Cancer Immunology Research

Chronic stimulation of CD8 T cells within the tumor microenvironment (TME) induces a hypofunctional state characterized by diminished cytotoxicity and functionally impaired anti-tumor function, known as exhaustion. Exhaustion is associated with epigenetic changes that remain relatively stable despite interventions like immune checkpoint inhibition (ICI). Although epigenetic changes are potentially reversible, reports of therapeutic strategies to effectively restore function in exhausted CD8 T cells remain limited. Here, we report DNA methyltransferase 1 (DNMT1) inhibition (DNMT1i) in counteracting CD8+ T cell dysfunction during the anti-tumor response. We show that DNMT1i synergizes with ICI to rescue the tumor cell killing activity of chronically stimulated CD8 T cells in a melanoma model. DNMT1i mitigates transcriptional features of exhaustion while inducing a divergent effector program. DNMT1i attenuates the global increase in chromatin accessibility associated with exhaustion and enables epigenetic remodeling of the exhausted chromatin landscape upon restimulation. Finally, DNMT1i enhances the effector function of melanoma patient-derived tumor infiltrating lymphocytes after prolonged ex vivo expansion. These studies establish DNMT1 targeting as a promising strategy to counteract CD8 T cell exhaustion and potentiate ICI efficacy.

Brain metastases (BrM) are associated with poor prognosis. A better understanding of anti-tumor immune responses in the context of immune specialized microenvironment of the brain is required to develop improved therapeutic strategies for this disease. We demonstrate that the conventional dendritic cells type 1 (cDC1) gene signature positively correlates with a prolonged BrM-dependent survival in melanoma and breast cancer patients. Furthermore, intracranial anti-tumor immune responses in preclinical BrM models consistently rely on cDC1s for tumor growth control, BrM-dependent survival and maintenance of the intra-tumoral CD8+ T cell pool, in contrast to variable, cancer type-dependent cDC1 roles in extracranial tumors. This is underpinned by tumor site-specific cDC1 molecular profiles with distinct Toll like receptor repertoires, upregulation of co-stimulatory molecules and IL-12, and enhanced type-I-IFN signaling in intracranial cDC1s, with the latter driving increased cDC1 activation. cDC1s also promote the conversion of progenitor exhausted CD8+ T cells to transient effectors, which is further enhanced by immune checkpoint blockade therapy. These findings pinpoint cDC1s as a major cell population of interest in the development of future immunotherapies for BrM.

Advanced BRAF-mutant cutaneous melanoma can be treated with targeted therapy when immune checkpoint inhibitors (ICIs) fail or are not a feasible option. Nevertheless, most patients do not achieve a durable response, highlighting the critical need for therapeutic partners that enhance the long-term efficacy of targeted therapy. Transcriptomic analysis of a BRAF-mutant melanoma model of acquired resistance identified P-selectin glycoprotein ligand-1 (PSGL-1) as a top-upregulated immune mediator upon resistance acquisition. PSGL-1 is a key regulator of CD8+ T cell exhaustion and differentiation, and its inhibition has been shown to enhance T cell function across multiple disease models. Based on these observations, we hypothesized that combined targeting of BRAF/MEK and PSGL-1 would improve anti-tumor responses. Here, we demonstrate that dual inhibition of BRAF/MEK and PSGL-1 elicits durable tumor control in a preclinical model of PD-1-refractory cutaneous melanoma. Single-cell RNA sequencing of the tumor microenvironment reveals robust reprogramming of intratumoral CD8+ T cells toward a less terminally differentiated, memory-like phenotype following combined BRAF/MEK and PSGL-1 targeting. Consistent with these findings, CD8+ T cells in the tumor-draining lymph nodes of PSGL-1-/- mice exhibit enhanced functionality and a less differentiated state of exhaustion when compared with wild-type mice. To extend these observations to a translationally relevant setting, we further show that antibody-mediated blockade of PSGL-1, in combination with BRAF/MEK inhibition, yields superior anti-tumor activity compared with either monotherapy. Collectively, these findings identify PSGL-1 as a promising therapeutic target to enhance the durability of targeted therapy and provide a strong rationale for future clinical evaluation.

