Cancer Immunology Research

Understanding how immune checkpoint inhibitors (ICIs) reshape human T-cell responses requires models that move beyond static transcriptomic snapshots and discrete cell-state classifications. Here, we present an integrative AI-enabled virtual cell framework that represents human CD8 T-cell responses as dynamic and computable systems during ICI therapy. By integrating single-cell RNA sequencing with paired T-cell receptor sequencing within the C2S-scale foundation model, we construct a virtual representation of individual T cells, in which each cell is encoded by a unique functional identity that captures its transcriptional, signaling, and clonal characteristics. Using this framework, we identify a previously unrecognized dynamic latent effector state of CD8 T cells characterized by intermediate expression of effector genes, distinct signaling activity, and ongoing clonal expansion. Across independent patient cohorts, the virtual cell model consistently indicates that ICI therapy mainly acts by unmasking pre-existing effector potential rather than inducing de novo effector differentiation. Notably, this latent effector population remains transcriptionally restrained despite active signaling and clonal expansion, revealing a hidden reservoir of antitumor immune capacity. More broadly, our study demonstrates how AI-enabled virtual cell modeling can reconstruct latent cellular states and their dynamic transitions from multidimensional single-cell data. By incorporating functional identity into virtual cell model, this framework uncovers biologically meaningful yet non-obvious T-cell effector program during cancer immunotherapy and provides a generalizable approach for studying immune dynamics in human disease.

Solid tumors are often surrounded by abnormal vasculature and a dense collagen-rich extracellular matrix that severely restrict the infiltration of T cells, including tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR)-T cells. These physical barriers represent a major obstacle to the efficacy of adoptive T cell therapies in solid tumors. We previously identified Formin-like 1 (FMNL1) as a cytoskeletal regulator critical for T cell extravasation and migration through restrictive environments, making it a promising target to improve T cell infiltration into tumors. Here, we developed a bioengineering platform to enhance T cell cytoskeletal dynamics by overexpressing FMNL1 in TILs and CAR-T cells. FMNL1 overexpression significantly increased T cell migration through restrictive pores in transwell assays, supporting enhanced migratory capacity of T cells under mechanically constraining conditions. Importantly, FMNL1 overexpression did not impair T cell reactivation or cytotoxic function in vitro. In murine models of melanoma and lung carcinoma characterized by limited effector T cell infiltration, FMNL1-overexpressing TILs and CAR-T cells had significantly increased accumulation at tumor sites compared to controls. Importantly, enhanced tumor accumulation resulted in improved therapeutic activity, as adoptive transfer of FMNL1-overexpressing CAR-T cells limited tumor growth and prolonged the survival of tumor-bearing mice in multiple melanoma models. Together, our findings identify FMNL1 as a broadly applicable cytoskeletal engineering target to enhance T cell accumulation and persistence in restrictive tumor microenvironments, thereby overcoming a fundamental limitation of adoptive cellular immunotherapy in solid tumors.

