Fig 2: Deletion of genes in naive T cells with minimal off-target editing using CHIME. a Quantification of thymic subsets (CD4– CD8–, CD4– CD8+, CD4+ CD8–, CD4+ CD8+) from WT or Cas9 + control sgRNA chimeric mice. b Representative flow cytometry plots of CD44, CD62L, and c CD69 from splenic CD8+ T cells. d Quantification of naïve status of CD8+ T cells in (b, c). e Flow cytometry plots of PD-1 expression in CD4+ T cells (top panel) and CD8+ T cells (bottom panel) from representative non-targeting control sgRNA or Pdcd1 sgRNA chimeras following aCD3/CD28 stimulation. f Quantification of PD-1 expression for two control and three Pdcd1 sgRNAs from (e). g TIDE assay on naïve CD4+ and CD8+ T cells for three Pdcd1 targeting sgRNAs. h, i TIDE assay on naïve (h) CD4+ and (i) CD8+ T cells designed to detect the top three predicted off-target sites (1st, 2nd, 3rd) for three Pdcd1 targeting sgRNAs. Dashed line represents the aberrant sequence (%) when comparing two non-targeting control sgRNAs (background aberrant sequence). All experiments had at least three biological replicate animals per group and are representative of two independent experiments. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed among the replicate bone marrow chimeras by one-way ANOVA (a, d, f) (*p < .05, **p < .01, ***p < .001, ****p < .0001). See also Supplementary Fig. 2. Source data are provided as a source data file

Fig 3: (A) FACS analysis showing the percentage of F4/80+macrophage population out of CD45+alive cells, major histocompatibility complex class II–positive and CD206+population out of F4/80+cells, Npk46+ population out of CD45+cells, and Ly6g+population out of CD45+in Ldha WT and KO tumors. *P < .05. (B) FACS analysis illustrating the % of T helper CD4+ and cytotoxic CD8+ cells performed on CD45+/CD11b– lymphocytes. (C) The % of Foxp3+/CD25+ T regulatory population out of CD4+ cells, and PD-1+ cells out of CD8+ population in Ldha WT and KO tumors. Results are expressed as means ± SD of 3 livers per group (t test). *P < .05; **P < .01. (D, E) Immunofluorescence staining showing CD8+ cells in Ldha WT and KO tumors and in peritumoral area: blue is DAPI, red is CD8+. (F) Quantification of immunofluorescence results. ns, not significant.

Fig 4: Recombinant IL-37 treatment reduces PD-1 surface expression and improves the function of aged T-cells. Aged (24 months old) C57BL/6 mice were treated with control immunoglobulin (Control Ig) or recombinant IL-37 (rIL-37) every other day for 2 weeks. Naïve CD4+ T-cells and CD8+ T-cells were purified from treated mice using MACs selection and stimulated in vitro with aCD3/aCD28. On day 3 post-stimulation, (a–c) the mean surface expression of CD44 and PD1 (mean fluorescence intensity [MFI]) and (d–g) the percentage and MFI of IL-2/IFN-?-producing T-cells were determined using flow cytometric analysis. Means ± SD are shown in (b, c, e, f, and g) with **p < 0.01, ***p < 0.001, and ****p < 0.0001 determined using a Student's t test relative to aged T-cell responses from Control Ig treated mice. n = 9 mice/group with 3 independent experiments conducted. The red boxes in (a) and (d) denote functional parameters of interest

Fig 5: Functional analysis of ICOS+CD38+ CD4+ and CD8+ T cells and response to SARS-CoV-2 Spike antigen.PBMCs collected from study participants (n = 10) pre-vaccination (day 0), day 28 post-vaccination (7 days post-boost), and day 105 post-vaccination were examined for ICOS, CD38, and other markers by fluorescence flow cytometry individually in one setting to avoid batch variation. A CD4+ T cells were gated from lymphocytes and defined as ICOS+CD38+ or ICOSloCD38lo. Percentages of CD4+ICOS+CD38+ T cells were quantified and data from each participant is connected by lines. B Mean fluorescence intensity (MFI) of CCR7 within CD4+ICOS+CD38+, CD8+ICOS+CD38+, CD4+ICOSloCD38lo, or CD8+ICOSloCD38lo T cells. C, D PBMCs were stimulated with PMA/ionomycin and examined for cytokine production. T cells defined as ICOS+CD38+ (red) or ICOSloCD38lo (blue) were examined for C IFN-? or D TNF. Representative samples on the left and individual participants are shown on right. E Day 28 PBMC samples were stimulated with activation beads or T cells were sorted based on CD38 and ICOS expression. Sorted T cells were labeled with CellTrace Violet (CTV) to track and stimulated with T-cell (CD3)-depleted autologous PBMCs ± SARS-CoV-2 Spike peptide pool (+ peptide, blue and red). Sorted T-cell populations were analyzed after 48 h for IL-2, TNF, and Granzyme b in F. BCL6 expression in sorted T-cell populations, stimulated as in E. G Tfh markers CXCR5 and PD-1 compared on day 0 and 28 samples. H Unstimulated days 0 and 28 ICOS+CD38+ and ICOSloCD38lo T cells were analyzed for BCL6 expression (FMO; fluorescence-minus-one control for BCL6). I CXCR5 MFI within BCL6+ T cells. FMO control for BCL6 in ICOSloCD38lo T cells is shown by the blue dotted line, and by the red dotted line for ICOS+CD38+ T cells. B Significance was determined by paired two-way ANOVA mixed-effects model. E, F Repeated measures one-way ANOVA. C, D, G–I Two-way ANOVA with Sidak’s multiple comparisons test. Representative samples are shown and each data point represents an individual participant. All error bars ± SD. ns = not significant, *p < 0.5, **p < 0.005, ***p < 0.0005, ****p < 0.00005.