Macrophages in the tumor microenvironment are known to upregulate PD-L1 expression, thereby suppressing T cells through PD-1 ligation. However, the manner in which PD-L1 expression intrinsically impacts macrophages and their immunomodulatory phenotype is less clear. Clarifying this knowledge gap would yield insight into the mechanisms of immunosuppression within the tumor microenvironment. To characterize the macrophage intrinsic role of PD-L1, we used complementary genetic and pharmacological approaches by analyzing primary murine bone marrow-derived macrophages (BMDMs) with complete genetic PD-L1 deletion and wildtype BMDMs treated with anti-PD-L1 blocking antibodies. Macrophages were evaluated across naive, pro-inflammatory (M1), and tumor conditioned (TCM) polarization states in vitro. Unlike prior reports, neither genetic deletion nor antibody blockade dramatically altered the expression of macrophage polarization markers or in vitro phagocytic capacity. Both conditions consistently reduced surface levels of the M1-associated costimulatory molecule CD80, prompting further analysis of T cell interacting and antigen presenting proteins, in which we revealed disparate effects of genetic deletion and antibody blockade on the surface levels of MHCI, MHCII, PD-1, and PD-L2. These findings suggest that PD-L1 deletion and antibody-mediated blockade contribute to macrophage immune regulatory profiles in distinct manners. This difference supports a model in which PD-L1 functions in macrophages beyond its canonical role as a ligand for PD-1, influencing antigen presentation and checkpoint molecule levels and playing a broader role in immune regulation in the tumor microenvironment.

Transforming Growth Factor {beta} (TGF{beta}) is a potent immunosuppressor and a primary driver of resistance to cancer immunotherapy. While preclinical models have long suggested that TGF{beta} inhibition could synergize with immune checkpoint inhibitors, these effects have proven difficult to replicate in clinical settings. The highly regulated TGF{beta} pathway can be inhibited through various mechanisms, including neutralizing activated ligands or inhibiting upstream activators, such as integrins. Recent structural data demonstrated that integrin v{beta}8 can enable TGF{beta}1/3 signaling without releasing the active cytokines from their Latency-Associated Peptides , suggesting that ligand-blocking antibodies may have limited access to their epitopes. Here, we show that integrin v{beta}8 blockade, while achieving anti-tumor responses similar to those of anti-TGF{beta} antibodies, does so through a distinct mechanism of action. Anti-v{beta}8 is 3 orders of magnitude more potent at inhibiting v{beta}8-mediated TGF{beta} activity than a commonly used antibody against the mature form of the cytokine. Whereas TGF{beta} ligand inhibition has little effect on TGF{beta} signaling in tumor-draining lymph nodes (tdLN) and requires IFN{gamma}for its anti-tumor effects, v{beta}8 blockade strongly inhibits TGF{beta} signaling in tdLN and, in combination with PD-L1 blockade, drives tumor control through an IFN{gamma} -independent mechanism that strictly requires T cell egress from tdLN. Combined v{beta}8 and anti-PD-L1 blockade enhances antigen presentation in dendritic cells (DCs) and, unlike TGF{beta} ligand blockade, improves the efficiency of DC-induced T cell activation in response to cross-presented antigen. These findings suggest that v{beta}8 blockade can disable an immunologically critical source of TGF{beta} signaling that is not addressed by antibodies targeting TGF{beta} ligands, suggesting a promising new approach to TGF{beta} pathway modulation.

Immune effector cell-associated neurotoxicity syndrome (ICANS) is a major complication after CAR T cell therapy, but its underlying mechanisms remain poorly understood. We performed longitudinal immune profiling of paired whole blood and serum samples from patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL) treated with CD19 CAR T cells. At peak neurotoxicity, high-dimensional mass cytometry and serum proteomics identified the expansion of CD163 monocytes and immature CD10lowCD101low neutrophils correlated with elevated serum ST2 and IL-2RA concentrations. Integrative immune module analysis identified these features among the strongest predictors of ICANS severity. Independent single-cell transcriptomic profiling validated the emergence of immunoregulatory CD163 monocytes and identified CD177 as a biomarker of ICANS-associated immature neutrophils. Together, these findings reveal a coordinated myeloid inflammatory network associated with ICANS and nominate candidate biomarkers and therapeutic targets for improving the safety of CAR T cell therapy. Significance: We demonstrate that immunoregulatory CD163+ monocytes and immature, activated CD177hiCD10lowCD101low neutrophils emerge in patients with moderate to severe ICANS at peak toxicity following CD19 CAR T cell therapy. These findings identify an uncharacterized myeloid network potentially contributing towards ICANS pathogenesis.