Immune checkpoint inhibitors (ICI) that are antibodies against CTLA-4 have achieved therapeutic effects on multiple types of cancers 1-4, but the mechanisms underlying the therapy remain incompletely understood. In contrast to the initial theory that the antibody blockades CTLA-4 on T cells is of central importance, it has recently been demonstrated that selective reduction of CD4+Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment by the antibody plays a key role in the anti-tumor effects5-8, although this phenomenon remains under debate in human patients9-12. We show here that anti-CTLA-4 antibody engages CTLA-4 in Tregs specifically in the tumor tissues to reduce their stability and survival and weaken their suppressive function through upregulating TGF-{beta} signaling. This leads to the antibody-mediated - upregulation of CD4+ and CD8+ effector T cell anti-tumor immunity and consequently cancer immunotherapy. Specifically, anti-CTLA-4 antibody directly stimulates CTLA-4 in intratumor Tregs to enhance their TGF-{beta} receptor I (T{beta}RI)-mediated TGF-{beta} signaling13. This results in reduction of IL-2 receptor CD25 expression and downregulation of lactate metabolism in intratumor Tregs to reduce their stability and survival 14-16, and also compromises their suppressive function by inhibiting Foxp3 expression. This activity requires high levels of CTLA-4 expression in the intratumor Tregs and the presence of antibody Fc receptors in the tumor tissues. Strikingly, deletion of T{beta}RI specifically in Tregs completely prevents the reduction of intratumor Tregs and abrogates the anti-CTLA-4-mediated cancer immunotherapy. In contrast to intratumor Tregs, anti-CTLA-4 antibody treatment blocks CTLA-4 in the intratumor CD4+ Foxp3- and CD8+ T effector cells and in the peripheral Tregs to decrease their T{beta}RI expression and increase their expansion due to their relatively low levels of CTLA-4 in the same tumor bearing mice. Significantly, the engagement of CTLA-4 by anti-human CTLA-4 antibody (ipilimumab) also upregulates TGFBR1 but decreases Il2RA expression and lactate metabolism in human Tregs in vitro and in humanized mice in vivo, leading to suppression of tumor progression. We have provided additional mechanism underlying anti-CTLA-4-mediated anti-tumor effects through destabilizing intratumor Tregs by CTLA-4 engagement-mediated TGF-{beta} signaling. This could lay a theoretical foundation for designing optimal immunotherapy based on anti-CTLA-4 antibody in cancer patients.

Adoptive T cell therapies have transformed the treatment of selected haematological malignancies but remain limited in many cancer settings by biological barriers that restrict effective and durable antitumour responses. Here, we investigated whether pharmacological blockade of the endothelin receptor pathway, which regulates vascular, stromal and immune processes relevant to antitumour responses, could enhance T cell-based cellular immunotherapy. Using orthotopic 4T1 triple-negative breast cancer and A20 B cell lymphoma models, we evaluated endothelin receptor blockade with non-engineered adoptive T cell therapy and anti-CD19 CAR T cell therapy, respectively, with or without immune checkpoint inhibition. Endothelin receptor blockade substantially increased objective responses across both therapeutic platforms, while the addition of immune checkpoint inhibition further increased response frequency and promoted maintained complete responses, resulting in the most durable tumour control. Mice achieving maintained complete responses resisted tumour rechallenge, consistent with long-term antitumour immune protection. High-dimensional T cell profiling identified distinct intratumoral T cell states associated with tumour control and elevated CD2 expression as a recurring feature across these populations. Together, these findings identify endothelin receptor blockade as a rational combinatorial strategy for enhancing T cell-based cellular immunotherapy and provide a strong rationale for the clinical evaluation of this therapeutic strategy.

Whether human tumor-specific CD4 T cells can drive antitumor immunity in cancers lacking tumor-cell MHC class II expression has not been established. In MHC class II-negative melanoma and breast cancer, CD4 T cells with a transcriptional signature of tumor specificity correlated with myeloid and CD8 T cell activation. Using a patient-derived NY-ESO-specific, HLA-DP04-restricted TCR, we tested whether human CD4 T cells could control tumors through indirect antigen presentation. In vitro, CD4 T cells inhibited HLA-DP-negative melanoma growth through recognition of tumor-derived antigen on antigen presenting cells (APC). In humanized mice, CD4 T cell transfer induced regression of HLA-DP-negative tumors, and control required HLA-matched human APCs. CD4 T cell transfer induced endogenous CD8 T cell proliferation and myeloid activation that mirrored immune programs correlated with the presence of these cells in patients. These findings establish that human tumor-specific CD4 T cells coordinate antitumor immunity through APC without direct tumor recognition, supporting their therapeutic relevance in MHC class II-negative cancers.

