Unknown,Transcriptomics,Genomics,Proteomics

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Longitudinal scRNA-Seq of immune cells from GL261 murine brain Tumors and MHC class II deficient tumor-mircoenvironments

ABSTRACT: Longitudinal phenotyping of T cells and myeloid cells in early (day 14) and late (day26) GL261 tumors. Single cell RNA sequencing of CD45+ immune cell from intracranial murine GL261-SIINFEKL tumors 20 days after inoculation. SIINFEKL-reactive T cells were sorted based on dextramer staining. Using time-resolved scRNA-seq of murine tumor-infiltrating immune cells from early (d14) and late stage (d26) syngeneic GL261 gliomas we observed constant MHCII expression by murine microglia over time and overall high but decreasing MHCII expression in late stage bbm indicating a dynamic process of MHCII expression. We show that loss of major histocompatibility complex (MHC) class II (MHCII)-restricted antigen presentation on bbm drives dysfunctional intratumoral tumor-reactive CD8+ T cell states through increased expression of Tox, a critical regulator of T cell exhaustion. By calculating the normalized abundance of T cell states along the pseudotime trajectory of SIINFEKL-reactive CD8+ T cells retrieved from myeloidMHCII-proficient and -deficient microenvironments, we found an enrichment of a distinct T cell state in myeloidMHCIIKO CD8+ T cells that was defined by peak expression of exhaustion markers. Mechanistically, MHCII-dependent activation of CD4+ T cells restricts myeloid-derived osteopontin that triggers a chronic activation of nuclear factor of activated T cells (Nfat)2 in tumor-reactive CD8+ T cells. In summary, we provide evidence that MHCII-restricted antigen presentation on bbm is a key mechanism to directly maintain functional cytotoxic T cell states in brain tumors.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

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PROVIDER: E-MTAB-12523 | biostudies-arrayexpress |

SECONDARY ACCESSION(S): ERP144083

REPOSITORIES: biostudies-arrayexpress

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Project description:Innate memory phenotype (IMP) CD4+ T cells are non-conventional Î±Î² T cells exhibiting features of innate immune cells, characterized as CD44high and CD62Llow in periphery. It is recently reported by our group that bone marrow chimeric mice lacking thymic MHCI expression develop predominantly IMP CD8+ T cells, while those lacking hematopoietic MHCI develop predominantly naÃ¯ve CD8+ T cells. Here we perform hirarchical clustering analysis and found that CD4+ T cells share similar property: chimeras lacking thymic MHCII gave rise to predominantly CD4+ T cells that resemble IMP CD4+ T cells observed in WT mice, and vice versa, chimeras lacking hematopoietic MHCII had a majority of naÃ¯ve-like CD4+ T cells resembling naÃ¯veCD4+ T cells seen in WT mice. We used microarrays to compare the global programme of gene expression to determine whether the hematopoietic MHCII selected CD4+ T cells are IMP, and whether the thymic MHCII selected CD4+ T cells are naÃ¯ve CD4+ T cells as observed in WT mice. Through hierarchical clustering and analysis of global gene differential expression, we determined that hematopoietic MHCII dependent IMP CD4+ T cells generated from WT bone marrow transplanted into irradiated MHCII-/- recipients, resemble IMP CD4+ T cells in WT mice, while naÃ¯ve CD4+ T cells generated from MHCII-/- bone marrow transplanted into irradiated WT recipients, resemble naÃ¯ve CD4+ T cells in WT mice. Cell Sorting was performed using a Cytopeia Influx Cell Sorter. Chimeric IMP (CD45.1+TCRÎ²+CD4+CD44highCD62Llow) CD4+ T cells were sorted from splenocytes of CD45.1+WTâCD45.2+MHCII-/- chimeras (WM IMP CD4), and chimeric naÃ¯ve (CD45.2+TCRÎ²+CD4+CD44lowCD62Lhigh) CD4+ T cells were sorted from splenocytes of CD45.2+MHCII-/- â CD45.1+WT chimeras (MW naÃ¯ve CD4) respectively, 8 weeks post transplantation. WT IMP (TCRÎ²+CD4+CD44highCD62Llow) and naÃ¯ve (TCRÎ²+CD4+CD44lowCD62Lhigh) CD4+ T cells were sorted from splenocytes of 8-week old WT mice.

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Longitudinal scRNA-Seq of immune cells from GL261 murine brain Tumors and MHC class II deficient tumor-mircoenvironments

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