Combination targeted therapy with BRAF/MEK inhibitors and immune therapy show promising therapeutic outcomes in melanoma; however, the development of drug resistance still represents a formidable challenge. Remaining unexplored is the possibility that BRAF/MEK inhibitors themselves inadvertently compromise the tumor immune microenvironment, limiting the efficacy of immunotherapy when it is used in combination with targeted inhibitors. Herein, we profiled the landscape of the BRAF regulatome identifying a novel transcription factor, TFAP2A, newly linking BRAF/MEK drug resistance to antitumor immunity. Specifically, we found that BRAF/MEK inhibitors significantly upregulate TFAP2A. Further, genetic disruption of TFAP2A overcomes BRAF/MEK-inhibitor resistance, promotes stromal enrichment, and enhances intratumoral infiltration of macrophages in an immune-compromised mouse model. In a syngeneic mouse model, TFAP2a knockout not only suppresses tumor growth but also induces potent anti-tumor tertiary lymphoid structures (TLSs). Single cell transcriptomics revealed that the absence of TFAP2A shapes the antitumor microenvironment with an influx of M1-like macrophages, CD8+ T cells and mature dendritic cells. By identifying TFAP2A as a shared driver of both targeted therapy resistance and immunosuppression, our work offers a one-stone-two-bird strategy to overcome drug resistance and elicit antitumor immunity.

Colorectal cancer (CRC) remains a leading cause of cancer mortality, with most cases refractory to immunotherapy. Distinguishing tumor-induced from steady-state mucosal T cell responses has been a critical barrier to understanding antitumor immunity in CRC. Using orthotopic transplantation of CRC organoids with and without metastatic potential, combined with temporal T cell fate-mapping, we show that non-metastatic tumors elicit early recruitment of CD8{beta}+ and CD4+ T cells that acquired cytotoxic and Th1-like programs, whereas pro-metastatic tumors induce a naive-like, hypoactivated state. Tumor-infiltrating CD4+ T cells underwent clonal expansion, including clones recognizing microbial and dietary antigens. T cells in physical contact with tumor cells, identified by uLIPSTIC, were enriched for expanded and cytotoxic clones. Fate-mapped T cells from non-metastatic tumors suppressed tumor growth in an IFN-{gamma}-dependent manner, whereas pro-metastatic tumor-derived T cells failed to do so. Mechanistically, pro-metastatic tumors downregulated MHCII, and Ciita targeting in non-metastatic organoids reduced CD4+ clonal expansion and led to tumor progression. Together, these findings define divergent early T cell trajectories associated with CRC metastatic potential, indicating that ineffective local immune engagement precedes metastatic dissemination.

Regulatory T (Treg) cells accumulate in the tumor microenvironment (TME) to suppress anti-tumor immunity, but the transcriptional regulators stabilizing their immunosuppressive state remain poorly defined. Here we show that transcriptional factor TOX is selectively upregulated in tumor-infiltrating (TI) Treg cells across human cancers and mouse tumor models, while remaining low in peripheral and naive Treg populations. Treg-specific deletion of TOX reduced tumor burden, impaired TI Treg-mediated immune suppression, and enhanced effector functions of CD8 and CD4 T cells. In mosaic mice, TOX-deficient Tregs were selectively depleted from tumors, accompanied by increased apoptosis. Single-cell RNA sequencing and TCR clonotype analysis linked TOX expression to an effector-like TI Treg state with clonal expansion, whereas TOX loss shifted cells toward a TCF7-associated progenitor-like phenotype. ATAC-seq revealed enrichment of AP-1 motifs in TOX-sufficient TI Tregs. In contrast, TCF7, LEF1, and FOXO1 motifs in TOX-deficient counterparts, uncovering the opposing transcriptional networks downstream of TOX. Furthermore, TOX deficiency augmented CD8 T cell responses to PD-1 blockade. Together, these findings establish TOX as a key regulator of TI Treg fitness and stability, and identify it as a potential therapeutic target to enhance the efficacy of PD-1-based immunotherapy.

