OncoImmunology

BackgroundTumor-infiltrating lymphocyte (TIL) therapy has demonstrated clinical efficacy in malignant melanoma; however, inefficient ex vivo expansion remains a major limitation. We previously showed that stimulation of tumor-infiltrating B cells via CD40-CD40L axis improves TIL expansion, and that direct activation of the 41BB-41BBL pathway on T cells enhances CD8 T cell outgrowth. We hypothesized that adding simultaneous targeting of both pathways would augment the growth and activity of CD8+ cytotoxic T cells. We conducted a study with the objective of determining the feasibility of dual stimulation with human tumors as justification for a Phase I trial. MethodsCD40L variants were generated by yeast display selection and evaluated for B cell binding and activation. The effects of CD40L variants on TIL expansion were evaluated using tumors derived from standard of care resections using fragment method. Based on these findings, a bi-specific molecule was designed and generated fusing a CD40L variant and 41BB to the N- and C-termini of a trimeric leucine zipper. The effects of the bi-specific molecule (termed CD40LEPC6-41BBL) on TIL expansion were evaluated in TIL cultures derived from lung tumor and melanoma fragments. TIL phenotypes were assessed by flow cytometry, including high-dimensional FlowSOM analysis, and tumor reactivity by autologous tumor co-culture assays. ResultsEach of our engineered CD40L variants bound B cells and induced CD80/CD86 expression at levels comparable to wild-type CD40L. Supplementation of TIL cultures with CD40L variants increased the success rate of TIL expansion compared to control. We then developed a bi-specific CD40LEPC6-41BBL molecule capable of binding to both B and T cells. Addition of CD40LEPC6-41BBL significantly increased total TIL yield and improved expansion success rates in both lung tumor and melanoma cultures. In particular, CD40LEPC6-41BBL promoted preferential expansion of CD8 T cells. High-dimensional analysis revealed enrichment of CD8 T cell clusters expressing CD39, CD69, TIM3, and CD56 in cultures supplemented with CD40LEPC6-41BBL. Furthermore, treated cultures displayed increased frequencies of CD27 CD4 T cells. Functional assessment suggested a trend toward enhanced tumor reactivity in melanoma-derived TIL products expanded with CD40LEPC6-41BBL. ConclusionsSimultaneous stimulation of CD40 and 41BB pathways using a novel bi-specific molecule resulted in qualitative and quantitative enhancement of TIL products. These findings support dual targeting of tumor-infiltrating B cells and T cells as a promising strategy to optimize TIL manufacturing for adoptive cell therapy in Phase I trials.

Tumor-draining lymph nodes (tdLNs) are critical hubs of anti-tumor immunity but are also vulnerable to tumor-mediated immunosuppression. We analyzed T cell and B cell receptor (TCR/BCR) repertoires and transcriptomes from sentinel and non-sentinel lymph nodes of patients with melanoma from a historical pre-immune checkpoint inhibitor cohort (1994-2002) and an independent contemporary validation cohort (2022-2024). Melanoma-positive lymph nodes exhibited increased immune receptor clonality compared with tumor-free nodes. While average clonality showed no consistent association with outcome, the presence of extreme high-clonality outliers in individual lymph nodes was strongly associated with poor melanoma-specific survival. These outliers were characterized by a loss of lymphocyte-related genes and activation markers, an enrichment of melanocytic transcripts, and the suppression of immune signaling pathways, consistent with local immune dysfunction. Increased clonality was confined to lymph nodes and not observed in the peripheral blood. T cell responses to melanocyte differentiation antigens were infre-quently shared between lymph nodes and peripheral blood, highlighting immune compartmentalization.

Although the tumor immune microenvironment has been studied in endometrial cancer, the systemic immune alterations associated with disease progression and their potential prognostic significance remain poorly defined. In this study, peripheral blood immune subsets were characterized by multiparametric flow cytometry in 67 patients with EC and 20 healthy controls, including dendritic cells, MDSCs, T-cell subsets, NK cells, and exhaustion and senescence associated markers. Immune profiles were similar between healthy controls and patients with early-stage disease, whereas advanced tumors showed marked changes, including dendritic cell expansion, reduced CD4+ T-cell proportions, and increased frequencies of CD8+CD27-CD28- and CD8+CD57+ populations. Among clinicopathologic features, MDSC levels were associated with tumor grade and myometrial infiltration, while regulatory T cells were increased in TP53-mutated and microsatellite-stable tumors. In the advanced cohort (n=31), non-responders frequently displayed elevated CD8+, CD8+CD27-CD28-, and CD8+CD57+ levels alongside decreased CD27+CD28+ proportions. In multivariable Cox models, higher baseline CD8+ (HR 1.13), CD8+ CD27-CD28- (HR 1.04), and CD8+CD57+ proportions (HR 1.07; all p<0.05) were independently associated with shorter PFS, whereas higher CD27+CD28+ levels were associated with improved PFS. CD27-CD28- proportions were also linked to worse PFS by Kaplan-Meier analysis (HR 5.1, log-rank p=0.005). Longitudinal analysis (n=28) showed that senescent-like lymphocyte levels remained associated with progression across timepoints (OR 18.80), whereas total CD8+ proportions diverged progressively, reaching significance only from 6 months onward. Together, these findings identify systemic immune remodeling as a characteristic of advanced endometrial cancer and support the potential of circulating immune profiling for patient stratification and prognostic assessment, pending validation in larger prospective cohorts.

