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 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)

Immune checkpoint inhibitor–based therapies often produce heterogeneous survival responses, including early risk, delayed treatment benefit, and durable long-term survival in a subset of patients. In these settings, conventional summary measures such as the hazard ratio may not adequately describe how treatment effects evolve over follow-up. We propose a milestone-based framework that separates long-term survival beyond a clinically meaningful time point from earlier outcomes and provides a practical way to characterize patient heterogeneity in treatment response. The framework summarizes treatment differences through milestone survival probabilities and, among patients who do not reach the milestone, characterizes short-term treatment ordering over time using a tau-based summary that helps identify hazard reversal. We illustrate the approach using reconstructed individual-level data from three landmark phase III trials: CheckMate 067, CheckMate 227, and CLEAR. Across these examples, the framework captures patterns that are difficult to summarize with conventional measures, including settings in which early disadvantage coexists with later durable benefit. It also helps clarify when treatment benefit begins to emerge and how short-term and long-term effects differ within the same trial. This approach provides a clinically interpretable and statistically principled way to evaluate heterogeneous and time-varying treatment effects in oncology trials with nonproportional hazards.

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).

Alterations in tumor infiltrating lymphocytes and myeloid cell populations following treatment with PSMAi-C’ dots and ICB in the Hi-Myc model (Day 10 post treatment). (a) Schematic of prostate cancer development and treatment before immunophenotyping of Hi-Myc GEM models. (b – o) Bar charts illustrating distinct T cell (b – i) and myeloid (j – o) populations harvested from Hi-Myc tumor-bearing mice (n = 3/group) treated with a multi-dose regimen (three doses every three days) of saline, ICB, PSMAi-C’dots or Dual Tx. Tumors were harvested 10 days after the final dose and dissociated to single cells, which were analyzed via multi-color flow cytometry. Data in (b – o) are presented as mean ± s.e.m. and a 1-way ANOVA with Tukey's multiple comparisons test was performed. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns, not significant.

Abstract The limited efficacy of immunotherapies in advanced prostate cancer stems from a tumor microenvironment (TME) in which myeloid-driven immune suppression, stromal remodeling, and metabolic barriers converge to limit antitumor immunity. In this study, we characterized the immunometabolic properties of an ultrasmall prostate-specific membrane antigen–targeting silica particle therapy as a first-in-class strategy to reprogram the Toll-like receptor (TLR)–ferroptosis axis in MYC-driven prostate cancer. As single agents, these particles suppressed lipid and steroid biosynthesis, disrupted lipid peroxidation control, and impaired nutrient flux, sensitizing tumors to ferroptosis. Coordinated redox remodeling, stromal reprogramming, and innate immune activation reversed myeloid suppression and promoted CD8+ T-cell infiltration. When combined with CSF-1R inhibition and immune checkpoint blockade, the particles suppressed tumor growth, extended survival beyond 100 days, and achieved up to 50% complete remission in MYC-overexpressing models. These findings position TLR–ferroptosis axis remodeling as a mechanistic blueprint for rational, particle-driven immunotherapies with broad translational potential in prostate cancer and other immunologically refractory malignancies. Significance: Clinically validated, PSMA-targeted ultrasmall core-shell silica particles reprogram immunometabolic pathways via a TLR–ferroptosis axis, enabling tumor microenvironment remodeling and potentiating checkpoint blockade in prostate cancer, with translational implications for treatment-resistant disease.

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 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).

PSMAi-C' dots trigger proinflammatory responses in prostate cancer and immune cells. (a) Gene expression levels, as fold changes over controls, using qRT-PCR and (b) normalized cytokine and chemokine expression levels by Proteome Profiler in Myc-CaP cells and supernatants, respectively, following incubation with 15 uM of PSMAi-C’ dots over 72 h. (c) IFN-a/b reporter B16 cell luminescence (OD620) after incubation with supernatants from particle-treated and untreated Myc-CaP cells. Percentage (%) of surface-expressed (d) IFNGR1, (e) MHC-I, (f) PD-L1, and (g) CD73 on vehicle- and particle-treated Myc-CaP cells by flow cytometry at 72-hour post-exposure. (h) Flow cytometry-based analyses of the %M1 and %M2 markers in vehicle- and particle-treated bone marrow-derived macrophages (BMDMs) over 72 h. (i) IFN-a/b reporter B16 cell luminescence (OD620) after incubation of BMDMs with supernatants from (h). Percentage of CD8+ T cell-specific (j) IFN-g and (k) TNF-a secretion from splenocytes by flow cytometry following a 72-hour incubation with or without PSMAi-C’ dots. (L) T cell cytotoxicity (expressed as luciferase (luc+) activity) following co-culture of mouse T cells with luc + Myc-CaP cells, with and without PSMAi-C’ dots for 48 h. All samples were run in triplicate. Numerical data are presented as mean ± s.e.m. 1-way ANOVA with Kruskal-Wallis test was performed in (a). Unpaired t-tests were performed in (d–g) and (i–l). 2-way ANOVA with Šídák's multiple comparisons test was performed in (c) and (h). *p < 0.05, **p < 0.01, ****p < 0.001.

