Supplementary Figure 2. MEK inhibition with CKI27 increases MHC and checkpoint ligand expression. Murine tumor cell lines were treated with DMSO or CKI27 for 72 hr and either with or without IFNγ (5ng/mL) for the last 24hr; n=3. FACS analysis of representative histograms for the MFI of MHC-I (H2Kb/Kd and H2Db/Dd), MHC-II, PD-L1, CD80 and CD86 are shown.

Supplementary Table 1. Flow cytometry antibody details:

Supplementary Figure 9. Intermittent CKI27 treatment and GITR engagement relieves suppressive effects of MEK inhibition on T cell proliferation, cytokine production, and effector function. (A-C) Human PBMCs were labelled with CTV, sub-optimally stimulated with 1:25 or 1:100 CD3/CD28 Dynabeads, and treated with DMSO, continuous CKI27 (96hr), washout CKI27 (24hr on, 72hr off), and/or GITR-L; n=2-3. (A) % proliferation of CTVlow CD8+ and CD4+ T cells. (B) FACS analysis of co-inhibitory, co-stimulatory, and activation markers on CD8+ and CD4+ T cells. Heatmaps represent fold changes of positive percentages of each marker normalized to DMSO. (C) Cytokine analysis of supernatants collected from all PBMCs. Heatmap represents fold changes of concentrations (pg/mL) of proteins normalized to DMSO. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Supplementary Figure 13. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while destabilizing Tregs in LLC tumor. (A) Schema of LLC tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Numbers of cells/mg of TILs; n=4-5. (C) Phenotypes of TILs; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 12. The triple combination increases activation of CD8+ T cells, CD4+ Teffs, and Tregs in CT26 TDLN. (A) Schema of CT26 tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Absolute number of immune cell populations in the TDLN; n=4-5. (C) Phenotypes of T cells from the TDLN; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplemental Figure 11. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while Tregs remain unaffected in LLC TDLN. (A) Schema of LLC tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Image of TDLNs from LLC tumor bearing mice. (C) Gating strategy for all in vivo flow experiments. (D) Absolute number of immune cell populations in the TDLN; n=4-5. (E) Phenotypes of T cells from the TDLN; n=9-10. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 14. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while destabilizing Tregs in CT26 tumor. (A) Schema of CT26 tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Tumor weights, numbers of cells/mg, and CD8:Treg ratio of TILs; n=4-5. (C) Phenotypes of TILs; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 5. Intermittent CKI27 allows for immune cell recovery in the spleen, increases frequencies in the TDLN, and inhibits TILs similarly to continuous treatment. (A) Schema of LLC tumor bearing mice treated with vehicle, daily 2mg/kg CKI27, or intermittent 5mg/kg 4on/3off CKI27. Mice were treated in a staggered schedule and all timepoints were harvested on day 23. (B-D) All fold changes were calculated by normalizing to DMSO. (B) Fold changes of absolute number (cells/uL) of spleen immune cell populations. (C) Fold changes of absolute number (cells/uL) of TDLN immune cell populations. (D) Fold changes of tumor weights (mg) and TILs (cells/mg); n=4-5. Data are shown as mean±SEM. Unpaired, nonparametric Mann-Whitney test was used to compare each time point. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Supplementary Figure 1. MEK inhibition with CKI27 increases MHC and checkpoint ligand expression. (A-B) Murine tumor cell lines were treated with DMSO or CKI27 for 72 hr and either with or without IFNγ (5ng/mL) for the last 24hr; n=3. FACS analysis of (A) MHC-I (H2Kb/Kd and H2Db/Dd) and MHC-II and (B) PD-L1, CD80 and CD86 surface expression. Median fluorescence intensity (MFI) values were normalized to DMSO and log transformed. Data are shown as mean±SEM.

Supplementary Figure 7. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of co-stimulatory markers. Representative dot plot data for FACS analysis of co-stimulatory markers expressed by CD8+ T cells.

Supplementary Figure 15. The triple combination favorably alters the genetic profile of immune cells in the TDLN. LLC tumor bearing mice were treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. TLDNs were harvested on day 21 (7 days post treatment). Live CD45+ cells were FACS sorted and processed for sc-RNA sequencing. (A-B) Heatmap showing top genes expressed by each cluster for (A) CD8+ and (B) CD4+ T cells. (C) UMAPs of each treatment groups showing different clusters and annotations. (D) UMAPs of CD8+ and CD4+ T cells from each treatment group showing specific activation genes and proteins.