Background: The T-cell receptor {beta} (TCR{beta}) repertoire reflects antigen-driven adaptive immune responses and provides insight into tumor-immune interaction. In prostate cancer (PCa), the immunosuppressive tumor microenvironment limits effective T-cell activation, and the antigenic drivers shaping intratumoral TCR repertoires remains poorly defined. This study aimed to characterize matched tumor and peripheral TCR{beta} repertoires from treatment-naive PCa patients and to identify shared clonotypes and antigenic specificities associated with disease severity. Methods: Next-generation sequencing was used to profile TCR{beta} repertoires from matched tumor biopsies and peripheral blood mononuclear cells obtained from treatment-naive PCa patients. Repertoires clonality, diversity, and was assessed using established metrics. Antigenic convergence was evaluated using GLIPH2 to identify shared CDR3{beta} motifs and predicted tumor-associated antigen (TAA) recognition, followed by functional validation using IFN-{gamma} ELISpot and T-cell expansion assays. Results: Tumor-derived TCR{beta} repertoires displayed reduced richness and increased clonality compared with peripheral blood mononuclear cells, consistent with local antigen-driven expansion. High-grade tumors demonstrated greater interpatient clonotype sharing and motif-level convergence, indicative of recognition of common TAAs. GLIPH2 analysis associated expanded clonotypes with epitopes derived from prostate-specific G-protein coupled receptor (PSGR), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA). Functional validation confirmed that peptide pools containing PSGR- and PSMA-derived epitopes induced IFN-{gamma} production and antigen-specific T-cell proliferation in vitro. Conclusions: These findings reveal an oligoclonal, antigen-driven intratumoral TCR{beta} landscape and identify PSGR and PSMA as immunogenic, potentially actionable targets. Integration of TCR profiling with antigen discovery pipelines may support the development of TCR-based biomarkers and precision immunotherapeutic strategies in prostate cancer.

Immune checkpoint therapies have transformed clinical practice; however, reliable biomarkers to predict response remain limited. Tumor mutational burden (TMB) has emerged as an important biomarker because it is thought to reflect neoantigen load, yet its predictive utility has been inconsistent. This limitation may partly arise because TMB primarily captures tumor-intrinsic immunogenicity, which is heterogeneous and does not fully reflect the state of antitumor immunity. To identify transcriptomic surrogates that capture both high mutational burden and antitumor immune activation, we investigated whether mRNA expression of mutagenic APOBEC3 family members could serve as surrogates for high TMB and T cell-rich tumors. Using a pan-cancer computational framework, we evaluated the association of four APOBEC3 genes with mutational burden, neoantigen load, immune infiltration, and immune checkpoint blockade response. Among APOBEC3A, APOBEC3B, APOBEC3G, and APOBEC3H, APOBEC3G emerged as the strongest and most consistent marker of a TMBhighCD8high and NeoantigenhighCD8high tumor phenotypes. Single-cell analyses further demonstrated that APOBEC3G is enriched in both malignant cells and T cells compared with other APOBEC3 family members, with APOBEC3G-positive CD8+ T cells exhibiting elevated activation markers including GZMB and IFNG. Importantly, retrospective analyses of 50 immune checkpoint blockade cohorts showed that APOBEC3G had the most consistent association among APOBEC3 family members with treatment response and clinical outcomes. Together, these findings identify APOBEC3G as a candidate transcriptomic marker of a TMB-associated, T cell-inflamed tumor state linked to immune-checkpoint blockade benefit, warranting further prospective validation.