Tumor-associated macrophages (TAMs) contribute to tumor immune evasion and therapeutic resistance. However, the epigenetic and transcriptional regulators that control TAM function remain largely unidentified. Here we investigated the role of the SWI/SNF chromatin remodeling complex in TAMs and whether disruption of SWI/SNF function in TAMs could improve immunotherapy. Pharmacologic inhibition of SWI/SNF ATPase activity improved the efficacy of checkpoint blockade immunotherapy, slowing tumor growth and reprogramming transcription broadly in tumor cells, tumor-infiltrating lymphocytes, and TAMs. To define the role of SWI/SNF in TAMs specifically, we genetically deleted the SWI/SNF subunit Arid1a in myeloid cells and found this was sufficient to suppress tumor progression and enhance checkpoint blockade response. Epigenomic and single cell analyses indicated that SWI/SNF inhibition and ARID1A deletion in TAMs reduced accessibility at enhancers of genes associated with poor prognosis, such as Spp1, and increased accessibility at promoters of interferon-stimulated genes (ISGs). CD86 was elevated on ARID1A-deficient TAMs and the enhanced immunotherapy response required CD86 costimulation and CD8+ T cells. These findings establish ARID1A-dependent chromatin remodeling as a determinant of TAM gene expression programs and show that disruption of myeloid SWI/SNF function improves checkpoint blockade immunotherapy via TAM reprogramming.

Personalized neoantigen (neoAg) vaccines have shown clinical promise in solid tumors1-8, yet their efficacy and mechanism of action in hematopoietic malignancies remain poorly defined9-11. Herein, we establish an immunocompetent syngeneic A20 B-cell lymphoma platform to test the efficacy of neoAg vaccines used either as mono- or combinatorial therapies with other immunotherapies12-17. Whereas subcutaneous A20 tumors were refractory to single-agent PD-1 or CTLA4 therapy, they were eradicated in a T cell-dependent manner in 90% of syngeneic hosts treated with dual immune checkpoint therapy (dual ICT, i.e., PD-1 + CTLA4). By mapping antigen specificity of dual-ICT-elicited T cells, we identified and validated dominant endogenous A20 MHC-I and MHC-II neoantigens and designed therapeutic synthetic long peptide (SLP) vaccines containing these neoepitopes. This vaccine (A20 neoVAX) promoted robust neoAg-specific CD4{square} and CD8{square} T cell responses in naive syngeneic BALB/c mice and induced tumor rejection in [~]70% of subcutaneous tumor-bearing mice. In addition, nearly all mice rejected their subcutaneous A20 tumors when A20 neoVAX was combined with PD-1. To render the results of this study more physiologic, we developed a systemic A20 lymphoma model and found that dual ICT failed to control tumor progression and A20 neoVAX delayed tumor progression and prolonged animal survival but did not induce tumor rejection. In contrast, A20 neoVAX plus dual ICT achieved durable systemic tumor elimination. Mechanistically, the combination of A20 neoVAX plus dual ICT amplified priming of A20 neoAg-specific T cells, prevented T cell dysfunction, sustained the cytotoxic capacity of tumor-specific CD8+ T cells, and induced Th1-skewing of CD4+ T cells in tumor and peripheral compartments. To increase the clinical relevance of these findings and to minimize potential adverse events in tumor-bearing, therapeutically treated individuals, we substituted CD8-targeted cytokine muteins (CD8-IL2 or CD8-IL21) for CTLA4. These agents represent genetically modified forms of IL-2 or IL-21 that selectively stimulate CD8+ T cells but have significantly reduced capacity to activate chronic inflammation and immunosuppressive functions of other immune cells. Whereas mice bearing systemic A20 lymphoma treated with either nothing, A20 neoVAX, or A20 neoVAX + CD8-IL2 failed to control tumor outgrowth, 66.7% of tumor-bearing mice treated with A20 neoVAX + CD8-IL2 + PD-1 rejected their tumors. In similar experiments in which CD8-IL21 was substituted for CD8-IL2, tumor clearance was also observed in two-thirds of A20-bearing mice but now rejection occurred in the absence of PD1. Together, these data define a framework for optimal personalized neoAg vaccination in B-lymphoma and demonstrate that neoAg vaccines can safely synergize with CD8+ T cell-selective immunotherapies to prevent T-cell dysfunction and generate durable systemic anti-tumor immunity.

