Autoimmune diseases in Treg-αζDKO mice.

Female Dgka-/-zf/f-Foxp3YFPCre/YFPCre or male Dgka-/-zf/f-Foxp3YFPCreand WT control mice were analyzed. A. Body weights of 5–9-month-old mice. B. Representative pictures of indicated organs in a pair of 6-month-old mice. C. Total cell numbers in the indicated organs in 2–9-month-old mice. D. Representative H&E staining of paraffin thin-sections of indicated organs. E. Seral autoantibodies titers in 7-month-old mice. F. Representative images of detection of seral antinuclear antibodies against fixed HEp-2 cells. G. Seral autoantibody IgG subtypes. H. Detection of IgG deposition in cyro-sections of kidneys with fluorescence confocal microscopy. Data shown are representative of or pooled from at least six experiments. Each circle or square represents one mouse of the indicated genotypes. Each line connecting the circle and square represents one pair of age- and sex-matched mice examined in one experiment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 determined by two-tail pair-wise (Figure 1A, data with lines connecting WT and αζDKO mice) or unpaired Student t test.

Enhanced Treg expansion in Treg-αζDKO mice.

A–G. Analysis of Dgka-/-zf/f-Foxp3YFPCre/YFPCreand WT-Foxp3YFPCre/YFPCre control mice. A. Representative FACS plots showing Foxp3 and CD25 staining in CD4+ SP thymocytes (top panels) and CD4 and Foxp3 staining in splenic and LN CD4+ T cells. B. Foxp3+ Treg and CD25+Foxp3−CD4+ pre-Treg percentages and numbers in the thymus. C. Foxp3+ Treg percentages and numbers in the spleen and mLNs. D. Death rate of Tregs. E. Percentages of Ki67+ cells within Tregs. F. BrdU incorporation in Tregs 8–10 hours after intraperitoneal (i.p.) injection of BrdU. G. Overlaid histograms showing Helios and Nrp1 expression in Tregs. H–M. Analysis of female Dgka-/-zf/f-Foxp3YFPCre/+and WT-Foxp3YFPCre/+ mice. H. Representative FACS plots showing intracellular Foxp3 and YFP staining in CD4+ T cells. I. YFP+Foxp3+ and YFP−Foxp3+ Treg percentages in CD4+ T cells. J. YFP+Foxp3+/YFP−Foxp3+ ratios in individual mice. K. Total, YFP+, and YFP− Treg numbers in the spleen and mLNs. L. Representative FACS plots showing BrdU incorporation in LN YFP+ Tregs. M. Percentages of Ki67+ cells in YFP+ and YFP− Tregs. N, O. Analyses of mixed BM chimeric mice. CD45.1+CD45.2+ WT mice were lethally irradiated and intravenously (i.v.) injected with a mixture of CD45.1+ WT with either CD45.2+ WT-Foxp3YFPCre/YFPCreor Dgka-/-zf/f-Foxp3YFPCre/YFPCre BM cells. Recipient mice were analyzed 6–8 weeks after reconstitution. N. Intracellular staining of Foxp3 in CD4+TCRβ+ T cells. O. CD45.1+CD45.2− and CD45.1−CD45.2+ Treg percentages in individual mice. Data shown are representative of or pooled from 5–23 experiments except F, L, N, and O. F is pooled from four experiments. L represents two experiments. N and O are representative or pooled from three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 determined by two-tail unpaired Student t test.

Altered signaling, metabolism, and transcriptional programs in αζDKO Tregs.

