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. Author manuscript; available in PMC: 2023 Aug 15.
Published in final edited form as: J Immunol. 2022 Jul 29;209(4):660–664. doi: 10.4049/jimmunol.2200317

Promoting TCF1+ T-cell self-renewal to improve PD-1 blockade

Peter H Wang *, Robert Washburn *, Rohan Maniar †, Michael Mu *, Olivia Ringham *, Radomir Kratchmarov *,2, Brian S Henick †, Steven L Reiner *,‡
PMCID: PMC9387677  NIHMSID: NIHMS1820664  PMID: 35905999

Abstract

Immune checkpoint blockade is limited by resistance to treatment, with many patients not achieving durable anti-tumor responses. Self-renewing (TCF1+)3 CD8+ T cells have recently been implicated in efficacy of anti-PD-13. Mice challenged with syngeneic tumors were treated with anti-PD-1 and/or a reversible iPI3Kδ3, designed to promote T cell self-renewal. Growth of tumors in untreated mice was characterized by waning proportions of TCF1+ T cells, suggesting self-renewing T cells become limiting for successful immunotherapy. Higher proportions of TCF1+ T cells in tumor and blood correlated with better control of tumor growth. Combining anti-PD-1 and iPI3Kδ conferred superior protection compared to either monotherapy and was associated with higher frequency of TCF1+ T cells in tumor and blood compared to anti-PD-1 alone. These findings reveal predictive importance of self-renewing T cells in anti-tumor immunity and suggest that resistance-directed strategies to enhance T-cell self-renewal could potentiate the efficacy of PD-1 blockade.

Introduction

T cells are crucial in the control of infections and cancer. Recent advances have supported a model in which antigen-specific T cell responses are expanded and maintained through a process of cellular regeneration. Quiescent precursor cells are activated to yield proliferative progenitors, whose further activation yields irreversibly differentiated effector T cells (1–4). The stem cell-like ability of T cells to self-renew while producing differentiated progeny appears essential for continuous production of functional T cells.

In contrast to acute and persistent low-level infections, the repetitive antigenic activation associated with chronic-active viral infections and cancer is often characterized by progressive dysfunction of the effector T cell response (5). In the setting of T cell dysfunction, it is sometimes possible to intervene therapeutically using blockade of inhibitory signals in order to intensify the T cell response. Mounting evidence, however, has suggested that the primary mechanism-of-action of PD-1 blockade is not to reverse dysfunction of the most differentiated T cells, but rather to drive proliferation and differentiation of self-renewing (TCF1+)3 progenitor T cells to produce a large wave of fresh effector-like T cells (1, 3, 6–9). In this study, we provide evidence that limiting availability of self-renewing T cells represents a category of resistance to immunotherapy. We examined the nature of the responding CD8+ T cell subsets during syngeneic tumor challenges in mice. Results of therapeutic PD-1 blockade, with and without an intermittent administration of an inhibitor of phosphoinositide 3-kinase delta (iPI3Kδ)3 to enhance T-cell self-renewal suggests a possible strategy to overcome some forms of resistance to immune checkpoint blockade.

Materials and Methods

Tumor challenges

C57BL/6J (Jackson Laboratories) mice aged 8–12 weeks were used for tumor challenges with MC38 colon carcinoma (a gift from Dr. Arnold Han, Columbia U.) or CMT167 Kras mutant lung carcinoma cells (ATCC). Tumor cells (2.5×105) were inoculated subcutaneously into the right flank and tumors were measured every other day with metric calipers in two dimensions. Volume was estimated as the longer dimension (length) multiplied by the square of the shorter dimension (width) multiplied by π/6. Mice were euthanized if tumor volume exceeded 4 cm3. Tumor-challenged mice were randomized to treatment with anti-PD-1 [200 μg (female); 250 μg (male)] (J43 clone, BioXcell) or isotype control (Armenian Hamster IgG, BioXcell) intraperitoneally weekly beginning at day 3 (MC38) or day 5 (CMT167) until day 19 (CMT167) or day 24 (MC38). A subset of anti-PD-1 treated mice were randomized to treatment with iPI3Kδ 200 μg (Idelalisib; Selleckchem) or vehicle by oral gavage weekly starting 2 days before the second anti-PD-1 treatment. In experiments evaluating tumor infiltrating lymphocytes, mice were treated with anti-PD-1 on days 4, 7, 10, 13 and/or iPI3Kδ on days 8, 9, 11, 12. Animals were housed in a specific pathogen-free facility accredited by Association for Assessment and Accreditation of Laboratory Animal Care, and experiments were performed in accordance with Columbia University’s Institutional Animal Care and Use Committee.

