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In this study, involving melanoma patients and a mouse model for melanoma, an optimal anti-tumour response was induced by using a combination of radiation with anti-CTLA4 and anti-PD-L1 antibody therapies, each attacking the tumour from a different angle. Three-way treatment of melanoma This study, involving melanoma patients and a mouse model for melanoma, demonstrates that an optimal anti-tumour response involves a combination of the three tested treatment modalities: high-dose radiation, together with two different types of immune checkpoint inhibitors (anti-CTLA4 and anti PD-L1), each attacking the tumour from a different angle. Immune checkpoint inhibitors 1 result in impressive clinical responses 2 , 3 , 4 , 5 , but optimal results will require combination with each other 6 and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (T reg cells), thereby increasing the CD8 T-cell to T reg (CD8/T reg ) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/T reg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

Resistance to immunotherapy is one of the biggest problems of current oncotherapeutics. While T cell abundance is essential for tumor responsiveness to immunotherapy, factors that define the T cell-inflamed tumor microenvironment are not fully understood. We used an unbiased approach to identify tumor-intrinsic mechanisms shaping the immune tumor microenvironment (TME), focusing on pancreatic adenocarcinoma because it is refractory to immunotherapy and excludes T cells from the TME. From human tumors, we identified ephrin-A receptor 2 (EPHA2) as a candidate tumor-intrinsic driver of immunosuppression. Epha2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy. We found that prostaglandin endoperoxide synthase 2 (PTGS2), the gene encoding cyclooxygenase- 2, lies downstream of EPHA2 signaling through TGF-[beta] and is associated with poor patient survival. Ptgs2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy; pharmacological inhibition of PTGS2 was similarly effective. Thus, EPHA2/PTGS2 signaling in tumor cells regulates tumor immune phenotypes; blockade may represent a therapeutic avenue for immunotherapy-refractory cancers. Our findings warrant clinical trials testing the effectiveness of therapies combining EPHA2/TGF-[beta]/PTGS2 pathway inhibitors with antitumor immunotherapy and may change the treatment of notoriously therapy-resistant pancreatic adenocarcinoma.

Multiplexed imaging has transformed our ability to study tissue organization by capturing thousands of cells and molecules in their native context. However, these datasets are enormous, often comprising tens of gigabytes per image, and require complex workflows that limit their broader use. Current tools are often fragmented, inefficient, and difficult to adopt across disciplines. Here we show that SPACEc, a scalable Python platform, streamlines spatial imaging analysis from start to finish. The platform integrates image processing, cell segmentation, and data preprocessing into a single workflow, while improving computational performance through parallelization and GPU acceleration. We introduce innovative methods, including patch proximity analysis, to more accurately map local cellular neighborhoods and interactions. SPACEc also simplifies advanced approaches such as deep-learning annotation, making them accessible through an intuitive interface. By combining efficiency, accuracy, and usability, this platform enables researchers from diverse backgrounds to gain deeper insights into tissue architecture and cellular microenvironments. SPACEc offers a user-friendly, expert-curated workflow for multiplexed image analysis, enabling researchers to easily perform advanced spatial biology studies and streamline the path from experiment to discovery.

BACKGROUNDNeoantigens derived from KRASMUT have been described, but the fine antigen specificity of T cell responses directed against these epitopes is poorly understood. Here, we explore KRASMUT immunogenicity and the properties of 4 T cell receptors (TCRs) specific for KRASG12V restricted to the HLA-A3 superfamily of class I alleles.METHODSA phase 1 clinical vaccine trial targeting KRASMUT was conducted. TCRs targeting KRASG12V restricted to HLA-A*03:01 or HLA-A*11:01 were isolated from vaccinated patients or healthy individuals. A comprehensive analysis of TCR antigen specificity, affinity, crossreactivity, and CD8 coreceptor dependence was performed. TCR lytic activity was evaluated, and target antigen density was determined by quantitative immunopeptidomics.RESULTSVaccination against KRASMUT resulted in the priming of CD8+ and CD4+ T cell responses. KRASG12V -specific natural (not affinity enhanced) TCRs exhibited exquisite specificity to mutated protein with no discernible reactivity against KRASWT. TCR-recognition motifs were determined and used to identify and exclude crossreactivity to noncognate peptides derived from the human proteome. Both HLA-A*03:01 and HLA-A*11:01-restricted TCR-redirected CD8+ T cells exhibited potent lytic activity against KRASG12V cancers, while only HLA-A*11:01-restricted TCR-T CD4+ T cells exhibited antitumor effector functions consistent with partial coreceptor dependence. All KRASG12V-specific TCRs displayed high sensitivity for antigen as demonstrated by their ability to eliminate tumor cell lines expressing low levels of peptide/HLA (4.4 to 242) complexes per cell.CONCLUSIONThis study identifies KRASG12V-specific TCRs with high therapeutic potential for the development of TCR-T cell therapies.TRIAL REGISTRATIONClinicalTrials.gov NCT03592888.FUNDINGAACR SU2C/Lustgarten Foundation, Parker Institute for Cancer Immunotherapy, and NIH.