Non-targeted C’ dots induce tumor-specific inflammatory responses in the Hi-Myc prostate cancer model. Ex vivo gene expression profiling of (a) tumor and (b) spleen harvested from Hi-Myc mice 4 days after the final dose of vehicle or non-targeted particles. Specific classes of genes (i.e., iron/ferroptosis-related, DAMPs, antigen presentation, and immune-related) were profiled. Transcripts were normalized to Gapdh and represented as fold changes, with significant changes indicated by values above 2 (dashed lines). Gene expression changes represented by bar plots at (c) 24 h and (d) 96 h post-particle administration in Hi-Myc mice, shown as log2 fold change transcript alterations relative to saline; significance indicated by values > 1 (solid lines).

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.

LBA9505 Background: EA6174 (STAMP) is the first randomized phase III trial of adjuvant pembrolizumab (pembro) in resected Merkel cell carcinoma (MCC), a rare, aggressive skin cancer with high relapse risk after surgery. This abstract presents new analyses of MCC-specific outcomes and radiation therapy (RT) effects. Methods: Patients (pts) with resected stage I without a sentinel lymph node biopsy to IIIB MCC were randomized 1:1 to pembro 200 mg IV q3 weeks ×17 doses (n = 147) or standard of care (SOC) (n = 146). Randomization was stratified by stage (I/II vs III) and intended RT. Co-primary endpoints were relapse-free survival (RFS) and overall survival (OS). MCC-specific outcomes were captured as relapse due to recurrence or death due to any cause (RFS_a), and MCC-specific recurrence and death (RFS_b). Distant metastasis-free survival (DMFS) was defined as time to distant recurrence or death from MCC. 280 pts provided 80% power with one-sided type 1 error rate of 0.05 to evaluate the hazard ratio (HR) of 0.56 hierarchically (RFS before OS). One-sided p-values from the log-rank test are reported in the analysis (data as of 2/19/26, median follow-up 47 months). Results: 293 pts enrolled (67.9% male; 84.3% stage III, median age 69, > 50% accrued during the COVID-19 pandemic). Overall RFS (RFS_a) was numerically improved with pembro but not statistically significant between arms (p = 0.10). DMFS was improved in pts treated with pembro (p = .05). Significant improvement was observed in MCC-specific outcomes (RFS_b) in pts treated with pembro, with HR = 0.66 [90%CI (0.45,0.96)], resulting in 1-year and 2-year RFS_b rates of 84% [90%CI(78,88)] and 77% [90%CI(70,82)] in the pembro arm, compared with 73% [90%CI( 67,79)] and 67% [90%CI (60,74)], in the SOC arm (p = 0.032). The impact of pembro on RFS_b was maintained (p = 0.039) among the subset that received any RT (n = 131 pembro, n = 123 SOC). For pts treated concurrently with RT, pembro did not affect outcomes; however, for pts treated sequentially with RT prior to randomization (n = 68 pembro and n = 65 SOC), pembro improved RFS_a (P = 0.014), RFS_b (p = 0.009), and DMFS (p = 0.004). Conclusions: EA6174 demonstrates that pts treated with pembro experienced significantly fewer MCC-related events than pts who received SOC and that adjuvant pembro improves DMFS. RFSa may have been influenced by the average age of enrolled pts and by disruptions and morbidity associated with patient enrollment during the COVID-19 pandemic; as such RFSb may be a more accurate indicator of pembro efficacy. These findings support a meaningful impact of pembro on MCC-specific outcomes, particularly for distant relapse. Pembro may be of particular benefit when RT is delivered sequentially rather than concurrently with pembro, similar to other studies of RT and immunotherapy in solid tumors. Mature OS and MCC-specific survival data will further guide use of adjuvant immunotherapy for MCC. Clinical trial information: NCT03712605 .

Serum cytokine/chemokine analyses demonstrate PSMAi-C’ dots maintain a safe inflammatory profile relative to controls in the presence and absence of ICB. Proteome profiles of detectable cytokines and chemokines in serum from mice treated with saline, PSMAi-C' dots, ICB, and Dual Tx (a) 4 days and (b) 10 days following the final dose. All data are presented as mean ± s.e.m. (c) Corresponding body weights of mice over time (n = 5 per cohort). 1-way ANOVA with Tukey's multiple comparisons test was performed (***p < 0.005).

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.