Background Endometrial cancer exhibits marked molecular and immune heterogeneity that is only partially explained by established genomic biomarkers. We investigated whether T cell receptor (TCR) repertoire architecture captures complementary dimensions of antitumor immunity beyond conventional molecular classification. Methods Paired tumor and peripheral blood samples from eight patients with molecularly characterized endometrial cancer underwent TCR repertoire profiling. Diversity, clonality, and tumor blood overlap metrics were integrated with genomic variables, including tumor mutational burden (TMB), genomic instability metric (GIM), and POLE status. Principal component analysis and correlation analyses were used to identify major dimensions of repertoire organization. Composite Immune Focusing and Immune Sharing Scores were derived to summarize dominant repertoire patterns. Results The first two principal components explained 70.1% of total repertoire variance and revealed substantial heterogeneity independent of histological subtype. TMB was strongly associated with reduced repertoire diversity and increased clonal dominance, resulting in a robust association with the Immune Focusing Score ({rho} = 0.88, p = 0.004). POLE mutated tumors occupied the extreme end of this focusing continuum. In contrast, genomic instability was associated with increased tumor blood repertoire overlap and preserved diversity, reflected by a strong correlation between GIM and the Immune Sharing Score ({rho} = 0.76, p = 0.027). The two immune scores showed minimal correlation with each other ({rho} = -0.24, p = 0.57), indicating that they capture largely independent aspects of immune organization. Conclusion Integrative analysis of TCR repertoire architecture and tumor genomics identifies distinct immunogenomic states in endometrial cancer that are not fully captured by conventional molecular classification. If validated in larger cohorts, immune focusing and immune sharing metrics may provide complementary biomarkers for patient stratification and immunotherapy-oriented precision oncology

Background The clinical significance of clonal hematopoiesis of indeterminate potential (CHIP) in melanoma remains incompletely defined, particularly with respect to CHIP genotype, clone size, and somatic mutations (e.g BRAF mutations). We integrated human cohort data and a syngeneic melanoma mouse model to evaluate whether CHIP is associated with melanoma risk, tumor growth, and differential clinical outcomes. Methods We analyzed CHIP prevalence and survival in a large treatment-unselected melanoma cohort (n=2,480), evaluated tumor growth in a syngeneic BRAF-mutant (BRAFmut) melanoma murine model of TET2-CHIP and DNMT3A-CHIP, and assessed survival outcomes in an immune checkpoint inhibitor (ICI)-treated advanced melanoma cohort (n=361). Associations with progression-free survival (PFS) and overall survival (OS) were evaluated using Kaplan-Meier analyses and multivariable Cox proportional hazards models. Results CHIP was enriched among patients with treatment-unselected melanoma compared with age/sex-matched healthy controls, and larger CHIP clone size showed an age-adjusted association with inferior OS. In a syngeneic BRAFmut melanoma murine model, TET2-CHIP, but not DNMT3A-CHIP, was associated with significantly increased primary melanoma tumor growth. Among patients with ICI-treated advanced melanoma, CHIP was associated with worse OS compared with patients without CHIP. TET2-CHIP had the strongest adverse association with survival, whereas DNMT3A-CHIP was not significantly associated with PFS or OS. Conclusions CHIP is enriched in melanoma and exploratory analyses demonstrate genotype-specific differences in melanoma tumor growth and clinical outcomes. These findings support further investigation of genotype-specific CHIP profiling as a potential biomarker for melanoma risk stratification and immunotherapy outcomes.

Abstract Introduction: IL-15 is one of the most promising candidate cytokines in cancer immunotherapy due to its ability to promote the activity of different cytotoxic innate immune cell subsets such as NK, ILC1 and gammadelta T cells. During biosynthesis, IL-15 associates with IL-15alpha and is transported to the cell surface where IL-15Ralpha trans-presents IL-15 to target neighboring cells expressing the beta chain (IL-2Rbeta) and the common gamma chain. Our group previously showed that in autoimmune type 1 diabetes and early innate immune responses to infections trans-presentation by IL-15Ralpha is dispensable. Here we addressed the relative roles of IL-15 and trans-presented IL-15 in the control of established tumors and spontaneous tumor development. Methodology: Growth kinetics of tumor cell lines were monitored in WT, Il15-/- and Il15ra-/- mice. Spontaneous fibrosarcoma was induced with Methylcholanthrene (MCA) in WT, Il15-/- and Il15ra-/- mice. Cell lines were established from MCA-induced tumors to characterize their immunogenicity. Results: Growth of established tumor cell lines were comparable in the three genotypes. MCA-induced tumor incidence was reduced in Il15ra-/- mice when compared to WT and Il15-/- mice. In vitro, MCA tumor-derived cell lines expressed MHC-I and PD-L1 and had comparable proliferation rates. In vivo, MCA tumor-derived cell lines established from the 3 genotypes showed comparative growth in WT mice suggesting that IL-15 does not impact immunoediting. Nonetheless, NLRC5 expressing B16-F10 tumors were contained in WT and Il15ra-/- mice but not in Il15-/- mice. Conclusions: Taken together, these results show that in the absence of trans-presentation by IL-15Ralpha, IL-15 can better control spontaneous tumor development and that IL-15 signaling plays a minor role in immunosurveillance in this model. IL-15 signaling, independent of IL-15Ralpha has a significant role in the control of solid tumors.