Tumor-infiltrating lymphocyte and blood collection

Tumor tissue was resuspended in enzymatic mix of 5 mg/ml collagenase type I (MP Biomedical), 1.5mg/ml hyaluronidase Type I-S (Sigma-Aldrich), and 0.01 mg/ml DNase I (Worthington Biochemical) and incubated at 37C for 1 hour, vortexing periodically. Digests were filtered through a 70 μm cell strainer (Thermo Fisher) and washed twice with FACS buffer. Lysates were stained with Live/Dead Fixable Near-IR Dead cell stain kit (Thermo Fisher). Cells were then washed once with FACS buffer. Ante-mortem blood samples were collected in phosphate buffered saline in 5 μM EDTA mice. Red blood cells were lysed by ACK lysis (Thermo Fisher) for 5 minutes, and cells were washed twice with phosphate buffered saline.

Flow cytometry, intracellular staining

Single cell suspensions from murine peripheral blood or tumor cell suspensions were incubated with anti-mouse CD16/32 (clone 13; BioLegend) to block Fc receptors and stained with fluorescent-labeled antibodies. Prior to staining intracellular proteins, cells were fixed and permeabilized by FoxP3/Transcription Factor Staining Buffer Kit (TONBO). FACS antibodies were purchased from BioLegend, Cell Signaling Technology, Invitrogen, or BD Biosciences and included: CD3 (145–2C11), CD8b (YTS156.7.7), CD4 (RM4–5), CD62L (MEL-14), CD44 (IM7), TCF1 (C63D9), FoxP33 (MF-14), pS63 (D57.2.E). Flow cytometric data was analyzed with Flowjo V10.

In vitro T cell activation

Splenocytes from OT-I mice were activated with OVA peptide 10 μg/ml (GenScript)/APCs, recombinant huIL-2 100 units/mL (Thermo Fisher) in complete RPMI media (Gibco). In some experiments, cells were labeled with CFSE 5 μM (TONBO) to track cell division. Some cells were incubated with iPI3Kδ 10 μg/ml. Cells were collected after 3 or 4 days, washed with FACS buffer and stained for CD8 followed by fixation and permeabilization and staining of pS6 and TCF1. Glucose uptake was measured after collection by washing cells two times, and incubating cells in 2-NBDG3 100 μM (Cayman) at 45 mins, 37C in the dark.

Statistical analyses

One-way repeated measures ANOVA with Geisser-Greenhouse correction followed by a Tukey post-test for tumor growth curves; one-way ANOVA with Dunnett’s multiple comparison test for immune phenotypes grouped by treatment arms; and Pearson correlation between immune phenotype and tumor size as continuous variables were performed, all with Prism V9.

Results and Discussion

Waning fractions of self-renewing T cells during tumor progression

Progressive tumor growth is associated with increasing dysfunction of differentiated CD8+ T cells as well as waning fractions of self-renewing (TCF1+) progenitor CD8+ T cells (5). In mice challenged by flank injection of syngeneic melanoma cells or an autochthonous hepatocellular carcinoma model, the fraction of intratumoral antigen-specific TCF1+CD8+ T cells becomes substantially depleted after 2 weeks of tumor growth (8, 10). Our analysis of the polyclonal response of intratumoral CD8+ T cells from MC38 colon carcinoma tumors also revealed dramatic loss of TCF1+ T cells after 2 weeks of subcutaneous tumor growth (Fig. 1A, Supplemental Fig. 1A). This observation is compatible with a model supported by other findings wherein progressive chronic-active viral infection or substantial malignancy leads to a state of diminished T-cell regenerative capacity (7, 8, 10–13). Self-renewing progenitors, the critical source of freshly differentiated T cells, appear to become progressively depleted by the continual demand for production of effector T cells, while differentiated T cells become progressively dysfunctional as a result of chronic stimulation (5).

Figure 1. Ongoing intratumoral activation and PI3Kδ inhibition exert opposing effects on CD8+ T-cell self-renewal.

Figure 1.

