Egr2 and 3 control inflammation, but maintain homeostasis, of PD-1^high memory phenotype CD4 T cells

Chimeric mice were generated by reconstitution with mixed BM from GFP-Egr2 knock-in (CD45.1) and CD2-Egr2/3^−/− (CD45.2) mice. (A, B) Ki67 and CD44 expression in gated naïve, GFP-Egr2⁻ MP, GFP-Egr2⁺PD-1^high MP, and Egr2/3^−/−PD-1^high MP CD4 T cells from spleens and lymph nodes of chimeric mice 8–12 wk after reconstitution. (C) MP CD4 T cells (CD62L⁻CD44^hi) of GFP-Egr2 knock-in (CD45.1) and CD2-Egr2/3^−/− (CD45.2) origin were isolated from chimeric mice and mixed in equal numbers before labelling with CellTrace Violet. The labelled cells were adoptively transferred into wild-type recipients (CD45.1/2). 3 wk after transfer, CellTrace Violet was analysed on gated GFP-Egr2⁺PD-1^high, GFP-Egr2⁻PD-1^low (both CD45.1), and Egr2/3^−/−PD-1^high (CD45.2) donor cells. (A, C) are representative of three independent experiments. Data in (B) are the mean ± SD from groups of five recipient mice from one experiment and were analysed with Kruskal–Wallis tests, followed by Conover tests with Benjamini–Hochberg correction. N.S., not significant, *P < 0.05, **P < 0.01.

Naïve (CD62L⁺CD44^lo) and GFP-Egr2⁻ MP and GFP-Egr2⁺ MP from GFP-Egr2 knock-in and naïve and MP Egr2/3^−/− from CD2-Egr2/3^−/− mice were analysed by RNA-seq. (A) Unsupervised hierarchical clustering of selected genes showing expression patterns in naïve, GFP-Egr2⁻, GFP-Egr2⁺, and Egr2/3^−/− MP T cells. Selected genes relevant to MP T cell function are indicated. (B) Gene set enrichment analysis of Hallmark gene sets (Liberzon et al, 2015) for GFP-Egr2⁺ versus Egr2/3^−/− MP cells (left) and GFP-Egr2⁺ versus GFP-Egr2⁻ MP cells (right). Mean and 95% confidence intervals for selected pathways, colour coded to indicate Benjamini–Hochberg corrected P-values, are shown. The RNA-seq data are from three biological replicates, each with cells pooled from 10 mice, for each group.

CD4 T cells from GFP-Egr2 knock-in mice were stimulated for 24 h in vitro with anti-CD3 and anti-CD28 to induce GFP-Egr2 expression and then used for GFP-Egr2-ChIP-seq. (A) Most significant motif enriched in Egr2 ChIP-seq peaks (P = 1 × 10^1465). (B) Distribution of Egr2 binding sites in the genome. (C) Functional analysis of the genes bound in Egr2 ChIP-seq using the Hallmark gene sets (Liberzon et al, 2015). (D) Proportion of differentially expressed genes in RNA-seq (Fig 3) that are bound by Egr2; “+ versus −/−” and “+ versus −” indicate the GFP-Egr2⁺ MP versus Egr2/3^−/− MP and GFP-Egr2⁺ MP versus GFP-Egr2⁻ MP comparisons in RNA-seq, respectively. (E) Volcano plot of RNA-seq data for GFP-Egr2⁺ MP versus Egr2/3^−/− MP cells, with positive and negative log₂ fold changes indicating higher expression in GFP-Egr2⁺ or Egr2/3^−/− cells, respectively. Selected genes bound in GFP-Egr2-ChIP-seq are indicated. (F) ChIP-seq peaks (third track for each gene) associated with the indicated genes, together with RNA-seq reads from GFP-Egr2⁺ and Egr2/3^−/− MP cells (top two tracks), compared with signal from Input chromatin (fourth track). The ChIP-seq data are from three independent IPs each from an independent biological replicate.