Adeno-associated virus (AAV)-mediated gene therapy is currently being pursued as a treatment for the monogenic disorder α-1-antitrypsin (AAT) deficiency. Results from phase I and II studies have shown relatively stable and dose-dependent increases in transgene-derived wild-type AAT after local intramuscular vector administration. In this report we describe the appearance of transgene-specific T-cell responses in two subjects that were part of the phase II trial. The patient with the more robust T-cell response, which was associated with a reduction in transgene expression, was characterized more thoroughly in this study. We learned that the AAT-specific T cells in this patient were cytolytic in phenotype, mapped to a peptide in the endogenous mutant AAT protein that contained a common polymorphism not incorporated into the transgene, and were restricted by a rare HLA class I C alleles present only in this patient. These human studies illustrate the genetic influence of the endogenous gene and HLA haplotype on the outcome of gene therapy.

To determine the cellular and molecular basis of Castleman Disease (CD), we analyze the spatial proteome and transcriptome from a discovery ( n  = 9 cases) and validation ( n  = 13 cases) cohort of Unicentric CD, idiopathic Multicentric CD, HHV8-associated MCD, and reactive lymph nodes. CD shows increased stromal cells that form unique microenvironments. Interaction of activated follicular dendritic cell (FDC) cytoplasmic meshworks with mantle-zone B cells is associated with B-cell activation and differentiation. CXCL13+ FDCs, PDGFRA + T-zone reticular cells (TRC), and ACTA2-positive perivascular reticular cells (PRC) were the predominant source of increased VEGF expression and IL-6 signaling. MCD is characterized by increased TRC while UCD shows increased B-reticular cells (BRC). VEGF expression by FDCs is associated with peri-follicular neovascularization. FDC, TRC and PRC of CD activates JAK-STAT, TGFβ, and MAPK pathways via specific ligand-receptor interactions. Here, we show that stromal-cell activation and associated B cell activation and differentiation, neovascularization and stromal remodeling underlie CD. Castleman disease encompasses a group of disorders characterised by abnormal lymph node morphology. Here the authors use single cell and spatial transcriptomics to assess the stromal, immune and interaction architecture of different subtypes of Castleman disease, indicating potential ligand-receptor interactions between immune cells.

Highly multiplexed, single-cell imaging has revolutionized our understanding of spatial cellular interactions associated with health and disease. With ever-increasing numbers of antigens, region sizes, and sample sizes, multiplexed fluorescence imaging experiments routinely produce terabytes of data. Fast and accurate processing of these large-scale, high-dimensional imaging data is essential to ensure reliable segmentation and identification of cell types and for characterization of cellular neighborhoods and inference of mechanistic insights. Here, we describe RAPID, a Real-time, GPU-Accelerated Parallelized Image processing software for large-scale multiplexed fluorescence microscopy Data. RAPID deconvolves large-scale, high-dimensional fluorescence imaging data, stitches and registers images with axial and lateral drift correction, and minimizes tissue autofluorescence such as that introduced by erythrocytes. Incorporation of an open source CUDA-driven, GPU-assisted deconvolution produced results similar to fee-based commercial software. RAPID reduces data processing time and artifacts and improves image contrast and signal-to-noise compared to our previous image processing pipeline, thus providing a useful tool for accurate and robust analysis of large-scale, multiplexed, fluorescence imaging data.