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(A) Quantitation of % TCF1+ among intratumoral CD8+ T cells of untreated, MC38-challenged mice at indicated times post inoculation (all time points n ≥3 mice, except d19 and d25 [all n=2]). (B) OT-I transgenic CD8+ T cells stimulated as described in Materials and Methods were pulsed with 2-NBDG on d3 to index glucose uptake. Quantitation of relative 2-NBDG uptake in untreated (“ctrl”, grey) and iPI3Kδ-treated (black) cells (n=4/group). (C) Quantitation of pS6 intensity of activated, untreated (grey) and iPI3Kδ-treated (black) OT-I CD8+ T cells at d3 (n=9/group). (D) Left, representative flow cytometry of cell division versus TCF1 expression from CFSE-labeled, activated, untreated and iPI3Kδ-treated OT-I CD8+ T cells at d3 and d4. Right, quantitation of % TCF1+ among CD8+ T cells (untreated, grey; iPI3Kδ-treated, black). (n=9/group). In all graphs, mean and ±SEM error bars are shown. At least three replicate experiments were performed. Statistical significance was determined by unpaired two-tailed t-tests: **p <0.01, ****p <0.0001.

A strategy to maintain T cell self-renewal

The progressive shift from TCF1+ to TCF1− T cells presumably results from ongoing antigenic/costimulatory activation mediated by PI3Kδ signaling (4, 14–17). Consistent with prior studies of polyclonal and P14 transgenic T cells, we found antigenic activation of OT-I transgenic T cells in the presence of an inhibitor of PI3Kδ resulted in substantial reduction in anabolic activation as assessed by uptake of 2-NBDG, a glucose analog (Fig. 1B, Supplemental Fig. 1B), mTOR activation as indicated by pS6 protein (Fig. 1C, Supplemental Fig. 1C), and division-dependent production of TCF1− T cells (Fig. 1D, Supplemental Fig. 1D). These findings are consistent with the suggestion that anabolic/proliferative metabolism favors T cell differentiation and TCF1 silencing, while catabolic/quiescent metabolism favors self-renewal and maintenance of TCF1 expression (15–18).

Inhibiting PI3K signals during anti-cancer PD-1 blockade

In contrast to antigen and costimulatory signaling, which cooperatively activate PI3Kδ signaling and anabolic T cell activation (19), PD-1 signaling opposes anabolic T cell activation (20, 21). Consequently, blockade of PD-1 drives proliferation and differentiation of TCF1+ progenitors to produce large numbers of fresh TCF1− cell descendants during chronic-active infections and cancer (1, 3, 6–9). We, therefore, hypothesized that intermittent inhibition of PI3Kδ might improve the efficacy of PD-1 blockade if it could help to maintain the pool of self-renewing TCF1+ T cells in vivo.

Mice inoculated with MC38 colon carcinoma were randomized to weekly treatments of control antibody, anti-PD-1, iPI3Kδ, or combination therapy (Supplemental Fig. 1E). Treatment with anti-PD-1 or iPI3Kδ resulted in a statistically significant reduction in tumor volume (Fig. 2A), with substantial control of tumor growth in small number of anti-PD-1-treated mice (Supplemental Fig. 2A). The combination regimen, however, led to a greater and more uniform reduction in tumor growth compared to either monotherapy or control regimen. Untreated female mice exhibited increased tumor growth compared to male mice, with better responses to monotherapies and combination therapy than male mice (Supplemental Fig. 2A, 2D).

Figure 2. Addition of PI3Kδ inhibitor improves tumor control and T-cell renewal in anti-PD-1-treated MC38-challenged mice.

Figure 2.

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(A) Tumor growth curves of MC38-challenged mice treated with indicated agents (n=10–13 mice/group). Throughout tumor growth curve figures, vertical lines and significance symbols refer to comparison of combination therapy to other groups (indicated by color). Horizontal significance symbols refer to comparison of ctrl Ig to treatments of indicated color. (B) Quantitation of % TCF1+ among intratumoral CD8+ T cells at d14 in mice treated with regimen of shorter interval and duration (n=7–13 mice/group). (C) Representative flow cytometry of CD8 versus TCF1 from samples quantitated in (B). (D) Correlation between % TCF1+ among CD44hiCD8+ peripheral blood T cells at d19 and tumor volumes at indicated times after inoculation. Mean and ±SEM error bars are shown (A, B). Three replicate experiments were performed. Statistical significance was calculated by repeated measure one-way ANOVA with Geisser-Greenhouse correction followed by a Tukey post-test (A), one-way ANOVA with Dunnett’s multiple comparison test (B) and Pearson correlation (D) **p <0.01, ***p < 0.001; r = correlation coefficient.