Tregs in peripheral lymphoid organs from Dgka-/-zf/f-Foxp3YFPCre/YFPCreand WT-Foxp3YFPCre/YFPCre control mice were analyzed. A, B. Overlaid histograms show Erk1/2, S6, and Akt S473 phosphorylation (A) and CD71 and CD98 expression (B) in cTregs and eTregs. C. Overlaid histograms show 2-NBDG uptake in Tregs. D, E. Extracellular acidification rate (ECAR) measurements of sorted Tregs (n = 4 for both WT and DKO Tregs) following sequential treatment with glucose, oligomycin (OM, for mitochondrial perturbation), and 2DG (a glucose inhibitor). Representative ECAR profiles (D) and summary scatter plot of ECAR (E). F. Volcano plot comparison of gene expression between WT and DKO Tregs. Green-colored genes are differentially expressed with greater than 1.5-fold differences between WT and αζDKO Tregs (p < 0.05). Right panel shows total numbers of DEGs in DKO Tregs. G. Prominently changed KEGG pathways. H. Heatmaps show DEGs in TCR signaling, NFκB, cell cycle, and glycolysis. I. Hif1a and Mki67 mRNA levels. *, p < 0.05; **, p < 0.01; ***, p < 0.001 determined by two-tail unpaired Student t test.

Enhanced effector differentiation and altered properties of αζDKO Tregs.

A. Scatter plots show mean ± SEM of cTreg and eTreg percentages and numbers in Dgka-/-zf/f-Foxp3YFPCre/YFPCre and WT-Foxp3YFPCre/YFPCre mice.

B. Scatter plots show mean ± SEM of cTreg and eTreg percentages and numbers of YFP+ and YFP− CD4+Foxp3+ Tregs in female Dgka-/-zf/f-Foxp3YFPCre/+ and WT-Foxp3YFPCre/+ mice.

C, D. Analyses of mixed BM chimeric mice as described in Figure 2N. C. CD44 and CD62L expression in CD45.1+ WT and in CD45.2+ WT or αζDKO Tregs in mLNs. D. Scatter plots show mean ± SEM of cTreg and eTreg percentages in CD45.2+ and CD45.1+ Tregs.

E, F. In vitro contact inhibition assay. CTV labeled WT CD45.1+CD4+Foxp3YFP− Tcon were mixed with 2:1 ratio of CD45.1−CD45.2+ WT or αζDKO Tregs in the presence of mitomycin C treated splenocytes as antigen presenting cells (APCs) from TCRα-/- mice and were stimulated with an anti-CD3 antibody for 72 hours. E. Overlaid histograms show CTV dilution of CD45.1+ Tcon. Scatter plots show mean ± SEM of Tcon cells that were undivided, divided 1 – 4 times, and divided >5 times. F. Tcon, Treg, and APC populations revealed by CD45.1 and CD4 expression.

G. TCR-induced Treg proliferation in vitro. Overlaid histogram showing CTV dilution of Foxp3YFP+ Tregs from CTV-labeled splenocytes after anti-CD3 stimulation for 72 hours. H. Heatmap showing differentially expressed Treg effector molecules between WT and αζDKO Tregs (p < 0.05). I. mRNA levels of Il10, Tgfb1-3, and Foxp3 in Dgka-/-zf/f-Foxp3YFPCre/YFPCre and WT-Foxp3YFPCre/YFPCreTregs. J. Overlaid histograms comparing expression of indicated molecules in Dgka-/-zf/f-Foxp3YFPCre/YFPCreand WT-Foxp3YFPCre/YFPCre mice.

Data shown are representative of or pooled from at least three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 determined by two-tail unpaired Student t test and pairwise Student t test for E.

Increased effector lineages and gain-of-proinflammatory properties of αζDKO Tregs.

A–F. Analyses of WT-Foxp3YFPCre/YFPCre and Dgka-/-zf/f-Foxp3YFPCre/YFPCreTregs. A. Heatmap showing DE of TFs in Tregs by RNA sequencing. B. Intracellular staining of RORγt, T-bet, Bcl6, and GATA3 in splenic Tregs. C. Percentages and numbers (mean ± SEM) of Treg subsets. D. Heatmap showing DE of cytokines in Tregs by RNA sequencing. E. Intracellular staining of cytokines in Tregs after ex vivo PMA plus ionomycin stimulation in the present GolgiPlug for 5 hours. F. Percentages (mean ± SEM) of IFNγ+, IL17A+, and IL4+ Tregs.

