tavaré S, Caldas C, Aparicio S, METABRIC Group 2012. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. European Genome-Phenome Archive. EGAS00000000083 [DOI] [PMC free article] [PubMed]

    Supplementary Materials

    Figure 1—figure supplement 1—source data 1. Raw western blot images for Figure 1—figure supplement 1A, C.
    elife-85739-fig1-figsupp1-data1.pdf (594.2KB, pdf)
    Figure 1—figure supplement 2—source data 1. Nanostring PanCancer panel analysis of Met-1 Kin1-WT and NULL cells in vitro.
    elife-85739-fig1-figsupp2-data1.xlsx (101.5KB, xlsx)
    Figure 5—source data 1. Forward Phase Protein Array of Met-1 cells (raw values).
    elife-85739-fig5-data1.xlsx (29.7KB, xlsx)
    Figure 5—source data 2. Nanostring PanCancer Immune panel analysis of Met-1 Kin1-WT and NULL cells in vitro.
    elife-85739-fig5-data2.xlsx (99.4KB, xlsx)
    Supplementary file 1. List of antibodies used for immunophenotyping.
    elife-85739-supp1.xlsx (10.1KB, xlsx)
    MDAR checklist
    elife-85739-mdarchecklist1.docx (98.8KB, docx)
    Source data 1. NanoString PanCancer Immune panel analysis of bulk Met-1 tumors at day 10 (normalised expression), related to Figure 2 and Figure 5.
    elife-85739-data1.csv (101.9KB, csv)
    Source data 2. Nanostring immune exhaustion panel of isolated CD45+ cells from Met-1 tumors at Day 10, related to Figure 2, Figure 4 and Figure 6.
    elife-85739-data2.csv (93.6KB, csv)

    Data Availability Statement

    All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1, 2, 4, 5 and 6.

    The following previously published dataset was used:

    Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, Speed D, Lynch AG, Samarajiwa S, Yuan Y, Gräf S, Ha G, Haffari G, Bashashati A, Russell R, McKinney S, Langerød A, Green A, Provenzano E, Wishart G, Pinder S, Watson P, Markowetz F, Murphy L, Ellis I, Purushotham A, Børresen-Dale AL, Brenton JD, Tavaré S, Caldas C, Aparicio S, METABRIC Group 2012. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. European Genome-Phenome Archive. EGAS00000000083

    Articles from eLife are provided here courtesy of eLife Sciences Publications, Ltd

    RESOURCES