Self-renewing CD8+ T cell stabilization associated with combination therapy

To study the effect of interventions on CD8+ T cell dynamics without confounding differences of tumor size and antigen burden, mice were treated with regimens of shorter interval and duration (Supplemental Fig. 1E). Intratumoral T cells were analyzed at day 14 following challenge, before significant divergence in tumor volume between groups (Supplemental Fig. 2C). We found that greater stability of self-renewing T cells correlates with improved outcome from combination therapy. Mice treated with combination therapy exhibited a significant increase in frequency of intratumoral TCF1+CD8+ T cells compared to PD-1 blockade alone (Fig. 2B, 2C, Supplemental Fig. 1F), with a non-significant trend toward higher absolute numbers of TCF1+CD8+ T cells, but not of total CD8+ T cells (Supplemental Fig. 3A). Significant enhancement of the TCF1+CD8+ T cell fraction did not occur in mice treated with iPI3Kδ alone in vivo (Fig 2B), which might be due to the delayed administration and intermittent dosing schedule in vivo (Supplemental Fig. 1E) compared to the immediate and sustained application of drug in vitro (Fig. 1D, Supplemental Fig. 1D).

Blood-circulating TCF1+CD8+ T cells correlate with control of tumor growth

During local anti-tumor T cell responses, blood and lymphoid circulations are accessed by self-renewing TCF1+ precursor and progenitor T cells, as well as differentiated TCF1− T cells (22–27). Analysis of the TCF1+ fraction of antigen-experienced (CD44hi) CD8+ T cells as a continuous variable across treatment groups revealed an inverse correlation with both proximate and later tumor growth (Fig. 2D, Supplemental Fig. 1F). When grouped by treatment variable, non-significant trends of reduced TCF1+ frequency and absolute number were observed in the anti-PD-1 group compared to the control Ig group (Supplemental Fig. 3B). The combination treatment group exhibited a non-significant trend of increase in TCF1+ frequency compared to the anti-PD-1 group (Supplemental Fig. 3B). Although this approach does not distinguish between tumor-specific and non-specific antigen-experienced T cells in circulation, the findings suggest that non-invasive (blood) assessment of regenerative T cell dynamics may have predictive value for disease outcome.

Regenerative T cell dynamics during an independent tumor challenge

Treatment of mice subcutaneously challenged with a Kras-mutant lung cancer cell line, CMT167 also revealed improved outcome from combining anti-PD-1 and iPI3Kδ. Using a treatment schedule that began 2 days later and ended 5 days earlier than the regimen used in MC38 challenges (Supplemental Fig. 1E), combination treatment significantly enhanced control of CMT167 tumor growth compared to monotherapies and sham treatment (Fig. 3A, Supplemental Fig. 2B, 2E). Combination-treated mice in MC38 challenges responded better than in CMT167 experiments (Fig. 2A, 3A, Supplemental Fig. 1E), but further studies will be needed to determine if this was due to differences in treatment schedule or tumor-intrinsic factors.

Figure 3. Improved tumor control and T-cell renewal in CMT167-challenged mice treated with anti-PD-1-plus-iPI3Kδ.

Figure 3.

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(A) Tumor growth curves of CMT167-challenged mice treated with indicated agents (n=10–14 mice/group). (B) Quantitation of % TCF1+ among CD44hiCD62LloCD8+ blood T cells at d14 (colored symbols) compared to unchallenged mice (“naive”, black symbols). (C) Correlation between % TCF1+ values from blood shown in (B) and tumor volumes at indicated times after inoculation. (D) Quantitation of % TCF1+ among intratumoral CD8+ T cells at d14 in mice treated with shorter-interval regimen (n=12–15 mice/group). (E) Correlation between % TCF1+ intratumoral values shown in (D) and tumor volumes at d11. Mean and ±SEM error bars are shown (A, B, D). Three replicate experiments were performed with exception of two replicate experiments in B, C. Statistical significance was calculated by one-way repeated measures ANOVA with Geisser-Greenhouse correction followed by a Tukey post-test (A), one-way ANOVA with Dunnett’s multiple comparison test (B, D), and Pearson correlation (C, E) *p <0.05, **p <0.01, ***p < 0.001; r = correlation coefficient.