Background: Multiple myeloma (MM) progression is accompanied by remodeling of the bone marrow immune microenvironment. Local interactions among malignant plasma cells, stromal cells, myeloid cells, and immune cells not only support tumor cell survival, expansion, and immune escape, but are also closely associated with disease progression, therapeutic response, and clinical prognosis. Moreover, T cell exhaustion is a common T cells dysfunction in MM and limited efficacy of T cell-targeting therapies. However, the in situ organization and clinical significance of exhausted T cells in MM patients bone marrow remain insufficiently understood. Methods: In this study, we analyzed bone marrow Xenium 5K spatial transcriptomics data from control (Ctrl), monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SM), and MM samples. After canonical multi-sample integration and celltype annotation, we used Gaussian mixture model (GMM)-based spatial partitioning, and multilayer perceptron (MLP) machine learning for systematic characterization the T cell microenvironment in MM bone marrow. Results: Our results showed that exhaustion-like T cells increased during MM progression and formed spatially discrete T cell-enriched regions in the bone marrow, which we defined as exhaustion-like bone marrow T cell islands (eBM-TIs). These niches were mainly characterized by enhanced T cell-plasma cell communication associated with upregulated Galectin signaling. Pseudobulk analysis further showed enhanced IFN-related signaling in eBM-TIs, accompanied by upregulation of CXCR3 ligands such as CXCL9 and CXCL10, suggesting that the IFN-CXCL9/10 axis may contribute to T cell chemotaxis, maintenance of chronic inflammation, and formation of exhaustion-like states. By transferring spatial niche labels to scRNA-seq cohorts with available clinical staging information using MLP, we further found that the proportion of eBM-TI-like T cells was associated with higher disease risk and unfavorable prognostic outcomes. Conclusions: In summary, this study identifies eBM-TIs as a spatial niche in the MM bone marrow. These niches represent an important immune unit linking chronic inflammation, T cell exhaustion, and clinical risk, and may serve as a potential biomarker of MM disease progression.

BackgroundMutation-derived neoantigens, typically identified in primary tumors, are emerging therapeutic targets for personalized cancer vaccines and adoptive T-cell therapies. However, clinical efficacy of neoantigen-directed therapies in patients with metastatic disease remains limited, partly due to inter-site genetic heterogeneity. We investigated whether ubiquitous neoantigens-derived from mutations shared across all tumor sites-could provide more effective, durable targets, particularly in patients undergoing resection of metastatic lesions. MethodsWhole-exome and RNA sequencing were performed on 14 tumor samples (primary and 13 synchronous nodal metastases) from a treatment-naive patient with pancreatic neuroendocrine tumor (PNET). Ubiquitous mutations were identified bioinformatically, and their immunogenicity assessed using in-vitro stimulation of autologous peripheral blood mononuclear cells followed by IFN-{gamma} ELISpot assay. Neoantigen-specific T-cell clonotypes were further identified by HLA-tetramer staining and single-cell RNA/TCR sequencing. Neoantigen-reactive clonotypes identified in peripheral blood were tracked across multiple metastatic sites using bulk TCR{beta} repertoire sequencing. ResultsAmong 1,195 non-synonymous mutations detected, eight were shared across all 14 tumor sites. Of these, one encoded a neoantigen that elicited a reproducible IFN-{gamma} ELISpot response in peripheral blood, confirming its immunogenicity. Further, we identified the corresponding neoantigen-reactive TCR clonotypes in blood. Comparison with bulk TCR{beta} repertoires from eight metastatic sites showed that these clonotypes were present in every site analyzed, with evidence of local clonal expansion. ConclusionThis study provides direct evidence that a single ubiquitous mutation-derived neoantigen can generate systemic T-cell responses and clonotype expansion across multiple metastatic sites in a TMB-low, TIL-low tumor. Our findings support incorporating mutation-sharing status across metastases as a key criterion for neoantigen selection in cancer vaccines and adoptive T-cell therapies. This approach could inform the design of neoantigen-directed immunotherapies in metastatic PNET and potentially other metastatic solid tumors. What is already known on this topicNeoantigen-directed therapies, such as personalized cancer vaccines or adoptive T-cell transfer, can induce anti-tumor responses but have shown limited success in metastatic disease. One major barrier is genetic heterogeneity between tumor sites, suggesting that targeting ubiquitous mutations-those shared across all tumor sites-may improve the efficacy of such therapies. What this study addsIn one patient with metastatic pancreatic neuroendocrine tumor involving 13 lymph nodes, we identified eight ubiquitous mutations, one of which generated a detectable neoantigen-specific T-cell response in blood. The corresponding T-cell clonotypes were found across all metastatic sites analyzed and showed evidence of clonal expansion, providing direct evidence of systemic and local recognition of a shared neoantigen in a TMB-low/TIL-low cancer. How this study might affect research, practice or policyThese findings support incorporating mutation sharing across metastases as a key criterion in neoantigen selection for cancer vaccines and adoptive T-cell therapies. This strategy could enhance the relevance and durability of neoantigen- directed approaches in patients with metastatic disease.

