ABSTRACT: Comparison of gene expression by microarray in diabetes antigen specific (NRP-V7), LCMV antigen specific (GP33), and CD8+ T cells of similar phenotype from NOD mice
Project description:Type 1 diabetes mellitus (T1D) is caused by killing of insulin producing β cells by CD8+T cells. The disease progression accelerates as glucose tolerance deteriorates suggesting that acquired factors contribute. To identify how environmental factors may lead to the expansion and pathogenicity of the diabetogenic T cells, we analyzed diabetogenic NRP-V7-reactive CD8+ T cells that recognize a peptide from the diabetes antigen IGRP in prediabetic NOD mice. There was an increase in the frequency of the NRP-V7-reactive cells coinciding with the time of glucose intolerance. The T cells persisted in hyperglycemic NOD mice maintained with an insulin pellet despite destruction of beta cells. We compared gene expression in the NRP-V7-reactive T cells to others that shared their phenotype but not selected for reactivity to diabetes antigens viral antigen reactive cells. Compared to these other cells, the NRP-V7-reactive cells exhibited gene expression of memory precursor effector cells. They had reduced cellular proliferation and were less dependent on oxidative phosphorylation. When prediabetic NOD mice were treated with 2DG to block aerobic glycolysis, there was a reduction in the diabetes antigen vs other cells of similar phenotype and loss of lymphoid cells infiltrating the islets. In addition, treatment of NOD mice with 2DG resulted in improved cell granularity. These findings identify a link between metabolic disturbances and autoreactive T cells that promotes development of autoimmune diabetes.
Project description:CD8 T cells specific for beta cell antigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) were identified and isolated with NRP-V7 tetramers from the pancreatic draining lymph node and pancreas of non-obese diabetic (NOD) mice and used for single-cell RNA-seq and VDJ-seq.
Project description:miRNA profiling of Db-GP33-41 specific murine CD8 T cells following infection with LCMV Armstrong Various in-vivo subsets of antigen-specific CD8 T cells were FACS sorted and analyzed for miRNA expression profiling. Samples were purified from either uninfected animals or after days 4.5, 9 and >60 post infection with LCMV Armstrong.
Project description:Recent findings suggest that undifferentiated, stem-like, antigen specific T cells serve as an important long-term reservoir for autoimmune CD8 T cell responses. However, it is still unclear whether CD4 T cells exhibit a similar differentiation trajectory culminating in terminal differentiation, acquisition of an exhausted phenotype, and loss of stemness and function. We analyzed islet infiltrating T cells in 8- and 16-week old NOD mice by scRNAseq and flow cytometry and found that while CD4 T cells in autoimmune diabetes share many features of exhaustion with CD8 T cells, expression patterns of inhibitory receptors are distinct in autoimmune T cells compared to T cells in chronic LCMV infection.
Project description:Transcriptome profiling of lung tissues and lung CD8+ TRM during early phase of infection to investigate the mechanism of lung TRM cells during antigen re-exposure within 48hours. After P14 T cell transfer, PR8 infection would not induce P14 TRM cells, while PR8-gp33 would. Comparing the gene expression of lung tissues after LCMV Armstrong challenge at day 30 after PR8 or PR8-gp33 infection would help us understand the function of lung CD8+ TRM cells. Sorted TRM cells from mice challenged with LCMV Armstrong at day 30 after PR8-gp33 infection would reveal the activation mechanism of TRM cells.
Project description:T cells specific for a certain antigen are a heterogeneous population characterized by cells expressing different T Cell Receptors (TCRs). As the TCR determines the avidity of a T cell clone for its cognate antigen, this feature has also a significant impact on the fate of such clone during antigenic challenge. In the context of chronic viral infection, T cells specific for the chronic virus differentiate into exhausted cells with limited functionality. We find that, in the process of exhaustion following infection with LCMV-Clone13, certain clones of CD8 T cells specific for the LCMV immunodominant epitope GP33 are enriched compared to others.
Project description:Single-cell immune repertoire sequencing (gene expression and immune receptor repertoire) was performed under various contexts. This includes CD4 T follicular cells (sorted by surface expression of PD1+ CXCR5+ CD4+) following high dose LCMV clone 13 infection on days 10, 25, and 50 and low dose clone 13 infection on day 10 post infection. We additionally profiled virus-specific CD8 T cells that are specific to either GP33 or NP396. Gp33-specific CD8 T cells were sorted 15 months post infection with either low dose clone 13 LCMV, MCMV-ie2-gp33 or MCMV-m45-gp33. NP396-specific CD8 T cells were isolated either 10 days post challenge or 25 days post challenge using the viral model of dejavu (https://pubmed.ncbi.nlm.nih.gov/16604192/). B and T cells were isolated 14 days post induction of Experimental autoimmune encephalomyelitis (EAE).
Project description:During acute viral infections, naïve CD8+ T cells differentiate into effector CD8+ T cells and, after viral control, into memory CD8+ T cells. Memory CD8+ T cells are highly functional, proliferate rapidly upon reinfection and persist long-term without antigen. In contrast, during chronic infections, CD8+ T cells become “exhausted” and have poor effector function, express multiple inhibitory receptors, possess low proliferative capacity, and cannot persist without antigen. Exhuasted CD8+ T cells can be further segregated by their expression of the inhibitory cell surface receptor PD-1. We performed transcriptional profiling on both PD-1 High and PD-1 Intermediate H2-Db GP33-specific CD8+ T cells. H2-Db GP33-specific CD8+ T cells were sorted from C57BL/6 mice 30 days p.i. with LCMV clone 13. These cells were then segregated by their expression of the inhibitory cell surface receptor PD-1 into PD-1 High and PD-1 Intermediate subpopulations. We performed transcriptional profiling on these subpopulations.
Project description:Altered CD8 T cell differentiation and functional exhaustion prevent control of chronic virus infection and cancer. Yet, how fate commitment and exhaustion are determined and dynamically modulated throughout persistent infection are unclear. We compared the activation and differentiation of LCMV GP33-specific CD8 TCR transgenic cells (P14) primed at the onset versus in the midst of established persistent LCMV-Clone 13 viral infection. LCMV GP33-specific CD8 TCR transgenic (P14) cells were injected into naïve mice immediately infected with LCMV-Cl13 (Early priming) or into mice that had been infected 21 days earlier with LCMV-Cl13 (Late Priming). Sixty hours post-priming P14 cells were sorted from mice and subjected to RNA seq. We show early primed cells very rapidly exhibit a transcriptional profile of robust activation, effector differentiation and dysfunction, while late primed cells have increased expression of genes involved in memory differentiation and maintenance.