Examination of blood effector-memory phenotype (CD44hiCD62Llo) CD8+ T cells revealed a significant reduction in frequency and non-significant trend of reduction in absolute number of TCF1+ cells associated with PD-1 blockade, which were both corrected by combination therapy with anti-PD-1-plus-iPI3Kδ (Fig. 3B, Supplemental Fig. 3C, 3D). In less fractionated (CD44hi) antigen-experienced CD8+ T cells, a similar pattern was observed, albeit with lesser significance than in the CD44hiCD62Llo effector-memory subset (Supplemental Fig. 3E). Analyses of blood effector-memory TCF1+ frequency as a continuous variable across treatment groups revealed an inverse correlation with proximate and later tumor growth (Fig. 3C), as was observed for TCF1+CD44hiCD8+ T cells in MC38 challenges (Fig. 2D).

When treatments for CMT167 challenges were administered at shorter intervals and duration (Supplemental Fig. 1E), combination therapy resulted in higher frequency of intratumoral TCF1+CD8+ T cells (Fig. 3D). By contrast, absolute cell counts of TCF1+CD8+ T cells and CD8+ T cells did not increase as result of combination therapy (Supplemental Fig. 3F). As a continuous variable across treatment arms, intratumoral TCF1+CD8+ T cell frequency inversely correlates with tumor size (Fig. 3E), suggesting an association between T-cell regenerative status and disease course. Using an antigen-specific melanoma model, we confirmed that intratumoral TCF1+ frequency as a continuous variable is inversely related to tumor volume (Supplemental Fig. 1G, 3G).

Although efficacy of PD-1 blockade is CD8+ T cell-dependent (28), it has been suggested that the frequency of TCF1+CD8+ T cells, rather than absolute numbers of CD8+ T cells may predict response to immunotherapy in melanoma lesions from patients (13). The current correlation of TCF1+CD8+ T cell frequency with inhibition of tumor growth is consistent with other recent studies suggesting success of anti-PD-1 therapy specifically depends on the availability of self-renewing TCF1+CD8+ T cell progenitors (7, 8, 10–13).

It is likely that optimizing abundance of TCF1+CD8+ T cells by adding iPI3Kδ contributes to the observed benefit of combination therapy. While this manuscript was in preparation, other classes of PI3K inhibition combined with other inhibitory receptor blockade were reported to offer enhanced efficacy, at least partly owing to improved CD8+ T cell dynamics (16, 29). In contrast to prior observations (16, 29, 30), we did not detect a significant reduction in percentage or absolute number of FoxP3+CD4+ T cells in tumor or blood from iPI3Kδ treatment (Supplemental Fig. 3H, 3I, and data not shown), perhaps because intermittent dosing of iPI3Kδ lessens its negative effects on Tregs while still favorably impacting CD8+ T cell dynamics (31). Whether inhibition of myeloid suppressive cells contribute to efficacy of iPI3Kδ in these conditions also remains to be determined (32). PI3K-driven silencing of TCF1 expression and loss of self-renewal is emblematic of the entire constellation of anabolic cell metabolism (18). It is, therefore, likely that targeting other pathways involved in cellular anabolism might improve T-cell self-renewal, without vetoing differentiation, in order to improve the efficacy of immune checkpoint blockade (4, 14–19).

Supplementary Material

Supplementary Material

Key points.

Self-renewing T cells responsive to PD-1 blockade are depleted from growing tumors.

Stability of self-renewing TCF1+CD8+ T cells correlates with immunotherapy response.

PI3Kδ inhibitor added to anti-PD-1 boosts TCF1+ T cell pool and lessens tumor growth.

Acknowledgments

We thank Shana Coley, Arnold Han, Samhita Rao, Naiyer Rizvi, Andrea Schietinger, and Mark Stein for helpful suggestions.

This work was supported by National Institute of Health (AI076458, AI106711, CA20370), V Foundation, Charles H. Revson Foundation, and Conquer Cancer Foundation.

Footnotes

3.

Abbreviations used in this article: TCF1, T cell factor 1; PD-1, Programmed cell death protein 1; iPI3Kδ, inhibitor of phosphoinositide 3-kinase delta; FoxP3, Forkhead box P3; pS6, Phosphorylated S6 ribosomal protein; 2-NBDG, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose.

Disclosure

Intellectual property related to the use of metabolic manipulation to enhance immunotherapy is the subject of a US patent application filed with R.M., B.S.H., and S.L.R. listed as inventors.

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Supplementary Material
NIHMS1820664-supplement-Supplementary_Material.pdf (1.7MB, pdf)

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