Background CD19-directed CAR-T cell therapy can induce durable remissions in chronic lymphocytic leukemia (CLL), but response rates are lower than in other B-cell malignancies, in part because CLL is characterized by T-cell dysfunction, defective immune synapse formation, and impaired target-cell co-stimulation. Lenalidomide is an immunomodulatory drug with the potential to act on both sides of the CAR-T/CLL interface by improving T-cell fitness and modifying malignant B-cell susceptibility to immune engagement. Methods We are conducting an open-label, non-randomized phase I/II clinical trial VTB-CLL002 (ClinicalTrials.gov identifier: NCT06762431) evaluating the safety and efficacy of CD19 CAR-T cell therapy combined with concomitant lenalidomide in patients with relapsed or refractory CLL and small lymphocytic lymphoma followed by lenalidomide maintenance. The primary endpoint was safety. The secondary endpoint included overall response rate (ORR), complete response (CR), progression-free survival (PFS) and overall survival (OS). Results Twelve patients were treated. The median age was 60 years and the median number of prior lines of therapy was 2. All patients were BTK inhibitor-naive, and all had measurable disease at the time of infusion. CAR-T manufacturing was successful in all patients. All treated patients achieved complete remission, with a median time to response of 1 month. CAR T-cells expansion was observed in all patients, with a median peak expansion of 137 cells/L and a median time to peak expansion of 14 days. CAR T-cells remained detectable at the last follow-up in all patients, with persistence documented up to 24 months. At dose levels 2-3, eight of nine patients had ongoing MRD-negative responses at the time of analysis. Toxicity was clinically meaningful. Cytokine release syndrome (CRS) occurred in all patients, with severe CRS observed in 2 of 12 patients. ICANS occurred in 5 of 12 patients, including severe ICANS in 4 of 12 patients. One patient developed late grade 4 ICANS temporally associated with lenalidomide reintroduction and secondary CAR-T expansion. Early and late immune effector cell-associated hematotoxicity were common. In mechanistic studies, lenalidomide enhanced CAR-T proliferation and cytotoxicity, shifted CAR-T cells toward effector-associated phenotypes, reduced selected exhaustion markers during repeated antigen challenge, and increased IL-2 and IFN-{gamma} secretion. Lenalidomide also increased CAR-T/CLL conjugate formation and upregulated CD54/ICAM-1 on CLL target cells without broad induction of CD80, CD86, or CD40, consistent with improved adhesive target-cell engagement rather than classical co-stimulation. Transcriptomic profiling supported enhanced Th1/cytotoxic and T-cell activation-associated programs with lower T reg -associated genes in lenalidomide-treated CAR-T cells. Conclusions Lenalidomide-augmented CD19 CAR-T therapy demonstrated strong early clinical activity in relapsed/refractory CLL, characterized by deep responses, durable CAR-T persistence, and substantial incidence of immune effector-associated toxicities. These findings support further evaluation of lenalidomide as a rational CAR-T partner in CLL and suggest that its activity may involve both improved CAR-T fitness and enhanced target-cell engagement. Future studies should optimize lenalidomide timing and dosing to preserve response depth while reducing delayed immune-effector toxicity.

The role of UBE2N in myeloid cell-mediated immune suppression in cancer remains undefined. Here, we examined the function of UBE2N in myeloid cell-mediated tumor progression using a temporally inducible myeloid-specific knockout model (LysMCreERUbe2nfl/fl). Temporally induced deletion of Ube2n in myeloid cells (Ube2nMyeKO) significantly hindered growth of YUMM1.7 melanoma. This was accompanied by reduced myeloid cell burden within the tumor microenvironment. We observed altered abundance of PD-1, PD-L1, and SPP1 in the Ube2nMyeKO tumor microenvironment at the tissue level. In vitro analysis showed that knock-in expression of a catalytically deficient UBE2NC87S mutant in bone marrow-derived macrophages (BMDMs) markedly decreased expression of Spp1. We observed decreased SPP1 secretion in Ube2nMyeKO BMDM-conditioned media (CM). Treatment with Ube2nMyeKO BMDM-CM decreased co-expression of PD-1, TIM-3, and LAG-3 on chronically stimulated T cells. Antibody-mediated neutralization of SPP1 in Ube2nWT BMDM-CM decreased PD-1 expression on CD8+ T cells. Together, these findings suggest a role for myeloid UBE2N in YUMM1.7 progression.

