Abstract Although the role of neutrophils in modulating antitumor T-cell responses has been extensively studied, their direct effects on tumor cells remain less well understood. In this study, we investigated whether neutrophils have the capacity to directly kill tumor cells independently of T cells. We found that anti-CD40–based therapy, when combined with IL10 receptor blockade, initiates a Batf3-dependent pathway in which IL12 and IFNγ secretion results in oncolytic neutrophil activity. Using a combination of microscopy, single-cell, and functional assays, we observed that killing of tumor cells by neutrophils is dependent on physical contact and degranulation. This degranulation-mediated killing is associated with an atypical dynamic invasive neutrophil phenotype. In line with our preclinical findings, our phase I trial of anti-CD40 shows that circulating IL12, IFNγ, and IL10 increase in response to anti-CD40, whereas our phase Ib/2 PRINCE study shows that lower circulating IL10 is associated with favorable overall survival (OS) specifically among anti-CD40–treated patients. Finally, we found that neutrophil expansion with granulocyte colony-stimulating factor is associated with improved OS, specifically in patients treated with anti-CD40, suggesting that this pathway may be amenable to therapeutic intervention in patients with advanced cancer.

INTRODUCTION:Tumor-draining lymph nodes (tdLNs) are key hubs for antitumor T-cell priming. However, their contribution to PD-1 checkpoint blockade in bladder cancer remains poorly defined. In this study, we investigated whether tdLNs and lymphocyte trafficking are required for effective anti-PD-1 therapy in an orthotopic preclinical model of bladder cancer. METHODS:We used C57BL/6 mice bearing intravesical MB49-luciferase tumors. Lymphocyte trafficking was blocked pharmacologically using the sphingosine-1-phosphate receptor modulator FTY720, or surgically by iliac/obturator and lumbar lymphadenectomy prior to anti-PD-1 antibody administration. Tumor burden, survival, and immune cell phenotypes were assessed by flow cytometry. Lymphatic drainage of the bladder was mapped by intravesical Evans Blue dye. RESULTS:Anti-PD-1 monotherapy significantly improved survival (P = 0.0019) and reduced tumor weight (P = 0.019) and (P = 0.0029), effects that were abrogated with FTY720 or tdLN removal. Anti-PD-1 increased intratumoral CD8⁺ effector, proliferating (Ki67⁺), and stem-like (PD-1⁺ TCF1⁺) T cells, as well as CD40high cDC1 dendritic cells, consistent with enhanced cross-priming. Evans Blue tracing confirmed iliac and lumbar nodes as the dominant tdLNs. Study limitations include the restricted lymphatic architecture in mice compared to humans, which may underestimate redundancy in human tdLN networks. CONCLUSIONS:Effective PD-1 blockade in bladder cancer significantly depends on intact tdLNs and active lymphocyte trafficking. Preserving nodal integrity may optimize immunotherapy responses, supporting the rationale the rationale for neoadjuvant PD-1 blockade prior surgical disruption of lymphatic channels.

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 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 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).

Abstract Adoptive cell transfer (ACT) has demonstrated potent anti-tumor efficacy in melanoma, yet therapeutic resistance frequently emerges within immunosuppressive tumor microenvironments. Indoleamine 2,3 dioxygenase 1 (IDO1) is a tryptophan catabolizing enzyme that generates kynurenine (Kyn), an immunomodulatory metabolite known to suppress effector T cell function. Here, we show that tumor overexpression of IDO1 undermines ACT efficacy through dual mechanisms of T cell exclusion and cytotoxic impairment. Using murine B16 melanoma cells engineered to overexpress IDO1 (B16IDO1), we observed elevated Kyn levels, reduced CXCL9/10 and CCL5 chemokine expression, and decreased intratumoral T cell infiltration. In vitro, IDO1 expressing tumor cells exhibited resistance to killing by activated CD8 PMEL T cells, a phenotype dependent on soluble factors in conditioned media and reversible with pharmacologic IDO1 inhibition. In vivo, adoptive transfer of either PMEL or TRP1 T cells, CD4 T cells specific for tyrosinase-related protein 1, failed to control B16IDO1 tumors, correlating with decreased infiltration, function, and survival. Pharmacologic blockade of IDO1 enhanced T cell infiltration and improved ACT mediated tumor control. Together, these findings identify IDO1 as a regulator of ACT resistance by suppressing T cell trafficking and effector activity. Complementary analyses of patient TIL, tumor, and serum samples are underway to assess correlations between IDO1/Kyn levels and immune cell burden, underscoring clinical relevance. Further studies extend this framework to human models, including IDO1+ melanoma xenografts and CAR T cells, to explore the translational potential of targeting the IDO1-Kyn-AHR axis to enhance cellular immunotherapy efficacy. Citation Format: Mamadou Alpha Bah, Rachana Maniyar, Jonathan F. Khan, Anais Assouvie, Sadna Budhu, Parwiz Abrahimi, Inna Serganova, Gabrielle A. Rizzuto, Taha Merghoub, Jedd D. Wolchok. Tumor IDO1 drives resistance to adoptive cell transfer by suppressing T cell recruitment and effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6537.

SIRPα is a well-characterized inhibitory receptor on myeloid immune cells. However, human and mouse melanoma cells can also express high levels of SIRPα. Whether and how melanoma cell-intrinsic SIRPα contributes to tumor progression and anti-tumor immunity remains underexplored. Here, we identify a role of tumor cell-intrinsic SIRPα in suppressing immune recruitment and activation. SIRPα deletion in melanoma cells enhances tumor control and increases immune infiltration. Transcriptomic analyses reveal that loss of tumor cell-intrinsic SIRPα upregulates the chemokine CXCL10 in both human and mouse melanoma cells. Notably, Cxcl10 knockdown in SIRPα-deficient melanoma partially rescues tumor growth and reduces CD8+ T cell infiltration, mirroring the phenotype of SIRPα-expressing tumors and indicating that tumor cell-intrinsic SIRPα promotes immune evasion by suppressing Cxcl10-mediated T cell recruitment. Our study uncovers an unrecognized mechanism of SIRPα-mediated immune suppression and highlights SIRPα silencing as a potential therapeutic strategy to enhance immune infiltration and T cell-mediated tumor control across SIRPα-expressing cancers.

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).

Complete gene list for 6 cell types (Related to Figure 5F)

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)

Although the role of neutrophils in modulating antitumor T-cell responses has been extensively studied, their direct effects on tumor cells remain less well understood. In this study, we investigated whether neutrophils have the capacity to directly kill tumor cells independently of T cells. We found that anti-CD40-based therapy, when combined with IL10 receptor blockade, initiates a Batf3-dependent pathway in which IL12 and IFNγ secretion results in oncolytic neutrophil activity. Using a combination of microscopy, single-cell, and functional assays, we observed that killing of tumor cells by neutrophils is dependent on physical contact and degranulation. This degranulation-mediated killing is associated with an atypical dynamic invasive neutrophil phenotype. In line with our preclinical findings, our phase I trial of anti-CD40 shows that circulating IL12, IFNγ, and IL10 increase in response to anti-CD40, whereas our phase Ib/2 PRINCE study shows that lower circulating IL10 is associated with favorable overall survival (OS) specifically among anti-CD40-treated patients. Finally, we found that neutrophil expansion with granulocyte colony-stimulating factor is associated with improved OS, specifically in patients treated with anti-CD40, suggesting that this pathway may be amenable to therapeutic intervention in patients with advanced cancer.

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.