Blockade of inhibitory PD-1 signaling on T cells is a cornerstone of cancer immunotherapy, with current strategies targeting PD-1 or its ligand PD-L1. However, PD-1 engages an alternative ligand, PD-L2, whose role in tumor immunity remains poorly defined. Here, we show that PD-L2 is upregulated on intratumoral CCR7+ conventional dendritic cells (cDCs) in both mouse and human melanoma. Using genetic mouse models enabling selective ablation of PD-L1 or PD-L2 in cDCs, we identify a division of labor between these ligands: PD-L1 controls the size of the progenitor CD8 T cell pool in tumor-draining lymph nodes by modulating stem-like CD8 T cells, whereas PD-L2 limits progenitor exhausted CD8 T cell differentiation within the tumor microenvironment. Loss of PD-L2 in cDCs enhances cytotoxic CD8 T cell responses and suppresses tumor growth, particularly in tumors enriched for CCR7+ cDC1s. Consistent with this, increased CCR7+ cDC abundance is associated with poor prognosis in human cancers. Spatial transcriptomic analyses reveal co-localization of CCR7+ cDC1s and Tpex within CCL19hi niches, where cancer-associated fibroblasts serve as the predominant source of CCL19. Finally, intratumoral GM-CSF drives PD-L2 expression on CCR7+ cDCs, with Tpex and NK cells as major sources. Together, these findings establish cDC-associated PD-L1 and PD-L2 as spatially and functionally distinct checkpoints governing CD8 T cell differentiation. Our results suggest that the abundance of CCR7+ cDC1s may guide the choice between anti-PD-1 and anti-PD-L1 therapies and support the development of PD-L2-directed blockade.

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.

Immunotherapy transformed cancer treatment, yet precise correlates of response remain to be defined. While intratumoral follicular helper like (Tfhl) CD4 T cells and IgG1+ B cells have been associated with improved outcomes to PD-1 blockade for hepatocellular carcinoma (HCC), their mechanisms contributing to response are unclear. To address this question, we developed a murine model of HCC and examined CD8, CD4 and B cell responses. Increasing CD4-help sensitizes mice to PD-1 blockade, in a CD4- and CD8-dependent manner. Tumor-specific CD4 T cells contained Tfhl and Th1 populations, and both Bcl6 and T-bet were required for efficacy. Antigen-specific B cells were essential for CD4-helper expansion, yet secretion of antibodies was not required for long-term survival. Thus, B cells are critical for effective immunotherapy, but secreted antibodies are not.

Adoptive T-cell therapy (ACT) has demonstrated marked clinical benefit in hematological malignancies but has limited efficacy against solid tumors. Cancer vaccines can improve ACT, but the mechanisms underlying this combination are poorly understood. Here, we combined ACT with chimpanzee adenovirus ChAdOx1 (ChAd) and modified vaccinia Ankara (MVA) encoding tumor antigens. Whereas PD1 blockade failed to improve ACT, the combination with ChAd/MVA vaccination drove durable regression of established tumors, correlating with expansion of tumor-reactive T cells in lymphoid organs and increased tumor infiltration by T cells. Mechanistically, ChAd priming induced CXCR3 ligands (CXCL10 and CXCL9) in the vaccine-draining lymph node, thereby recruiting transferred T cells to this node independently of cognate antigen. CXCR3 expression on the transferred T cells, but not on host cells, was required for the anti-tumor effect of the combination therapy and favored T-cell accumulation in the vaccine-draining lymph node and the tumor. These findings identify CXCR3 on the transferred T cells as a determinant of ACT plus vaccination synergy and provide a rationale for combining viral-vector vaccines with TCR-T therapies, and for selecting or engineering T cells to retain CXCR3 expression.

