Project description:Adoptive T-cell Therapy (ACT) involves using tumor-infiltrating lymphocytes (TIL) isolated from metastatic melanoma and expanding them ex vivo prior to infusion into lympho-depleted patients. This is one of the most promising approaches to treat metastatic melanoma, with the rates of clinical response between 48-50% based on studies done at NCI, M.D. Anderson Cancer Center (Houston, TX), and Sheba Medical Center (Tel Aviv, Israel). In the Phase II ACT Trial at M.D. Anderson Cancer Center , our group has uncovered an association between positive clinical response and the amount of CD8+ tumor-infiltrating lymphocytes expressing B and T Lymphocyte Attenuator (BTLA), a reported inhibitory receptor on T-cells. We used microarrays to detail the differences in the global programme of gene expression between CD8+BTLA+ vs CD8+BTLA- TILs in order to understand the molecular basis of the clinical association. TILs were isolated by enzymatically digest the melanoma tumor fragments obtained from Stage IIIc/IV melanoma patients at M.D. Anderson Cancer Center. The TILs were expanded with high-dose IL-2 for two weeks prior to sorting by FACS (fluoresecence-activated cell sorter) for CD8+BTLA+ and CD8+BTLA- susbets. RNA was extracted from each sorted subsets and hybridized on Affymetrix microarrays
Project description:Adoptive T-cell Therapy (ACT) involves using tumor-infiltrating lymphocytes (TIL) isolated from metastatic melanoma and expanding them ex vivo prior to infusion into lympho-depleted patients. This is one of the most promising approaches to treat metastatic melanoma, with the rates of clinical response between 48-50% based on studies done at NCI, M.D. Anderson Cancer Center (Houston, TX), and Sheba Medical Center (Tel Aviv, Israel). In the Phase II ACT Trial at M.D. Anderson Cancer Center , our group has uncovered an association between positive clinical response and the amount of CD8+ tumor-infiltrating lymphocytes expressing B and T Lymphocyte Attenuator (BTLA), a reported inhibitory receptor on T-cells. We used microarrays to detail the differences in the global programme of gene expression between CD8+BTLA+ vs CD8+BTLA- TILs in order to understand the molecular basis of the clinical association.
Project description:Metastatic uveal melanoma generally responds poorly to immunotherapy. The aim here was to sequence tumor-infiltrating lymphocytes from uveal melanoma metastases to study their phenotypes and T-cell receptor (TCR) clonotypes. We performed paired single-cell transcriptome and TCR sequencing using the 10x Genomics platform of IL2-expanded tumor-infiltrating lymphocytes from 7 liver and 1 subcutaneous metastasis.
Project description:CD8+ T cells are the primary target of immune checkpoint inhibitor (ICI) therapy in the treatment of melanoma. ICI therapy only benefits a subset of patients and complicating this issue is a reliable prediction method that does not require invasive biopsies. In the hope of remedying this challenge, we conducted single-cell transcriptomic analyses of CD8+ T cells in peripheral blood lymphocytes (CD8-mPBLs) and, importantly, tumor-infiltrating lymphocytes (CD8-mTILs) from 8 patients with metastatic melanoma.
Project description:TET2-mutant clonal hematopoiesis (CH) is associated with reduced cancer metastasis in human cohorts. Using mouse models of colorectal cancer liver metastasis, we demonstrate that Tet2-deficient hematopoietic cells suppress metastatic tumor burden by preventing CD8+ T-cell exhaustion. Bulk RNA-seq of sorted CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) from Vav1CreTet2KO, Cd4CreTet2KO, and Tet2fl/fl control mice was performed to characterize transcriptional changes associated with Tet2 loss in tumor-infiltrating T cells.
Project description:TET2-mutant clonal hematopoiesis (CH) is associated with reduced cancer metastasis in human cohorts. Whole genome bisulfite sequencing (WGBS) of PD-1+CD8+ tumor-infiltrating lymphocytes (TILs) sorted from liver metastatic tumors of Vav1CreTet2KO and Tet2fl/fl mice was performed to characterize genome-wide DNA methylation changes associated with TET2 loss in exhausted CD8+ T cells.
Project description:To investigate the impact of Card11 on TIL differentiation, we isolated TILs from B16-F10 tumors of WT mice, E134G mice, and K215M mice and performed scRNA seq. We also sorted tumor-infiltrating lymphocytes (TILs) from melanoma(B16-F10) of WT mice and conducted scRNA and scTCR sequencing. To further investigate the impact of Card11 on TCR clonal diversity, we sorted CD8 T cells from melanoma WT mice or K215M mice and performed scRNA and scTCR sequencing.
Project description:The immune system can recognize and respond to tumors. However there are some conditions in which the genetic instability and heterogeneity of tumor cells leads to the development of variants that can escape the immune system. T cells have infiltrated inside many tumors (Tumor Infiltrating Lymphocytes or TILs), but generally these TILs have lost their functional capacity and are unable to eliminate tumor cells. We developed a model of autochthonous melanoma in mice that recapitulates some aspects of inflammatory melanoma in humans. These include a systemic Th2/Th17-oriented chronic inflammation, recruitment of immunosuppressive myeloid cells and acquisition by TILs of an M-bM-^@M-^\exhaustedM-bM-^@M-^] phenotype characterized by expression of receptors for multiple inhibitory molecules. To address the molecular bases for the M-bM-^@M-^\exhaustedM-bM-^@M-^] TILs phenotype, we performed transcriptomic analyses on sorted CD8 or T cells from the induced melanomas. These transcriptomes were compared to those of naM-CM-/ve CD8 T cells and of CD8 T cells immunized with a virus. 10 samples, 3 replicates for controls (untreated and infected with AdP1A), 4 replicates for TILs CD8 from melanoma
Project description:Transcriptional profiling of FACS-sorted and splenic control mouse cells, comparing splenic cells from FVBneuN vs Neu+ expressing FVBneuN mice with Gr1+ CD11b+ sorted tumor-infiltrating mononuclear or splenic myeloid-derived suppressor cells
Project description:While melanoma cells often express a high burden of mutated proteins, the infiltration of reactive T cells rarely results in tumor-eradicating immunity. We discovered that large extracellular vesicles, known as melanosomes, secreted by melanoma cells are decorated with MHC molecules that stimulate CD8+ T cells through their T-cell receptor (TCR), causing T cell dysfunction and apoptosis. Immunopeptidomic and TCR-seq analyses revealed that these melanosomes carry MHC-bound-Tumor-Associate- Antigens with higher affinity and immunogenicity, which compete with their tumor cell of origin for direct TCR–MHC interactions. Analysis of biopsies from melanoma patients confirmed that melanosomes trap infiltrating lymphocytes, induce partial activation, and decrease CD8+ T cell cytotoxicity. Inhibition of melanosome secretion in vivo significantly reduced tumor immune evasion. These findings suggest that MHC export protects melanoma from the cytotoxic effects of T cells. Our study highlights a novel immune evasion mechanism and proposes a therapeutic avenue to enhance tumor immunity.