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Gene expression in anergic and naïve CD4 T cells deficient for Nr4a1 and Nr4a3

ABSTRACT: The NR4A family of nuclear receptors are upregulated by acute and chronic antigen stimulation, and play redundant, tolerogenic roles in T and B cells. To bypass their obligate function in the Treg lineage, we generated competitive chimeras with Nr4a1-/-Nr4a3-/- (double knockout or DKO) bone marrow mixed with congenically marked WT bone marrow at a 1:5 ratio. As a result of a defect in Treg production by DKO cells, Treg in mixed chimeras are almost exclusively of WT origin. By contrast, we generate an approximately 1:1 DKO:WT ratio in the peripheral T cell compartment because DKO cells escape negative selection. After reconstitution, we observed pronounced accumulation of anergic DKO CD4 T cells, but a defect in functional tolerance resulting in autoantibody production. Based on these studies and published literature, we hypothesize that the NR4A family regulate TCR-induced transcripts in both naive and anergic CD4 T cells. To identify such targets, we sorted naive and anergic CD4 T cells of either WT or DKO genotype (CD45.1+ or CD45.1-) on the basis of CD73/FR4/CD44 expression from these chimeras directly into RLT buffer. In parallel, we stimulated sorted naive CD4 T cells of each genotype in vitro with plate-bound anti-CD3 and anti-CD28 stimulation for 3 hrs and then generated RLT lysates. We made biological triplicate samples of each condition (naive WT and DKO, naive stimulated WT and DKO, anergic WT and DKO) resulting in 6 different populations x 3 replicates = 18 samples. Samples were subjected to library prep and bulk RNA sequencing to identify DEG.

ORGANISM(S): Mus musculus

PROVIDER: GSE178782 | GEO | 2021/06/24

REPOSITORIES: GEO

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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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Gene expression in anergic and naïve CD4 T cells deficient for Nr4a1 and Nr4a3

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