Immunotherapy has emerged as a core therapeutic strategy for melanoma. Programmed death protein 1 (PD-1) is a critical immune checkpoint molecule that restrains host anti-tumor immunity, and therapeutic agents blocking the PD-1 signaling pathway have been widely deployed in clinical practice. Nevertheless, single-agent PD-1 blockade fails to elicit robust clinical responses in the majority of patients. Therefore, there is an urgent unmet need to develop combinatorial regimens capable of augmenting the anti-tumor efficacy of PD-1 inhibition. Gasdermin D (GSDMD), a pore-forming effector protein that orchestrates pyroptosis, exerts inherent anti-tumor activities upon overexpression. However, whether GSDMD can synergize with PD-1 blockade to enhance therapeutic outcomes against melanoma remains poorly defined. To address this question, we established an attenuated Salmonella engineered strain for targeted delivery of GSDMD, and further investigated the anti-melanoma therapeutic efficacy of combining this engineered bacterium with anti-PD-1 antibody via immunofluorescence staining, flow cytometry and other analytical approaches. Our in vivo results demonstrated that combinatorial treatment markedly suppressed melanoma progression in tumor-bearing mice relative to monotherapy with either GSDMD-expressing bacteria or anti-PD-1 antibody alone. Mechanistically, co-treatment upregulated intratumoral expression of GSDMD and the pro-apoptotic protein BAX, while simultaneously downregulating PD-1 expression. In addition, the GSDMD/anti-PD-1 combination significantly elevated the proportions of CD4 and CD8 T lymphocytes in both peripheral blood and splenic tissues, and facilitated robust tumor infiltration by these two T cell subsets. Compared with phosphate-buffered saline (PBS) and scramble control groups, combinatorial therapy promoted tumor infiltration of M1-type tumor-associated macrophages (TAMs) and repolarized TAMs away from the immunosuppressive M2 phenotype. Consistently, serum levels of the pro-inflammatory cytokines TNF- and IFN-{gamma} were markedly elevated following combined intervention. Collectively, this study verifies that attenuated Salmonella carrying GSDMD synergizes with anti-PD-1 antibody to elicit potent anti-tumor effects in melanoma-bearing mice by amplifying systemic and intratumoral anti-tumor immune responses, which provides a preclinical rationale for novel combinatorial therapeutic strategies against melanoma.

Tumor infiltrating CD8+ T cells (TILs) progress to a state of terminal exhaustion (Ttex) which have impaired functionality and are nonrenewable. However their precursors (Tpex) are renewable and can generate efficient effector cells. We started from the observation that melanoma patients undergoing therapy with checkpoint inhibitors show increased survival when their T cells have low BCL11B mRNA. In line with this, ablation of Bcl11b in CD8+ TILs conferred a superior anti-tumor response in murine melanoma and ovarian cancer models. Bcl11b KO TILs failed to progress to the Ttex state and retained elevated stemness. Bcl11b exerted its role by repressing expression of essential transcription factors (TF) controlling stemness, and conversely by promoting expression of exhaustion-associated TFs and inhibitory receptor genes, through complex epigenetic control. In addition, Bcl11b KO CD8+ T cells showed increased Ag-specific cytolytic activity and elevated Gzmb and Prf1 proteins, but no increase in their mRNAs, however presented higher expression of genes with role in translation. Furthermore, CRISPR-CAS9-mediated deletion of BCL11B in human TILs from a patient with poor response to adoptive cell therapy with autologous TILs, improved their cytolytic activity and promoted expression of the stemness-associated TF TCF1, underlying its potential therapeutic use. HIGHLIGHTS- Adoptive transfer of Bcl11b KO CD8+ TILs surpasses WT in tumor burden reduction - Bcl11b ablation reprograms TILs and impairs the progression to Ttex state - Bcl11b KO CD8+ T cells have elevated cytotoxicity and kill only Ag-MHCI targets - BCL11B deletion in nonresponder ACT-TIL improves cytolytic activity and elevates TCF1 GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/742578v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@10040d4org.highwire.dtl.DTLVardef@1a045caorg.highwire.dtl.DTLVardef@145f790org.highwire.dtl.DTLVardef@8012ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tumor-draining lymph nodes serve as critical sites for the generation, maintenance and differentiation of stem-like CD8 T cells during antitumor immune responses. Recent evidence has shown that delivering interleukin-2 to PD1 stem-like CD8 T cells using PD1-IL2v, an immunocytokine combining PD-1 blockade and IL-2R agonism, promotes their differentiation into potent effector cells with enhanced tumor-killing capacity. However, it remains unclear how targeted interleukin-2 therapies imprint early stem-like T-cell differentiation programs. Here, using single-cell transcriptomics and T-cell receptor sequencing in murine pancreatic tumor models, we demonstrate that PD1-IL2v induces an early bifurcation in the differentiation of stem-like CD8 T cells within tumor-draining lymph nodes. We identify an effector-primed stem-like population characterized by the expression of interferon-response genes, natural killer cell receptor genes, and Cx3cr1, consistent with activation of interleukin-2 and STAT5-associated programs. Clonal tracking revealed substantial overlap between these lymph node-derived cells and intratumoral effector populations, supporting a developmental relationship between early priming in lymph nodes and downstream effector differentiation. In contrast, an alternative stem-like state that displayed features associated with T-cell exhaustion, including increased Tox expression, was observed upon PD-1 therapy. Together, these findings identify an early branch point in stem-like T-cell differentiation and provide mechanistic insight into how PD1-IL2v circumvents exhaustion pathways to preferentially generate functional antitumor immunity.