Across cancers, tumor-infiltrating CD8+ T cells expressing the tissue-resident memory T cell (TRM) markers CD69 and CD103 are strongly associated with favorable clinical outcomes. However, a substantial fraction of these cells in human tumors are not tumor-specific, but instead recognize unrelated viral antigens. These virus-specific bystander TRM-like cells are prevalent in tumors and retain functional potential, raising interest in strategies that leverage pre-existing antiviral immunity for cancer immunotherapy. Yet their origins and differentiation states remain poorly defined, limiting both the interpretation of residency-based tumor-infiltrating lymphocyte (TIL) phenotyping and efforts to rationally harness these TRM-like cells. Here, using mouse models of GBM and melanoma, we demonstrate that resting circulating memory T cells trafficked into tumors via GPCR-dependent signaling and rapidly adopted a tissue-resident phenotype, independent of cognate antigen. Strikingly, in GBM, but not melanoma, pre-existing brain TRM contributed substantially to the bystander TIL compartment and were the dominant source of CD69+/CD103+ bystander T cells, revealing a tumor- and tissue-specific origin for this subset. These findings were further supported by transcriptional analysis of T cell receptor clones present in both paired patient GBM tumor and peritumoral brain, which identified shared features with TRM-derived TILs in mouse GBM. Overall, this work provides new insight into tumor immunosurveillance, inform the interpretation of CD69+/CD103- and CD103+ TIL populations, and lay a foundation for immunotherapeutic strategies aimed at harnessing circulating and pre-existing virus-specific TRM populations in tumors.

Cytotoxic T lymphocytes (CTLs) play a central role in antitumor immunity; however, metabolic reprogramming within the tumor microenvironment often compromises their effector function, making metabolic targeting crucial for the improvement of T cell function. Folate-dependent purine synthesis, a core pathway sustaining the nucleotide pool, is highly activated in tumors, yet its role in regulating tumor immune sensitivity remains unclear. Here, by establishing a co-culture system of melanoma cells and human T Cell Receptor (TCR)-engineered T cells, we systematically evaluated the effects of folate-dependent purine synthesis inhibitors on tumor cell response to CD8+ T cell cytotoxicity. We found that inhibition of key enzymes such as methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and glycinamide ribonucleotide transformylase (GART) markedly enhanced tumor cell sensitivity to T cell killing, an effect also observed with exogenous nucleoside supplementation. Mechanistically, inhibition of folate-dependent purine synthesis suppresses glycolysis by downregulating critical glycolytic enzymes, thereby reducing lactate production. Reduction in lactate further weakens lactylation and stability of the immune checkpoint protein PD-L1. In parallel, impaired purine synthesis disrupts uridine metabolism, blocks ribose salvage, and distally influences glycolysis. Collectively, our study identified the folate-dependent purine synthesis-glycolysis axis as key regulator of tumor immune response and highlights metabolic targeting as a promising strategy to improve cancer immunotherapy.

Natural killer (NK) cells infiltrate many solid tumours, yet the mechanisms that determine their functional heterogeneity across tumour types remain poorly understood. Differences in tumour immunogenicity, inhibitory signalling, and nutrient availability have all been implicated, but unifying explanations are lacking. Here, we compared four syngeneic tumour models implanted at identical anatomical sites to isolate tumour-intrinsic effects on NK-cell fate. Tumour-infiltrating NK cells displayed striking tumour-specific differences in cytokine production, cytotoxic protein expression, and persistence. These differences were not explained by cytokine availability or global features of the tumour metabolic environment. Instead, quantitative proteomics and time-resolved in vivo labelling revealed that NK cells enter tumours in a functionally competent state but rapidly diverge thereafter. In suppressive tumour microenvironments, NK cells undergo early mitochondrial loss, translational repression, and impaired proteostatic responses, accompanied by increased apoptotic priming. These defects result in reduced effector function and failure of intratumoural persistence despite preserved recruitment. In contrast, permissive tumours sustain NK-cell translational capacity, cytokine responsiveness, and long-term residency. Together, these findings identify disruption of translational and mitochondrial homeostasis as a central mechanism limiting NK-cell persistence in solid tumours. This work establishes early tumour-induced defects in protein synthesis and cellular fitness as key constraints on durable NK-cell immunity and provides a framework for restoring effective innate anti-tumour responses.

