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 supplement 1. Flow cytometry gating examples of T cell populations.

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.

(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-1^wt/wt and MMTV-Kin-1^fl/fl spontaneous tumor model.

(A) Spontaneous MT-Kin-1^wt/wt or MT-Kin-1^fl/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-1^wt/wt or MT-Kin-1^fl/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.