Mitochondrial DNA (mtDNA) instability is an emerging feature of cancer, yet its biological effects on the tumor microenvironment (TME) and tumor progression remain incompletely defined. Using melanocyte-specific conditional silencing of mitochondrial transcription factor A (TFAM) in an autochthonous BrafV600E/Pten-/- melanoma model, we demonstrate that tumor cell-intrinsic mtDNA instability accelerates melanoma growth and metastatic dissemination in a cGAS-dependent manner. Transcriptomic profiling of early and late tumor stages revealed that mtDNA instability drives temporal remodeling of the tumor microenvironment (TME), characterized by an early type I interferon (IFN)-rich inflammatory state that licenses immunoregulatory programs as tumors progress. Tumor-associated macrophages emerged as the principal immune cell type reshaped by tumor cell-intrinsic mtDNA stress signaling, with early enrichment of inflammatory states and a shift toward efferocytosis-associated, tissue-adaptive states. The early mtDNA instability-associated macrophage signatures mapped to human melanoma states associated with favorable survival and therapy response, whereas the late signatures mapped to states associated with poor prognosis and therapy resistance across independent cohorts. Anti-PD-1 treatment delayed progression of mtDNA-instable tumors, consistent with a therapeutic sensitivity window associated with the early IFN-rich immune state. These findings establish tumor cell-intrinsic mtDNA instability as a driver of temporal immune-stromal remodeling that promotes melanoma progression and shapes therapeutic response.

Reproducing a physiologically relevant tumor microenvironment in vitro is essential for developing effective immunotherapeutic treatments. By integrating the use of combinatorial receptor blockade and organoid models we provide a deep functional understanding of CD155 and CD112 receptors in solid tumors and their impact on cellular immunotherapy and infiltration. CD226 showed plasticity in response to the environment, being able to switch between CD155 and CD112 depending on the ligand availability. In addition, CD226 drove NK cell infiltration into tumor tissues via CD155 and CD112 ligation, with CD226-CD112 interaction specifically promoting migration from the periphery to the core. Downregulation of CD155 and TIGIT induced by the tumor microenvironment and previous drug exposure reduced the long-term efficacy of TIGIT blockade. Taken together, our findings point towards using CD112R blockade in primary tumors to simultaneously enhance NK cell killing activity and promote infiltration into the tumor core via CD226 and CD112 interaction. ONE SENTENCE SUMMARYTumors shape the hierarchy of CD155-CD112 receptors, reducing TIGIT blockade efficacy, while CD226 drives NK infiltration and shows binding plasticity

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.