Peritoneal carcinomatosis is a major cause of death in gastric cancer, yet effective therapies remain limited. Tumor-derived soluble factors are increasingly recognized as key regulators of the peritumoral microenvironment. Here, we nominate osteopontin (SPP1) as a tumor-derived mediator that orchestrates macrophage-driven immunoregulation in gastric peritoneal carcinomatosis. Using integrated analyses of human clinical datasets and murine models, we demonstrate that tumor-secreted SPP1 promotes macrophage recruitment and induces tolerogenic IL-10 production. Clinically, SPP1 correlated with inferior overall survival and progression-free survival in gastric cancer. In syngeneic murine models of gastric peritoneal carcinomatosis, intracavitary pharmacologic inhibition of SPP1 restricted peritoneal dissemination, impaired macrophage infiltration and suppressed IL-10 production. Consistent with these findings, macrophage depletion phenocopied antitumor effects of SPP1 inhibition, resulting in decreased metastatic burden. Collectively, these findings define a mechanism of tumor-macrophage crosstalk that promotes peritoneal dissemination and provide a rationale for therapeutic targeting of SPP1 in gastric peritoneal carcinomatosis.

EWSR1::FLI1, the oncogenic chimeric transcription factor driving Ewing sarcoma (EwS)induces expression of exquisitely EwS-specific neogenes (Ew_NGs) through neomorphic binding and transcription activation at GGAA microsatellites in genomic regions that are silent in normal tissues. We show that peptides encoded by Ew_NGs are presented on HLA-I complexes on EwS cells. The cytokine secretion of CD8+ T cells specific for Ew_NG-encoded HLA-I-bound peptides is activated by all HLA-I-matched EwS cells but not by non-EwS cells. These T cells kill EwS cells in an HLA-I restricted manner. This cytotoxicity is dependent on the expression of EWSR1::FLI1 and of the corresponding Ew_NG. It can be reproduced by transduction of the TCR into donor T cells (TCR-T) which kill EwS cells in vivo. Moreover, we show that neither off target nor allogeneic activation are observed with TCR-T thus paving the way for cell therapy in relapsed/resistant EwS patients for which therapeutic options are very limited. Statement of significanceThe chimeric transcription factor EWSR1::FLI1 generates tumor-specific neogenes encoding neoantigen presented by the HLA-I molecules of Ewing cells. Neoantigen-specific CD8+ T-cell clones and engineered TCR-T cells can selectively recognize and kill EwS tumor cells in vitro and in vivo.

Most high-dimensional studies of tumor-immune interactions focus on metastatic models, limiting insight into how immune remodeling in primary tumors shapes metastatic competence. Here, integrating single-cell RNA sequencing, CyTOF, and functional studies across metastatic (4T1) and non-invasive (EMT6) triple-negative breast cancer (TNBC) murine models, we define tumor state-specific immune programs that distinguish metastatic competence. Tumors with metastatic capacity uniquely drive early bone marrow expansion of CXCR2 neutrophils, which infiltrate primary tumors acquiring a CXCL2-producing phenotype that promotes EMT-associated cancer stem cell (CSC) plasticity. This program depends on TGF-{beta}/CEBPD-mediated induction of S100A9. Elevated CXCL2, together with G-CSF, establishes a feed-forward circuit that drives systemic neutrophil mobilization and recruitment to distant organs, where neutrophil-derived S100A8/A9 (calprotectin) promotes MET-driven CSC outgrowth and metastatic colonization. Clinically, gene signatures associated with CXCR2 neutrophils predict poor survival in TNBC patients, whereas monocyte/macrophage (CX3CR1) and T cell activation signatures correlate with improved outcomes. S100A9 ablation disrupts this cascade and enhances immunotherapy responsiveness, defining a TGF-{beta}/S100A9/CXCR2 axis linking immune remodeling, CSC plasticity and metastasis. HighlightsO_LIMetastatic TNBC engages a TGF-{beta}/C/EBP{delta}/S100A9 axis that expands CXCR2 neutrophils C_LIO_LINon-invasive EMT6 tumors retain a CX3CR1 monocyte/macrophage and T-cell landscape C_LIO_LICXCR2+ neutrophils in pre-metastatic niches suppress T cell response while promoting tumor cell proliferation C_LIO_LIS100A9 loss redirects myelopoiesis and potentiates anti-PD-L1 in TNBC models C_LI In BriefAlkan et al. dissect how tumor state programs the myeloid compartment in TNBC. Metastatic 4T1 tumors uniquely engage a TGF-{beta}/C/EBP{delta}/S100A9 axis driving CXCR2 neutrophil expansion and CXCL2/G-CSF-dependent systemic mobilization, coupling immune remodeling to EMT/MET cancer-stem-cell plasticity, while S100A9 loss restores CX3CR1 myeloid identity and unlocks checkpoint-inhibitor responsiveness.

