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Indolent non-Hodgkin’s lymphomas (iNHLs) are incurable with standard therapy and are poorly responsive to checkpoint blockade. Although lymphoma cells are efficiently killed by primed T cells, in vivo priming of anti-lymphoma T cells has been elusive. Here, we demonstrate that lymphoma cells can directly prime T cells, but in vivo immunity still requires cross-presentation. To address this, we developed an in situ vaccine (ISV), combining Flt3L, radiotherapy, and a TLR3 agonist, which recruited, antigen-loaded and activated intratumoral, cross-presenting dendritic cells (DCs). ISV induced anti-tumor CD8+ T cell responses and systemic (abscopal) cancer remission in patients with advanced stage iNHL in an ongoing trial (NCT01976585). Non-responding patients developed a population of PD1+CD8+ T cells after ISV, and murine tumors became newly responsive to PD1 blockade, prompting a follow-up trial of the combined therapy. Our data substantiate that recruiting and activating intratumoral, cross-priming DCs is achievable and critical to anti-tumor T cell responses and PD1-blockade efficacy.In situ vaccine recruits and activates cross-presenting dendritic cells and augments PD1 blockade efficacy in patients with indolent non-Hodgkin’s lymphoma.

It was recently proposed that T lymphocytes, which closely interact with APCs, can extract surface molecules from the presenting cells when they dissociate. These observations question the classical view of discrete interactions between phenotypically defined cell populations. In this review, we summarize some reports suggesting that membrane exchange at the immune synapse can be a vector for intercellular communication and envisage some consequences on the biology of T cells.

BackgroundHepatocellular carcinoma (HCC) is a major global health concern, causing over 700,000 deaths annually [1]. For decades, treatment for advanced HCC was restricted to broad-acting tyrosine kinase inhibitors that conferred limited survival benefits [2]. Recently, immunotherapies have revolutionized treatment of advanced HCC with objective response rates of around 30% [3]. However, with 70% of patients remaining insensitive, there is an urgent need to identify biomarkers of resistance and response and to design novel combination therapies that restore sensitivity in the resistant patients.MethodsTo understand mechanisms of HCC immune evasion, we performed transcriptomic analysis on a collection of immune-escaped murine tumors harboring MYC overexpression and loss of p53 (MYC;p53-/-) [4]. The immune-escaped tumors could be categorized into ”immune-inflamed” and ”immune-desert” through single sample gene set enrichment analysis. We further identified an enrichment signature of Notch signaling in immune-inflamed tumors that was associated with a specific deficit of CD8+ T cells – a result validated in HCC patient samples. To test the role of Notch in immune escape, we generated a novel murine model of HCC based on MYC overexpression, activated Notch1 Intracellular Domain (MYC;NICD1), and customized expression of tumor antigens.ResultsExpression of tumor antigens in MYC;p53-/- mice led to enhanced survival and active immune surveillance/tumor clearance by antigen-specific CD8+ T cells compared to control MYC;p53-/- mice without tumor antigens. However, this survival advantage conferred by tumor antigen expression was abrogated in MYC;NICD1 mice, demonstrating that Notch activation drives immune evasion in HCC in the presence of antigen expression. Mechanistically, Notch activation in the liver led to a reduced number of tumor antigen-specific CD8+ T cells within the tumor microenvironment compared to the MYC;p53-/- mice while the presence of dendritic cells was similar. Adoptive transfer experiments have revealed a potential impairment of CD8+ T cell priming and activation. Further, Notch1-driven tumors were resistant to anti-PD1 monotherapy as well as anti-PDL1/anti-VEGFR2 and anti-PDL1/anti-TGFB1 combination therapies, indicating a multifaceted immune resistance mechanism mediated in part by VEGF and TGFB1. Resistance to anti-PDL1/anti-VEGF combination therapy has been confirmed in HCC patients.ConclusionsTogether, Notch activation promotes a multifaceted immune escape program in HCC. Additionally, it promotes resistance to currently approved immunotherapies, establishing the Notch pathway as a putative biomarker for HCC patient stratification.AcknowledgementsThis study was funded by Damon Runyon-Rachleff Innovator Award and NCI-NIH R37 Award. KEL was supported by 5T32AI078892-12.ReferencesBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018 Nov;68(6):394–424.Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, Häussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J; SHARP Investigators Study Group. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008 Jul 24;359(4):378–90.Finn RS, Qin S, Ikeda M, Galle PR, Ducreux M, Kim TY, Kudo M, Breder V, Merle P, Kaseb AO, Li D, Verret W, Xu DZ, Hernandez S, Liu J, Huang C, Mulla S, Wang Y, Lim HY, Zhu AX, Cheng AL; IMbrave150 Investigators. Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma. N Engl J Med. 2020 May 14;382(20):1894–1905.Ruiz de Galarreta M, Bresnahan E, Molina-Sánchez P, Lindblad KE, Maier B, Sia D, Puigvehi M, Miguela V, Casanova-Acebes M, Dhainaut M, Villacorta-Martin C, Singhi AD, Moghe A, von Felden J, Tal Grinspan L, Wang S, Kamphorst AO, Monga SP, Brown BD, Villanueva A, Llovet JM, Merad M, Lujambio A. ß-Catenin Activation Promotes Immune Escape and Resistance to Anti-PD-1 Therapy in Hepatocellular Carcinoma. Cancer Discov. 2019 Aug;9(8):1124–1141.Ethics ApprovalAll mouse experiments were approved by the ISMMS Animal Care and Use Committee (protocol number IACUC-2014–0229).

Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment 1 , 2 . However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis 3 – 5 , whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions 1 . How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial–mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8 + T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions. Single-cell RNA sequencing and imaging of macrophages in human non-small cell lung cancer and in a mouse model of lung adenocarcinoma show that tissue-resident macrophages have a key role in early tumour progression.

CD4 + effector lymphocytes (T eff ) are traditionally classified by the cytokines they produce. To determine the states that T eff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic T eff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (T H ) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as T H markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ. Helper T cell subsets are characterized functionally by the cytokines they produce. Benoist and colleagues demonstrate that in vivo helper T cells do not manifest as discrete helper subsets but rather form a continuum shaped by microbial exposure.

BackgroundUnderstanding multicellular interactions within the tumor-immune microenvironment (TME) is essential for developing cancer therapies and precision medicine strategies. Tumors pose significant biological complexity due to patient and tissue heterogeneity, cell-cell interactions, and intricate signaling pathways. Macrophages are a heterogeneous cell population associated both positively and negatively with tumor progression and response to therapy, but the factors driving macrophage influence on the TME remain poorly understood. Spatial analysis of human tumors with machine learning offers a novel approach to deciphering the mechanisms underlying macrophage function.MethodsWe developed a spatial virtual cell foundation model, a custom multimodal transformer with 450M parameters, trained on CosMx spatial transcriptomics across over 40 million cells from 1399 non-small cell lung cancer (NSCLC) tumor resections.1 2 This model allows the simulation of gene expression in ‘virtual cells’ placed within spatially resolved TMEs and unlocks the ability to conduct in silico, cell-type specific experiments. To identify local cell-cell interactions between macrophages and particular cell types, we designed virtual ‘clonal neighborhood’ simulations, in which a virtual macrophage is surrounded by digital replicates of real tumor cells or fibroblasts sampled from NSCLC patients’ TME. We simulated 500 tumor or fibroblast clonal neighborhoods for each patient, leading to inferred gene expression on a total of 873,000 virtual macrophages. This allowed us to identify tumor-intrinsic and fibroblast-mediated transcriptional programs spatially associated with macrophage state. Topic modeling was applied to interpret the inferred macrophage transcriptional states, and predictive modeling analyses were used to identify tumor and fibroblast gene expression programs associated with different states.ResultsAnalysis of virtual macrophage revealed a diversity of transcriptional programs acquired from interacting with tumor cells or fibroblasts neighborhoods. A subset of these programs were associated with macrophage polarization towards immunosuppressive SPP1+ or immunogenic CXCL9+ states, known to correlate with patients‘ response to immunotherapy. We further identified programs in tumor cells and fibroblasts that correlate with these macrophage states. Specifically, our experiments unveiled collagen expressing fibroblasts promoting SPP1+ virtual macrophage phenotypes. This population of fibroblasts was enriched in KRAS STK11 