Tumor-associated macrophages (TAMs) promote tumor growth, inhibit effector lymphocytes, and induce resistance to immune checkpoint inhibitors (ICI). Therapies designed to deplete TAMs have had little success in the clinic, and strategies aimed to repolarize immunosuppressive TAMs to pro-inflammatory states have struggled with systemic toxicity. We have identified that the combination of low folate in the tumor microenvironment and TAM dependency on the high affinity folate receptor beta (FR{beta}) for folate uptake results in a unique metabolic dependency of TAMs necessary for their immunosuppressive function. FR{beta} (encoded by Folr2) is uniquely expressed on myeloid cells and upregulated on TAMs. FR{beta}+ TAMs exhibit an immunosuppressive phenotype in melanoma and are associated with worse clinical outcomes and resistance to ICI in melanoma patients. We generated a novel Folr2-/- mouse model to study why TAMs express a unique folate receptor and showed that tumor growth was slowed in the absence of Folr2 in a T-cell dependent manner. We observed a dramatic repolarization of Folr2-/- TAMs in vivo to pro-inflammatory states, resulting in increased cytotoxic T cell and NK cell infiltration into tumors. Importantly, we show that folate is low in the tumor microenvironment by performing metabolomics on melanoma tumors and adjacent normal tissue from warm autopsy patient specimens, and that we could increase tumor growth in Folr2-/- mice by increasing serum folate to supraphysiologic concentrations with a high folate diet. Integrated metabolomics and transcriptomic analysis demonstrated that Folr2-/- macrophages have impaired folate uptake and 1C metabolism-based reduction of oxidized glutathione in low folate conditions, resulting in increased mitochondrial reactive oxygen species (ROS) in Folr2-/- cells. Excess ROS results in leakage of mitochondrial DNA into the cytoplasm, activating cGAS-STING signaling and promoting pro-inflammatory macrophage polarization through TBK1 and NF-kB. Together, our data demonstrate that TAM expression of FR{beta} promotes their immunosuppressive functions by maintaining folate uptake in the low folate tumor microenvironment, suggesting that FR{beta} is a novel metabolic checkpoint on TAMs.

Glioblastoma (GBM) is a highly aggressive primary brain tumor that remains difficult to treat due in part to its disorganized and heterogeneous vasculature, known as the blood-tumor barrier (BTB), which limits therapeutic delivery and beneficial immune cell infiltration. Focused ultrasound (FUS) with microbubbles (MBs) can transiently disrupt the BTB to enhance drug delivery and may induce sterile inflammation (SI) that can beneficially remodel the tumor immune landscape. However, this concept has only been explored in implanted tumor models with modest immune effects. Here, we utilized a physiologically relevant genetically engineered mouse model (GEMM) generated via in utero electroporation targeting Nf1, Tp53, and Pten to study tumor-vascular-immune interactions. This 3x CRISPR-Cas9 GEMM recapitulates key features of human glioma, including infiltrative growth, histopathology, molecular alterations, and stage-dependent blood-brain barrier disruption. FUS+MBs were applied to transiently disrupt the BTB, and MRI confirmed increased vascular permeability in treated tumors. Flow cytometry revealed robust increases in tumor-infiltrating CD4+ helper and CD8+ effector T cells three days post-FUS treatment, without altering the CD8/Treg ratio. These findings were supported by immunofluorescence imaging. Double-negative and double-positive T cells were detected, but they were not significantly altered by FUS. Ki67 analysis indicated that increased T-cell accumulation was not driven by local proliferation. By seven days post-treatment, immune differences were no longer observed. Collectively, these results demonstrate that FUS-mediated BTB disruption selectively and rapidly enhances lymphocyte infiltration in a clinically relevant glioma model, supporting its potential as a temporally controlled immunomodulatory strategy for GBM.

Mucosal melanoma is a rare, lethal melanoma subtype with distinct genetics and limited benefit from immunotherapy. To define features associated with mucosal melanoma clinical outcomes, we assembled a large, multimodally profiled cohort spanning primary and metastatic disease. Compared with cutaneous melanoma, primary mucosal melanoma showed greater genomic heterogeneity, ploidy and aneuploidy, and a more dedifferentiated, proliferative transcriptional state. Analysis of genomic sequencing of 339 patients with mucosal melanoma identified four mutually exclusive genomic subtypes: CDK4/MDM2/CCND1, SF3B1-mutated, TP53/ATRX, and mucosal melanoma Triple WT, associated with distinct anatomic sites but similar outcomes. In the tumor microenvironment, we observed reduced T cell infiltration and increased CD163+ tumor-associated macrophages in mucosal melanoma relative to cutaneous melanoma. In matched longitudinal samples, CD163+ tumor-associated macrophages increased with disease progression and immunotherapy resistance. CD163+ tumor-associated macrophages engaged non-regulatory T cells through GAL9-TIM-3 interactions, nominating TIM-3 blockade as a therapeutic strategy for mucosal melanoma. These findings define the distinct aggressive biology of mucosal melanoma and highlight potential therapeutic targets and mechanisms of immune resistance.