BACKGROUND. Neoantigens derived from [KRAS.sup.MUT] have been described, but the fine antigen specificity of T cell responses directed against these epitopes is poorly understood. Here, we explore [KRAS.sup.MUT] immunogenicity and the properties of 4 T cell receptors (TCRs) specific for [KRAS.sup.G12V] restricted to the HLA-A3 superfamily of class I alleles. METHODS. A phase 1 clinical vaccine trial targeting [KRAS.sup.MUT] was conducted. TCRs targeting [KRAS.sup.G12V] restricted to HLA-A*03:01 or HLA-A*11:01 were isolated from vaccinated patients or healthy individuals. A comprehensive analysis of TCR antigen specificity, affinity, crossreactivity, and CD8 coreceptor dependence was performed. TCR lytic activity was evaluated, and target antigen density was determined by quantitative immunopeptidomics. RESULTS. Vaccination against [KRAS.sup.MUT] resulted in the priming of [CD8.sup.+] and [CD4.sup.+] T cell responses. [KRAS.sup.G12V] -specific natural (not affinity enhanced) TCRs exhibited exquisite specificity to mutated protein with no discernible reactivity against [KRAS.sup.WT]. TCR-recognition motifs were determined and used to identify and exclude crossreactivity to noncognate peptides derived from the human proteome. Both HLA-A*03:01 and HLA-A*11:01-restricted TCR-redirected [CD8.sup.+] T cells exhibited potent lytic activity against [KRAS.sup.G12V] cancers, while only HLA-A*11:01-restricted TCR-T [CD4.sup.+] T cells exhibited antitumor effector functions consistent with partial coreceptor dependence. All [KRAS.sup.G12V]-specific TCRs displayed high sensitivity for antigen as demonstrated by their ability to eliminate tumor cell lines expressing low levels of peptide/HLA (4.4 to 242) complexes per cell. CONCLUSION. This study identifies [KRAS.sup.G12V]-specific TCRs with high therapeutic potential for the development of TCR-T cell therapies. TRIAL REGISTRATION. ClinicalTrials.gov NCT03592888. FUNDING. AACR SU2C/Lustgarten Foundation, Parker Institute for Cancer Immunotherapy, and NIH.

To the Editor: Tran et al. (Dec. 8 issue) 1 describe a remarkable case of a patient with metastatic colorectal cancer treated with autologous T cells specific for mutant KRAS G12D and restricted to the major histocompatibility complex class I allele HLA-C*08:02. The authors hypothesize that in the United States alone, thousands of patients per year may be eligible for T-cell–based immunotherapy targeting KRAS G12D. To estimate how common this opportunity may be, we identified 151 patients with KRAS G12D mutations out of 6125 patients in the Cancer Genome Atlas. Of these, only 4 had the HLA-C*08:02 allele as determined by . . .

BackgroundChimeric Antigen Receptor (CAR) T cell therapy has shown strong antitumor potency in the context of hematological malignancies. However, CAR T cells face multiple barriers in treating solid tumors, such as trafficking to the tumor and a failure to expand and persist in the periphery. While ideally solid tumor target expression is restricted to the tumor to prevent on-target off-tumor toxicity, this scenario provides no opportunity for antigen-driven CAR T cell expansion in the periphery which can be correlated with response. To address this issue and increase the expansion and persistence of solid tumor-directed CAR T cells, we sought to design a dual-targeted CAR T cell against CD19 and the solid tumor antigen, mesothelin (MSLN), in order to maintain peripheral MSLN CAR T cell fitness and increase the number of CAR T cells which traffic to the tumor.MethodsLeveraging the paradigm of CD19 CAR T cells, which target a dispensable cell population and have exhibited success and safety in the clinic, we developed and optimized a CD19 and MSLN directed CAR T cell. We first assessed our technology in immunocompetent C57BL/6 mice where we established two syngeneic flank tumor models of pancreatic ductal adenocarcinoma (PDA) from KPC (KrasLSL.G12D/+p53R172H/+) mice, ranging in immunogenicity. In addition to assessing their antitumor efficacy, we also assessed their trafficking patterns compared to control CAR T cells. To determine the antitumor efficacy of human CAR T cells in NOD scid gamma (NSG) mice, we have developed a novel model to mimic CD19+ B cells in the periphery.ResultsWe have demonstrated specific cognate expansion of CD45.1+ dual murine CAR T cells and the induction of B cell aplasia in CD45.2+ C57BL/6 mice. Ultimately, we find enhanced B cell driven expansion in the blood, increased antitumor efficacy, and higher levels of early activation without increasing downstream exhaustion or toxicity compared to single CAR T cells. We have also identified alternative mechanisms of trafficking, likely owing to boosting in the spleen. Further, we have established a novel humanized model for CD19+ B cells in NSG mice and demonstrate that human dual CAR T cells expand in the periphery and demonstrate anti-tumor activity.ConclusionsDual-targeted CAR T cells are a successful strategy to enhance solid tumor-directed expansion in the periphery and tumor infiltration resulting in increased antitumor efficacy in syngeneic and humanized models of PDA.