Project description:This study shows a detailed analysis of the expression of CD45 isoforms on virus specific CD8 T cells after YFV vaccination (YFV-17D) longitudinally starting at two weeks after infection going out to over 10 years. As expected, effector CD8 T cells at day 14 express CD45RO but interestingly within 4-6 weeks all of the virus specific CD8 T cells become CD45RA and then remain CD45 RA for > 10yrs. The journey for these YFV specific CD8 T cells goes from naïve (CD45RA+ CCR7+) to effector/EM (CD45RO+ CCR7-) to TEMRA (CD45RA+ CCR7-) to TSCM (CD45RA+ CCR7+). These YFV specific CD8 T cells rarely acquire the canonical Tcm phenotype (CD45RO+ CCR7+). The few CM cell found in the YFV CD8 T cell response are effector cells in the first 11 days of the infection and their numbers become almost undetectable in subsequent samples. We found that CD8 T cells returned to expressing a mixture of CD45 isoforms similar but not identical to those found in naive T cells. CD45RA expression correlated closely with reduced expression of the splicing mediator hnRNP LL. This CD45RO to RA switch happens around one month post-infection and coincides with clearance of YFV, so we hypothesized that antigen may be playing a role in which CD45 isoform is expressed by the virus specific CD8 T cells. We addressed this issue by ex vivo analysis and provide evidence that this switch is indeed regulated by antigen. Sorted CD45RO positive YFV specific effector CD8 T cells re-express CD45RA when cultured ex vivo in the absence of antigen. We then show that SARS-CoV-2 spike specific CD8 T cells undergo the same CD45RA to RO to RA transition in vivo in a longitudinal analysis of a cohort of COVID-19 patients receiving the SARS-CoV-2 mRNA vaccine. These studies have important implications for T cell nomenclature and in particular for defining human memory CD8 T cell subsets. The re-expression of CD45RA in CD8 T cells likely explains the large number of TEMRA and absence of CM CD8 T cells. However, comparison to CD4 populations suggests that these findings are only applicable to CD8 T cells.

Project description:The goal of this study is to compare the transcriptional program and the TCR sequences of different subsets of CD8 T cells purified from the peripheral blood of 3 patienst with Amyotrophic Lateral Sclerosis 4 (ALS4) and 3 age-matched controls (unreleated). Peripheral blood mononuclear cells (PBMCs) were purified from 3 ALS4 patients and 3 age-matched healthy controls.By combining RNA and surface protein expression followed by multidimensional reduction using the Uniform Manifold Approximation and Projection (UMAP), we could identified 9 clusters corresponding to different CD8 T cell subsets: naïve/naïve like (clusters 0, 2, 5, 8), central memory (cluster 7), effector memory (clusters 4 and 6), TEMRA (clusters 1 and 3), and mucosal associated invariant T (MAIT) cells (cluster 9). ALS4 patients exhibited a higher proportion of TEMRA, and a decreased frequency of effector/effector memory cells, as compared to controls. Downregulation of IL7R and CCR7, and upregulation of GZMM and GZMB (encoding for Granzyme M and B, respectively), KLRB1 (Killer Cell Lectin Like Receptor B1), NKG7 (Natural Killer Cell Granule Protein 7), FGFBP2 (Fibroblast Growth Factor Binding Protein 2) and CCL4 (Chemokine (C-C motif) ligands 4), all of which correlate with a TEMRA phenotype are among the top deregulated genes in ALS4 CD8 T cells. The proportion of naïve cells seemed to be increased as well; however only 1 patient has an abnormal high frequency of CCR7+CD45RA+CD28+ cells, while the TEMRA signature was consistent among all 3 ALS4 donors. More than 90% of expanded clones (≥5) are TEMRA cells in ALS4 patients, in comparison to ~ 60% in healthy controls. By matching UMAP coordinates from RNA and TCR analyses, we observed a colocalization between the most expanded clones, i.e. CD8 T cells in which the same TCR sequence was shared by more than 20 cells, and the TEMRA subset that we found enriched in patients. We quantified this clonal expansion and found that over half of all TEMRA cells comprised of highly expanded clones in patients. Hyperexpanded (>100 clones) TEMRA cells were detected in 2 of the 3 patients, with one clone reaching as high as 407 cells. No hyperexpanded clones were found in other CD8 T cell subsets or in control patients. Comparison of surface protein expression from our CITE-seq analysis in “Low” and “High” (>20) expanded clones from ALS4 patients revealed a stronger TEMRA phenotype in the latter, as indicated by CD27 downregulation and CD45RA and CD57 upregulation.

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Homo sapiens

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