CD47 delivers a dominant "Dont Eat Me" signal that inhibits macrophage-mediated clearance of tumour cells. Using immune competent, chemoresistant neuroblastoma (NB) models, we tested a Fc-silent CD47 blocker (ALX301) with anti-GD2 antibody alone and in combination with a clinically aligned temozolomide/irinotecan chemoimmunotherapy backbone. Tumours expressed GD2 and CD47, and bound ALX301. In macrophage coculture assays, anti-GD2 antibody induced dose-dependent phagocytosis, whereas ALX301 or an anti-CD47 antibody alone did not. CD47 blockade in combination with a suboptimal concentration of anti-GD2 antibody showed an additive effect on phagocytosis in vitro. In vivo, however, ALX301 failed to improve tumour control or survival when added to anti-GD2 or to chemoimmunotherapy in two models. Toxicity was acceptable, showing only mild, expected red-cell changes without organ injury. This form of CD47 inhibition is therefore mechanistically active in vitro but insufficient to enhance anti-GD2 antibody-based therapy in immune competent mice bearing a chemoresistant NB, highlighting the potential need for myeloid-reprogramming partners.

BackgroundStrategies to define human monocytes by flow cytometry vary considerably across studies. Recently, toll-like receptor 2 (TLR2) has been proposed as a marker to identify "all monocytes" in human peripheral blood. However, the TLR2-defined monocytes also contained a previously ignored TLR2posCD14dim/negCD16neg population, which we termed the unclassified subset (UCS). MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors and patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS) were analyzed by multiparameter flow cytometry using TLR2pos gating. PBMCs from additional healthy donors were analyzed to characterize the UCS population, including the impact of using either TLR2pos or a negative selection-based gating strategy. ResultsThe TLR2pos CD14dim/neg CD16neg UCS population was present in healthy controls, MGUS, and MM patients. The UCS expressed the monocyte-macrophage scavenger receptor CD163 and was significantly reduced in MM patients compared to healthy donors (P<0.002). Further phenotypic characterization in healthy blood donors revealed that approximately 80% of UCS cells expressed CD163 at levels comparable to classical monocytes, yet phenotypically resembled CD163pos dendritic cells (DCs). Importantly, gating strategies influenced the composition of the UCS: negative selection-based gating captured all DC subsets, whereas TLR2pos gating primarily included CD1cpos DCs that were highly CD163pos. ConclusionsThese findings demonstrate that circulating CD163pos CD1cpos DCs are included in the TLR2pos cell population previously described as exclusively monocytes, highlighting the impact of gating strategy on monocyte subset identification. Further, the lower level of TLR2pos CD14dim/neg CD16neg CD163pos cells in MM patients may represent decreased levels of circulating DCs that may contribute to the immune dysregulation in this disease.

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by rapid progression and a poor prognosis. CAR-based cellular therapies are promising approaches, and CAR-T cells targeting GD2 have demonstrated transient efficacy. Identifying how tumors evade these treatments is essential for advancing therapy development. In this study, we investigated the mechanisms through which GBM cells evade GD2.chimeric antigen receptor (CAR)-T and CAR-invariant natural killer T (iNKT) in vitro and explored ways to overcome tumor escape. GD2-targeted CAR-T and CAR-iNKT cells were tested in a stepwise in vitro model that repeatedly exposed them to GD2+ cell lines. While CAR effector cells effectively killed GD2+ GBM cells in short-term assays, their anti-tumor efficacy declined after repeated antigen exposures. Tumor escape mechanisms included reduced CAR expression, impaired proliferation, reduced production of cytokine, granzyme, and perforin, tumor downregulation of GD2, trogocytosis, and upregulation of the HLA-E/NKG2A inhibitory compared to MICA-B/NKG2D activation pathways on tumor and immune cells. Increasing effector cell numbers or adding IL-15 +/- IL-7 partially improved CAR persistence but did not fully restore CAR effector functions. By contrast, IL-12 addition optimized tumor-killing capacity by increasing CAR effector cell proliferation, CAR surface expression, IFN-y production, and balancing HLA-E/NKG2A versus MICA-B/NKG2D pathways. In conclusion, GD2.CAR-T and GD2.CAR-iNKT cells effectively target GBM but are susceptible to repeated antigen exposure, which IL-12 could counteract. These findings encourage further development of armored IL-12 CAR-T or CAR-iNKT cells and further investigation of the roles of HLA-E and MICA-B pathways in immunotherapy against GBM.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and patient outcomes have remained largely unchanged despite advances in multimodal therapy. Immune checkpoint inhibitors (ICIs), which block the PD-1/PD-L1 axis to restore T cell-mediated anti-tumor immunity, have emerged as a promising treatment strategy. However, response rates remain below 20% in HNSCC, underscoring the need to better understand mechanisms of immune evasion within the tumor microenvironment. Syngeneic mouse models are essential for studying tumor-immune interactions, yet currently available HNSCC models are limited. Here, we report the development of a novel FVB/NJ-derived syngeneic HNSCC model generated from 7,12-dimethylbenz(a)anthracene (DMBA)-induced primary on floor of mouth/buccal tumors, designated FMOC1, FMOC2, and FMOC3 (FVB/NJ Mouse Oral Cancer). In vitro, all FMOC cell lines exhibited robust proliferative capacity with distinct proliferation kinetics. In vivo, all FMOC cell lines exhibited characteristic HNSCC histopathology, including cytokeratin 5 positivity, and were tumorigenic in immunodeficient NCG mice; however, in syngeneic immunocompetent mice, only FMOC1 demonstrated sustained tumor growth at orthotopic and flank sites, whereas FMOC2 and FMOC3 tumors underwent spontaneous regression within 2 weeks, indicating differential immune-dependent tumorigenicity among the lines. Consistent with this, depletion of CD4+ and/or CD8+ T cells restored tumor growth in FMOC2 and FMOC3 models, indicating a critical role for T cell-mediated immunity in tumor suppression. Notably, FMOC1 tumors were responsive to anti-PD-L1 and anti-CTLA-4 therapy, supporting their utility for evaluating immunotherapeutic strategies. Collectively, these findings establish the FMOC model as a novel and versatile platform to study tumor-immune interactions and immune evasion mechanisms in HNSCC, with potential applications in preclinical immunotherapy development.