mutant vs KRAS mutant tumors, suggesting genotype specific TME characteristics that may account for resistance to immune checkpoint therapy in KRAS STK11 mutant tumors.ConclusionsOur spatial virtual cell foundation model approach uncovers novel multicellular processes within the NSCLC TME, and helps elucidate how spatially-resolved events shape macrophage polarization. This approach identifies actionable targets, offering new insights to enhance immunotherapy efficacy and reverse immunosuppressive tumor environments.ReferencesLacey J Padron, et al. #1231 Foundation models of cell and tissue biology enabled by custom scaled data generation: insights from 1000 lung tumor samples. Journal for ImmunoTherapy of Cancer. 2024;12. Yubin Xie, et al. #3652 OCTO-virtual cell: a foundation model of cell and tissue spatial biology with application to patient stratification and target discovery. AACR. 2025.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense extracellular matrix (ECM) that sustains an immunosuppressive tumor microenvironment (TME). While this protective niche has been described, the molecular determinants orchestrating its formation and dictating its immune interactions are not well defined. Using Perturb-map, we determine how dozens of different gene perturbations shape the growth and cellular environments of PDAC clones through space and time. Our study reveals dynamic, gene-specific adaptations of immune neighborhoods during clonal selection. We identified (PAI2) and (PAI1) as key cancer-derived mediators of TME remodeling and immune evasion. These factors promote the deposition of a fibrin-rich ECM that shapes immune cell composition, locally retains and polarizes immunosuppressive macrophages and excludes cytotoxic T cells. Deletion of either or greatly enhanced tumor response to anti-PD1 immunotherapy in an aggressive PDAC model. Transcriptomic analysis further linked their expression to distinct PDAC subtypes and poor patient survival. Our findings demonstrate that and establish a permissive niche for tumor progression and show how PDAC cells exploit components of the fibrinolysis pathway to remodel the ECM, alter macrophage composition, and protect themselves from immune editing, ultimately reinforcing the role of extracellular factors in shaping an immune-privileged tumor niche.

Increasing evidence indicates oncogenes and tumor suppressors not only influence cell fitness but can also control the immunophenotype of cells. Here, we examined how 34 commonly mutated genes in colorectal cancer (CRC) may influence the expression of 8 key immunomodulatory proteins. To do this, we employed a functional genomics approach utilizing Pro-Code/CRISPR libraries for high-dimensional analysis. We introduced a library of 102 Pro-Code/gRNA combinations, targeting each of the 34 genes, in CT26 cells, a CRC cell model, and measured the expression of each of the immunomodulatory proteins by CyTOF mass cytometry. Notably, cells carrying a Pro-Code/CRISPR targeting the Trp53 lost expression of the immune co-stimulatory molecule CD80. Validation confirmed that Trp53 knockout resulted in the loss of CD80 and that activation of P53, through DNA damage or stabilization, resulted in CD80 upregulation. P53 ChIP-seq identified the CD80 promoter as a direct target of P53. CD80 regulation by P53 was identified in other cells, including normal epithelial cells and macrophages. Functionally, CD80 reduction caused by P53 loss led to a reduced capacity for CRC to prime antigen-specific T cells. These studies establish CD80, a canonical co-stimulatory molecule, as a direct target of the tumor suppressor and DNA damage response gene, P53. Competing Interest Statement The authors have declared no competing interest.