Regulatory CD8+ T-cells (CD8+ Treg) are a distinct yet understudied T-cell subset capable of simultaneous immunosuppression and cytolysis. Here, we characterized induced human CD8+ Treg (CD8-iTreg) generated from peripheral blood CD8+CD25- T-cells using anti-CD3e mAb-loaded artificial antigen presenting cells, IL-2, TGF{beta}, and Rapamycin. These CD8-iTreg differentiated into a stable, highly proliferative bifunctional population with suppressive activity comparable to CD4-iTreg while retaining cytolytic capacity similar to conventional CD8 cytotoxic T lymphocytes (CTL). Multi-parameter spectral flow cytometry and single-cell RNA-seq revealed a distinct immunoregulatory signature: a predominantly Treg-like profile marked by tissue-residency marker CD103 with increased canonical Treg markers (FoxP3, HELIOS, CD25, CD39, CTLA-4, CCR4, and IL-10) and reduced pro-inflammatory cytokines. A unique cytotoxic program was marked by elevated Granzyme-K (GzmK) and Thrombospondin-4 (Tsp-4), a thrombospondin family extracellular matrix glycoprotein upregulated in activated CD8+ T-cells. Cytolysis was primarily mediated by Perforin (Prf) and multiple Granzymes packaged into Tsp-4 supramolecular attack particles (SMAPs), with GzmK contributing to both cytotoxic and suppressive functions. After anti-CD19scFv CAR (CAR19) transduction, CAR19+ CD8-iTreg showed superior in vivo anti-tumor efficacy compared with CAR19-CTLs, significantly reducing tumor burden and prolonging survival in a CD19+ Nalm-6 human leukemia xenograft model while maintaining low pro-inflammatory cytokine production. In a xenogeneic graft-versus-host disease (GVHD) model with residual human leukemia, CAR19 CD8-iTreg inhibited GVHD lethality and controlled tumor growth without increasing systemic inflammation. Together, these findings support CD8-iTreg-based CAR therapies as a strategy to retain potent anti-leukemic activity while limiting inflammatory toxicities of conventional CAR T-cells, properties particularly beneficial in treating auto- and allo-immune diseases. One sentence summaryCD8-iTreg drive parallel tumoricidal and immunoregulatory functions mediated by releasing Tsp-4+ SMAPs containing granzyme K.

Interferon regulatory factor 8 (IRF8) is a master transcription factor of myeloid differentiation, but whether IRF8 intrinsically controls B cell function in tumors remains unknown. Using paired single-cell transcriptomic and chromatin accessibility profiling of tumors from wild-type and Irf8-deficient mice, we identify a B cell-intrinsic IRF8 axis regulating antigen presentation and sustaining anti-tumor CD8 T cell immunity. IRF8 establishes conserved chromatin accessibility programs across myeloid cells and plasmablasts centered on antigen processing and MHC class I presentation, but engages distinct motifs by lineage: myeloid cells preferentially utilize ISRE and ETS-composite elements, whereas plasmablasts are selectively enriched for EICE elements, reflecting B lineage-specific IRF8-IRF4 cooperation. Loss of IRF8 disrupts these programs, skews B cells toward plasmablast differentiation and reduces antigen presentation machinery. B cell depletion accelerated tumor growth, while CD40 agonism activated B cells, expanded T cells, and enhanced anti-tumor immunity. B cell-specific IRF8 deletion alone accelerated tumor growth, establishing a cell-intrinsic requirement independent of myeloid IRF8 function. The IRF8-regulated B cell signature was enriched in PD-1 blockade cancer patient responders, and plasmablast abundance correlated with response in pembrolizumab-treated cancer patients. These findings establish IRF8 as a lineage-adapted regulator of antigen presentation and define the IRF8-B cell axis as a determinant of anti-tumor immunity.