Small bowel gastrointestinal stromal tumors (GISTs) are more aggressive than gastric GISTs, yet the biologic basis for this difference remains poorly understood. We hypothesized that differential expression of immune checkpoints contributes to this site-specific behavior. Bulk RNA sequencing of 42 primary GISTs (36 gastric, 6 small bowel) revealed marked upregulation of VTCN1, which encodes the inhibitory checkpoint B7-H4, in small bowel tumors (log2FC = 7.95, adjusted P < 0.001). In contrast, expression of the therapeutically targeted checkpoints PD-L1, PD-1, and CTLA-4 was comparable between sites. Concordantly, B7-H4 enrichment was accompanied by an immunosuppressive tumor microenvironment, characterized by reduced antigen-presenting cells, fewer effector-memory CD8+ T cells, lower granzyme B expression, and suppression of interferon and inflammatory signaling pathways. Notably, the differences in B7-H4 expression were independent of imatinib-treatment status. These findings were corroborated in an external cohort of 77 untreated GISTs, in which VTCN1 was similarly enriched in small bowel tumors. Independent immunohistochemical analysis of a tissue microarray comprising 68 untreated primary GISTs confirmed the pattern, showing median B7-H4 positivity of 78.6% in duodenal, 20.5% in jejunal/ileal, and 0% in gastric tumors, with staining localized to tumor cells rather than stroma. Collectively, these data identify B7-H4 as a site-specific feature of small bowel GISTs and a potential therapeutic target for tumors that have not responded to conventional checkpoint blockade.

CD19-4-1BBL is a bispecific antibody fusion protein that targets CD19 and costimulates 4-1BB on T cells and other immune cells. Its antitumor activity has been reported in B-cell non-Hodgkin lymphoma with emphasis on its T-cell mediated cytotoxic activity. Its effect on other 4-1BB expressing immune cells is unexplored. Here, we investigated the molecular mechanisms and the antileukemic effect of CD19-4-1BBL in chronic lymphocytic leukemia (CLL), a B-cell malignancy profoundly marked by the immunosuppressive activity of myeloid-derived suppressor cells, tumor-associated macrophages and CD4+ regulatory T cells. We demonstrated that CD19-4-1BBL simultaneously mitigates the immunosuppressive phenotype and transcriptome machinery of these cells and promotes antitumor CD8+ T-cell immunity. Finally, in a preclinical, patient-derived xenograft model of CLL, we observed a favourable survival impact, especially in mice transplanted with immune cells from patients with high-risk/progressive leukemia. Our findings provide evidence that the CD19-4-1BBL treatment is a multifaceted, immune-based strategy that should be clinically explored in patients with chronic lymphocytic leukemia. KEY POINTSO_LICD19-4-1BBL sharpens the myeloid cell transcriptome and stimulates diverse memory CD8+ T cell clonotypic responses. C_LIO_LICD19-4-1BBL costimulation can be therapeutically exploited in high-risk chronic lymphocytic leukemia. C_LI