Mixed BM OT-II TCR retrogenic chimera models were created by adoptive transfer of an equal number of OT-II retrovirus transduced BM cells from wild-type (CD45.1) and CD2-Egr2/3^−/− (CD45.2) mice. (A, B) 8 wk after BM reconstitution, OT-II (I-A^b-OVA_329-337⁺CD4⁺) wild-type (CD45.1) and Egr2/3^−/− (CD45.2) cells from spleens and lymph nodes of chimeras were analysed for expression of CD62L and CD44. (C, D, E, F, G, H, I, J, K) GFP⁺CD62L⁻CD44^hi MP OT-II cells were isolated from the chimeras and equal numbers of wild-type (CD45.1) and Egr2/3^−/− (CD45.2) MP OT-II cells were adoptively transferred into wild-type mice (CD45.1/2). (C, D) The percentages (C) and absolute numbers (D) of donor cells of each genotype were assessed 24 h or 3 wk after transfer. (E) RT-PCR of the indicated genes in isolated OT-II wild-type or Egr2/3^−/− donor cells 3 wk after transfer. (F, G, H, I, J, K) 7 d after transfer, a group of recipient mice were infected with OVA-vaccinia virus i.p. and the percentage (F) and absolute number (G) of wild-type and Egr2/3^−/− donor cells were analysed before and 7 d after infection. (H, I, J, K) 7 d after infection, Ki67-positive (H, I) and IFNγ-producing (J, K) OT-II cells were analysed. (A) is representative of 15 recipient mice. (C, E, F, H, J) are representative of two to three experiments with similar results. Data in (B, D, G, I, K) are the mean ± SD of five recipient mice and were analysed with Mann–Whitney two-tailed tests. N.S., not significant, *P < 0.05, **P < 0.01.

(A, B) Naïve and GFP-Egr2⁺, GFP-Egr2⁻, and Egr2/3^−/− MP T cells from GFP-Egr2 knock-in and CD2-Egr2/3^−/− mice were analysed for CD127 and CD5 expression. (C, D) CD4 naïve and MP T cells were isolated from wild-type (WT) and CD2-Egr2/3^−/− mice and their TCRβ repertoires analysed. (C) Repertoire diversity was estimated using the Shannon entropy index normalized by the total number of unique amino acid clonotypes. Samples were downsampled to the size of the smallest repertoire 100 times and the Shannon entropy index calculated for each. The median of the 100 diversity estimates for each sample is plotted. (D) Rank frequency distribution of MP and naïve T cell clonotypes from wild-type and CD2-Egr2/3^−/− mice. Clonotype frequency was estimated using the three replicates for each condition using the Chao1 estimator. Clonotype rank against frequency in the repertoire is shown. The TCR-seq data are from three biological replicates, each with cells pooled from 10 mice, for each group. Data in (A) are representative of three independent experiments. Data in (B) are the mean ± SD from groups of mice and were analysed with Kruskal–Wallis tests, followed by Conover tests with Benjamini–Hochberg correction. N.S., not significant, *P < 0.05, **P < 0.01.

(A, B) GFP-Egr2⁺CD4⁺CD25⁻CD62L⁻CD44^hi and GFP-Egr2⁻CD4⁺CD25⁻CD62L⁻CD44^hi MP T cells were isolated from GFP-Egr2 knock-in and CD4⁺CD25⁻CD62L⁻CD44^hi MP T cells were isolated from CD2-Egr2/3^−/− mice and stimulated in vitro with 100 ng/ml IL-12 for 24 h before analysis of GFP-Egr2 and IFNγ by flow cytometry. (C, D) Naïve and GFP-Egr2⁺, GFP-Egr2⁻ and Egr2/3^−/− MP T cells from GFP-Egr2 knock-in and CD2-Egr2/3^−/− mice were analysed for GFP-Egr2 and T-bet expression. Data are representative of three to four experiments. Data in (B, D) are the mean ± SD of four samples and were analysed with Kruskal–Wallis tests, followed by Conover tests with Benjamini–Hochberg correction. N.S., not significant, *P < 0.05, **P < 0.01.

(A, B) CD3⁺CD4⁺ cells from PBMCs of healthy controls and rheumatoid arthritis patients were analysed for PD-1 and CD45RA expression by flow cytometry (A top panel) and the proportion of PD-1^highCD45RA⁻ cells was quantified (B, left panel). Egr2 and T-bet expression by these gated PD-1^highCD45RA⁻ cells was then analysed by flow cytometry (A, bottom panel) and the proportion of Egr2⁺ cells was quantified (B, right panel). (C, D) Patients in which more than 10% of PD-1^highCD45RA⁻ cells were T-bet positive were gated on Egr2⁻ and Egr2⁺ cells and analysed for T-bet and Granzyme B expression. Healthy controls in (C) are presented for comparison. Data in (B, D) are the mean ± SD and were analysed with Mann–Whitney two-tailed tests. N.S., not significant, *P < 0.05, **P < 0.01.