Supplementary Figure 10. The triple combination reduces tumor growth, is T cell dependent, and protects from re-challenge in LLC and CT26. (A-D) LLC tumor bearing mice were treated with vehicle, isotypes, GITR, CTLA-4, 5mg/kg 4on/3off CKI27, and/or CD8 for 4 weeks and tumor growth was monitored over time. (A) Average tumor growth (volume, mm3) of immunocompetent mice. (B) Average tumor growth (volume, mm3) of immunodeficient mice. (C) Average tumor growth (volume, mm3) of CD8 depleted mice. (D) Average tumor growth (volume, mm3) of mice from (A) that were re-challenged. (E-H) CT26 tumor bearing mice were treated with vehicle, isotypes, GITR, αCTLA-4, 2mg/kg 4on/3off CKI27, and/or αCD8 for 4 weeks and tumor growth was monitored over time. (E) Average tumor growth (volume, mm3) of immunocompetent mice. (F) Average tumor growth (volume, mm3) of immunodeficient mice. (G) Average tumor growth (volume, mm3) of CD8 depleted mice. (H) Average tumor growth (volume, mm3) of mice from (E) that were re-challenged. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 8. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of activation markers. Representative dot plot data for FACS analysis of activation markers expressed by CD8+ T cells.

Cancer treatment using immune checkpoint blockade (ICB) with anti-PD-1 and anti-CTLA-4 has been successful. However, primary and acquired resistance limits clinical benefit. To improve the effectiveness of ICB therapies, strategies that reorchestrate anti-tumor immunity through mechanism-based drug combinations are being actively explored. The alkylating chemotherapeutic agent cyclophosphamide (CTX) has direct tumoricidal and immunomodulatory properties, including the induction of homeostatic proliferation of T cells. Since ICB suppresses inhibitory signals in T cells, ICB might be able to augment CTX-induced homeostatic proliferation of antigen-specific T cells, thereby resetting the T cell receptor (TCR) repertoire in favor of tumor-specific T cells. Here, we showed that a single dose of CTX one day prior to starting αPD-1+αCTLA-4 treatment was sufficient to delay tumor progression in established melanoma and prolong survival in tumor-bearing mouse models. These effects extended to other lymphodepleting treatments, such as gemcitabine and radiation therapy. The anti-tumor immune response was mainly driven by the clonal expansion of activated/effector CD8+ tumor infiltrating lymphocytes. Furthermore, combined CTX and αPD-1+αCTLA-4 treatment demonstrated efficacy across additional preclinical tumor models, including colorectal cancer and triple negative breast cancer. Overall, these findings highlight that the combination of CTX and ICB represents a clinically relevant approach in the treatment of immunotherapy-refractory tumors.

Supplementary Figure 4. Intermittent CKI27 treatment partially relieves suppressive effects of MEK inhibition on T cell proliferation, cytokine production, and effector function. (A-C) Human PBMCs were labelled with CTV, sub-optimally stimulated with 1:25 or 1:100 CD3/CD28 Dynabeads, and treated with DMSO, continuous CKI27 (96hr) or washout CKI27 (24hr on, 72hr off); n=2-3. (A) Proliferation fold change of CTVlow CD8+ and CD4+ T cells was calculated by normalizing to DMSO. (B) FACS analysis of co-inhibitory, co-stimulatory, and activation markers on CD8+ and CD4+ T cells. Heatmaps represent fold changes of positive percentages of each marker normalized to DMSO. (C) Cytokine analysis of supernatants collected from all PBMCs. Heatmap represents fold changes of concentrations (pg/mL) of proteins normalized to DMSO. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Supplementary Figure 3. MEK inhibition with CKI27 increases HLA and checkpoint ligand expression. (A-B) Human tumor cell lines were treated with DMSO or CKI27 for 72hr and either with or without IFNγ (10ng/mL) for the last 24hr; n=3. FACS analysis of (A) HLA-ABC and HLA-DR and (B) PD-L1, CD80 and CD86 surface expression. MFI values are shown as mean±SEM.

Supplementary Figure 6. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of co-inhibitory markers. (A) Gating strategy for all T cell activation assays. (B) Representative dot plot data for FACS analysis of co-inhibitory markers expressed by CD8+ T cells.

Supplementary Table 2. Gene list associated with each cluster.

