Fibroblast orchestration of inflammaging via NF-kB activation (CD3+ T cell, CD8+ T cell)

Aging is associated with the accumulation of Gzmk+CD8+ T cells in the blood and multiple tissues that have features of exhaustion. We have found that by genetically activating NF-kB in fibroblasts (though the deletion of the NF-kB inhibitor Tnfaip3 with the pan-fibroblast Dermo1-cre, Tnfaip3 CKO) we are able to recapitulate this age-related accumulation of Gzmk+CD8+ T cells in the lungs of young mice. We additionally generated a Gzmk reporter mouse (GATOR) that allows the identification and isolation of these cells via tdTomato expression. In the first study associated with this record, we used scRNA and TCR sequencing to compare lung resident CD3+ T cells from young Tnfaip3 CKO mice with those from young control and aged wild-type mice. As a follow-up to this initial study we utilized scRNAseq to compare the transcriptome and TCRs of lung resident Gzmk+ CD8+ T cells from aged WT mice, young Tnfaip3 CKO;GATOR mice, and young GATOR mice chronically infected with LCMV clone 13 (as a "gold standard" for T cell exhaustion). Overall design: Lung resident CD3+ T cells were sorted from 1) 3 month old mice in which Tnfaip3 had been deleted from fibroblasts (Young_D1cA20), 2) 3 month old controls (Young_A20) and 3) 23 month old wild-type C57BL/6 (Aged) mice. All mice were male and on a C57BL/6 background. To identify resident vs circulating immune cells mice were injected with a fluorescently-labeled anti-CD45 antibody 3 minutes prior to sacrifice. Resident CD3+ T cells were identified as ivCD45-CD45+CD11b-CD3+. For each condition cells from 3 independent mice were combined. Prior to submission for 10x library preparation cells from each condition were labeled with Biolegend Totalseq C Antibodies for multiplexing. Barcodes for samples are as follows: Young_A20 (TotalSeq-C0301 anti-mouse Hashtag 1 Antibody), Young_D1cA20 (TotalSeq-C0302 anti-mouse Hashtag 2 Antibody) and Aged (TotalSeq-C0303 anti-mouse Hashtag 3 Antibody). Samples were then submitted in one well for 5' gene expression and TCR sequencing. For the study of lung resident Gzmk+ CD8+ T cells we performed scRNAseq on the following populations: 1) Total CD8+ T cells from 22-month (2 males, 1 female) C57BL/6 (Aged WT) mice, 2) CD8+tdTomato+ T cells from 4-month (2 males, 2 females) Dermo1-cre/+;Tnfaip3f/f;GATOR/+ (Young Tnfaip3 CKO) mice, and 3) CD8+tdTomato+ T cells from 3-month GATOR (2 males, 2 females) mice 4 weeks post-infection with LCMV clone 13 (Young LCMV). For each of the 3 conditions cells were pooled from the indicated animals. To identify resident vs circulating immune cells mice were injected with a fluorescently labeled anti-CD45 antibody 3 minutes prior to sacrifice. Resident CD8+ T cells were identified as ivCD45-CD45+CD3+CD4-CD8+. For the young Tnfaip3 CKO mice and Young LCMV mice Gzmk+(tdTomato+) cells were specifically selected from within the CD8+ population. Prior to submission for 10x library preparation cells from each condition were labeled with Biolegend Totalseq C Antibodies for multiplexing. Barcodes for samples are as follows: Young Tnfaip3 CKO (TotalSeq-C0301 anti-mouse Hashtag 1 Antibody), Aged WT (TotalSeq-C0302 anti-mouse Hashtag 2 Antibody) and Young LCMV (TotalSeq-C0303 anti-mouse Hashtag 3 Antibody). Samples were then submitted in one well for 5' gene expression and TCR sequencing.