Small cell lung cancer (SCLC) is a highly aggressive malignancy with limited therapeutic advances. Unlike many other cancers, its immune landscape, particularly immune competence and T cell recognition, remains poorly characterized. Here, we generate a single-cell transcriptome atlas of the SCLC immune microenvironment with paired T cell receptor (TCR) sequencing. By linking T cell states with clonality and a multilayered functional screening, we identify 6 tumor-reactive TCRs that recognize and eradicate autologous SCLC cell lines. We delineate a novel SCLC-reactive CD8+ T cell signature (SCLC_TR), enabling the identification of 47 further SCLC-reactive TCRs. The SCLC_TR signature performs extremely well in pancreatic ductal adenocarcinoma (PDAC), another immune-cold tumor indication, and, most strikingly, patients with elevated SCLC_TR signature scores exhibited significantly improved survival, underlining its prognostic potential. Comparative cell-cell interaction analyses implicate several immunosuppressive mechanisms, with myeloid cells and CD4+ regulatory T cells possibly acting as counterbalances to effector T cell activity in SCLC. In summary, our study challenges the prevailing notion of SCLC as an immune-cold tumor type by providing direct evidence of tumor-reactive T cell responses and introduces the SCLC_TR signature as a tool to identify tumor-specific T cells and their microenvironmental restraints and escape mechanisms, ultimately shaping next-generation immunotherapeutic strategies.

BackgroundChimeric Antigen Receptor (CAR) T cell therapy targeting B-cell maturation antigen (BCMA) has demonstrated impressive clinical efficacy in relapsed/refractory multiple myeloma (MM). Nonetheless, disease relapse limits durable response for most patients. Trogocytosis of target antigen by effector cells has emerged as a potential contributor to reduced surface antigen density, CAR T cell dysfunction, and fratricide. Although {gamma}-secretase inhibitors (GSI) significantly increase cell surface BCMA density and decrease soluble BCMA (sBCMA), their effects on BCMA trogocytosis and the resulting impact on CAR T-cell function remain incompletely understood. MethodsWe investigated the effects of GSI on BCMA-directed CAR T cell function and trogocytosis using in vitro co-culture systems with MM cell lines across a spectrum of BCMA expression. We validated findings using confocal microscopy and cytotoxicity assays. Trogocytosis and fratricide were assessed in time-resolved functional studies. Phenotypic and functional differences between trogocytosis-positive (CAR T Trogo+) and trogocytosis-negative (CAR T) cells were evaluated using multiparametric flow cytometry, proteomic profiling, single-cell RNA sequencing (scRNA-seq), T-cell receptor (TCR) sequencing, and in vitro rechallenge assays. We also interrogated clinical samples from two Phase I trials (NCT03338972 and NCT03502577) which employed the identical CAR T cell construct with or without GSI respectively, to evaluate the relationship between trogocytosis, CAR T cell persistence, and treatment outcome. ResultsGSI driven increases in BCMA density on MM cell lines enhanced CAR T cell cytotoxicity but concomitantly increased trogocytosis, particularly in high-antigen-density cell lines-(H929+GSI vs H929; 30 min (P<0.0001), 1 h (P<0.0001), 2 h (P<0.0001), 6 h (P<0.0001), and 24 h (P<0.0001) and in CD4 CAR T cells (K562mCherry+GSI, CD4 vs CD8 CAR T cells;10 min (P=0.01), 2 h (P=0.01), and 6 h (P=0.004). Following BCMA acquisition, CAR T cells (CAR T Trogo) exhibited reduced proliferative capacity, diminished cytotoxic function (CAR T Trogo+ vs CAR T; (P=0.01), and an increase in markers of exhaustion/activation (CD4+ CAR T Trogo+ vs CD4 CAR T and CD8+ CAR T Trogo+ vs CAR T; PD-1+LAG-3+, PD-1+TIM-3+, and TOX+TIM-3 co-expression, (P=0.007, P<0.0001, P=0.006 and P=0.004, P=0.03, P=0.006). In fratricide assays, CAR T Trogo cells were susceptible to killing by naive CAR T cells. Single cell RNA-seq supports the phenotypic findings revealing transcriptional features of heightened activation and accelerated exhaustion in CAR T Trogo+ cells. Clinical phase I trial data confirm BCMA trogocytosis in patient samples. ConclusionsOur findings highlight the paradoxical effects of increased BCMA density on BCMA CAR T cell therapy: enhancement of initial tumor targeting and promotion of trogocytosis-associated dysfunction. Trogocytosis may contribute to antigen modulation, CAR T cell exhaustion, and fratricide, potentially muting the therapeutic benefits of enhanced antigen density. To optimize GSI and mitigate trogocytosis-associated resistance mechanism, future clinical trial designs should incorporate early time-point sampling, a sample size providing sufficient statistical power to determine an impact on CAR T cell persistence and treatment response, and mechanistic assessments.