Introduction: Multiple suppressive mechanisms within the tumor microenvironment (TME) contribute to blunt anti-tumor T cell responses. Among them, tumor-associated T cells have been phenotypically described to be functionally exhausted (or dysfunctional), reflecting to a hyporesponsive state of chronically stimulated T cells that express multiple inhibitory receptors (or immune checkpoint molecules), such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT), and exhibit a compromised cellular cytotoxicity. While immune checkpoint blockade therapies aimed at reversing the dysfunctional state T cell effector function have demonstrated clinical effectiveness, not all cancer patients achieve long-term disease control. This is due, at least in part, to the refractory nature of what are categorized as terminally exhausted CD8+ T cells to PD-1/PD-L blockade, for example. Considering that CD8+ T cell exhaustion and/or dysfunction is one of the major therapeutic challenges, we aim to uncover pathways that contribute to program T cells toward exhaustion/dysfunction during cancer progression. Methods and experimental procedures: CD47 (a.k.a. integrin-associated protein, IAP) is known for its role as a ‘‘don’t-eat-me’’ signal in malignant, transformed cells. Our recent study found that both human and murine tumor-associated CD8+ T cells derived from lesions of human and murine melanoma, and ovarian cancers exhibit a significant elevation of surface CD47. Surprisingly, the population of tumor-associated CD8+ T cells that express high level of CD47 also express high levels of TOX, the critical factor that drives differentiation of exhausted T (Tex) cells, as well as other known immune checkpoint molecules. Through naïve T cell adoptive transfer experiments paired with single cell-RNA sequencing analysis, we demonstrated that CD47 plays a key role in driving T cell exhaustion. We demonstrated that extracellular matrix protein thrombospondin-1 (TSP-1) is the ligand for CD47-mediated T cell exhaustion during tumor progression by selectively disrupting the interaction between TSP-1 and CD47. Mechanistically, we demonstrate through immunoblot experiments that engagement of TSP-1:CD47 results in activation of calcineurin-NFAT axis, a key modulatory pathway upstream of TOX that drives T cell exhaustion and dysfunction. Summary of new, unpublished data: Our study uncovered a novel role of TSP-1:CD47 interaction, besides chronic antigen stimulation, in driving CD8 T cells exhaustion via the calcineurin-NFAT pathway. Our findings have implications for the mechanism of action in CD47 targeting therapies. Concluding statement: Extracellular matrix protein thrombospondin-1 and CD47 expressed on T cells contributes to promote TOX-driven T cell exhaustion program in cancer. Citation Format: Chien-Huan Weng, Anais Assouvie, Lauren Dong, Jean-Christophe Beltra, Sadna Budhu, Levi Mangarin, Yacine Marouf, Lucia Morgado-Palacin, Cailian Liu, Sébastien Monette, Jonathan Khan, Isabell Schulze, Dmitriy Zamarin, Linda Hamadene, Fadi Samaan, Daniel Hirschhorn, Stephane Pourpe, David Schroder, Roberta Zappasodi, Pamela Holland, Niroshana Anandasabapathy, John Wherry, Jedd D Wolchok, Taha Merghoub. Thrombospondin-1:CD47 signaling contributes to the development of T cell exhaustion in cancer [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B056.

T cell exhaustion is a major barrier to effective cancer immunotherapy. Although immune checkpoint blockade can reinvigorate exhausted T cells, not all patients achieve long-term responses, partly due to the refractory nature of terminally exhausted T cells. Beyond persistent antigen stimulation, the environmental drivers of exhaustion remain to be thoroughly characterized. Here we identify CD47 upregulation in tumor-infiltrating exhausted CD8+ T cells in both human and murine tumors. We reveal a novel role for the extracellular matrix protein thrombospondin-1 (TSP-1) in engaging CD47 on T cells to promote exhaustion. This interaction activates calcineurin–NFAT signaling, inducing upregulation of TOX and expression of inhibitory receptors, and impairing effector function during tumor progression. Importantly, disrupting the TSP-1–CD47 axis prevents T cell exhaustion and enhances tumor control. Our findings identify a novel pathway promoting T cell dysfunction and suggest that targeting the TSP-1–CD47 axis is a promising strategy to enhance T cell immunity and immunotherapy efficacy. Merghoub, Wolchok and colleagues reveal a role of the extracellular matrix protein thrombospondin-1 (TSP-1) and CD47 in promoting T cell exhaustion during tumor progression in mice and humans.