Advancements in immunogenomics and immuno-oncology have enabled the development of personalized cancer vaccines (PCVs) that target cancer cell-specific somatic variants. A subset of these variants produce neoantigens that, when presented on tumor cells by MHC molecules, have the potential to elicit a robust and specific immune response. To date, there are over one hundred interventional studies listed on clinicaltrials.gov that explore the use of PCVs. We have supported a number of these trials through the creation of bioinformatic pipelines, tools, and procedures for the identification of patient-specific neoantigen candidates. While many of these steps have been automated, the final selection of neoantigen candidates often relies on expert manual review, creating a bottleneck that limits scalability and full automation of PCV workflows. Addressing this challenge, we introduce NEAT (Neoantigen Evaluation & Automated Triage), a machine learning-based approach that enables automated neoantigen candidate prioritization and supports the transition toward more scalable and reproducible PCV design. We implemented a prediction model trained and tested on existing vaccine design results from 33 patients and 1,943 peptides, across 3 clinical trials, including 439 peptides prioritized for PCV inclusion. This model uses features such as tumor variant allele frequency, RNA expression, driver gene status, binding/presentation scores, and transcript support level to automatically predict whether a peptide will be accepted, rejected, or require further human review before inclusion in a vaccine. The model achieved a sensitivity of 0.847 and specificity of 0.924, with an area under the curve of 0.955. The model predictions have been incorporated in pVACtools version 7. By integrating this model into the vaccine development pipeline, we foresee a significant reduction in the time required to transition from patient sample collection to vaccine manufacturing, thereby enhancing the efficiency and scalability of PCV production.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with limited therapeutic options, underscoring the need for innovative treatments. Chimeric antigen receptor (CAR) therapy has transformed hematologic malignancies but faces key challenges in solid tumors, particularly on-target/off-tumor toxicity and antigen heterogeneity. Adapter CAR (AdCAR) platforms offer enhanced control by decoupling antigen recognition from CAR activation, enabling controllable, reversible, and multi-antigen targeting. Recent studies suggest AdCARs can function as an AND-gate using combinations of adapter molecules at controlled surface densities. This defines activation thresholds, termed the Surface Activation Matrix, that restricts full activation to tumor cells overexpressing the target antigen combination, thereby reducing off-tumor toxicity. In this study, we evaluated its applicability to PDAC using adapters targeting CD318, TSPAN8 and CD66c. We systematically evaluated single and combinatorial adapter dosing in co-culture assays with AsPC1 cells, in a donor-dependent manner. Low concentrations of individual adapters were non-cytotoxic, whereas combining them at identical sub-threshold doses restored potent tumor killing, demonstrating that AdCAR activation depends on cumulative adapter density rather than total amount. However, the activation threshold required for AND-gate cytotoxicity varied between donors, highlighting the need for patient-specific titration to achieve selective tumor killing. These findings validate that AdCAR T cell activity in PDAC can be finely tuned through adapter concentration and combinatorial targeting, enabling selective tumor recognition while minimizing on-target/off-tumor toxicity. This flexible, safety-oriented strategy supports targeting heterogeneous PDAC tumors, though donor-dependent variability remains a critical consideration for clinical implementation.

Over one million patients receive cancer immunotherapy annually, yet the mechanisms underlying life-threatening immune-mediated toxicities remain poorly understood. Checkpoint inhibitor pneumonitis (CIP) is the leading cause of immunotherapy-related mortality, with a case fatality rate approaching 10%, and no genetic risk factors have been described to date. We identified Dipeptidyl-peptidase 9 (DPP9) as the first genetic susceptibility gene for CIP in a clinico-genomics cohort of 4,397 patients treated with immune checkpoint inhibitors. Mechanistically, DPP9 suppresses CARD8 inflammasome activation and IL-18 secretion in human monocytes, a pathway which is engaged prior to CIP onset, with IL-18 selectively elevated in the plasma of patients who subsequently develop CIP. Myeloid-restricted ablation of Dpp8 and Dpp9 in mice recapitulated the pulmonary histopathological and immunological hallmarks of CIP, including granuloma formation, accumulation of IFN{gamma}-producing T cells and monocyte-derived macrophages. Each of these phenotypes were driven by excessive IL-18 secretion. Together, these findings establish DPP9 as a genetic determinant of CIP and nominate IL-18 blockade as a mechanistically rational therapeutic strategy.