G–I. Analyses of female WT-Foxp3YFPCre/+ and Dgka-/-zf/f -Foxp3YFPCre/+ Tregs. G. Percentages (mean ± SEM) of Treg effector sublineages in YFP+ and YFP− Tregs. H–I. IL17A and IFNγ expression in Tregs after ex vivo PMA plus ionomycin stimulation in the present GolgiPlug for 5 hours. H. Representative FACS plots of Tregs. I. Scatter plots show mean ± SEM of IFNγ+ and IL17A+ in YFP+ and YFP− Tregs.

J. Analyses of mixed BM chimeric mice described in Figure 2N. Scatter plots show mean ± SEM of Treg sublineage percentages in CD45.1+ WT (red circle) and CD45.2+ WT or αζDKO Tregs (blue square).

Data shown in B–J are representative of or pooled from at least three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 determined by two-tail unpaired Student t test.

Enhanced effector function of CD4+Foxp3− Tcon and CD8 T cells in Treg-αζDKO mice.

Dgka-/-zf/f-Foxp3YFPCre/YFPCreand WT-Foxp3YFPCre/YFPCre control mice were analyzed. A. CD4+Foxp3− Tcon and CD8+ T cell percentages and numbers in the spleen and mLNs. B. Ki67+ cells in Tcon and CD8 T cells. C. BrdU+ cells in Tcon and CD8 T cells. D. Representative FACS plots showing CD44 and CD62L expression in splenic CD4+Foxp3− Tcon and CD8+ T cells. E. Naïve, CM, and EM percentages and numbers of splenic CD4+Foxp3− Tcon and CD8 T cells. F. Representative FACS plots showing intracellular T-bet and RORγt staining in LN CD8 T cells. G. Percentages and numbers of T-bet+ CD8 T cells. H. Intracellular IFNγ and IL17A staining in CD8 T cells after PMA and ionomycin stimulation. Scatter plot represents mean ± SEM of IFNγ+ CD8 T cells from 6–12-month-old mice. I. Volcano plot comparing mRNA expression in WT and Treg-αζDKO CD4+Foxp3− Tcons after RNA-seq analysis. Genes colored in green are differentially expressed with greater than 1.5-fold differences between WT and αζDKO Tregs (p < 0.05). Table shows numbers of DE genes (p < 0.05). J. Top enriched KEGG pathways between WT and Treg-αζDKO CD4+Foxp3− Tcons. K. Heatmap showing mRNA levels of TFs and cytokines that were differentially expressed between WT and Treg-αζDKO CD4+Foxp3− Tcons (p < 0.05). L. T-bet, RORγt, Bcl6, and GATA3 proteins in splenic Tcons detected by intracellular staining. M. Percentages and numbers of T-bet+, RORγt+, Bcl6+, and GATA3+ cells in Tcons. N. IFNγ, IL17A, and IL4 protein levels in WT and Treg-αζDKO CD4+Foxp3− Tcons detected by intracellular staining after PMA and ionomycin stimulation for 5 hours. O. Serum cytokine levels in 5–12-month-old WT and Treg-αζDKO mice. Data shown are representative of or pooled from 4–22 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 determined by two-tail unpaired Student t test.

Enhanced Tfh/GC-B cell responses in Dgka-/-zf/f-Foxp3YFPCre/YFPCremice.

Splenocytes and LN cells from Dgka-/-zf/f-Foxp3YFPCre/YFPCreand WT-Foxp3YFPCre/YFPCre mice were analyzed. A. CXCR5 and PD-1 expression in CD4+Foxp3− Tcons. B. Percentages and numbers of CXCR5+PD-1+ Tfh-cells in 2–14-month-old mice. C. Heatmap shows DE of key Tfh/Tfr genes from transcriptomic analyses of Tcons described in Figure 6I. D. Overlaid histograms show expression-indicated molecules in Tfh cells. E. Representative FACS plots show GATA3 and Bcl6 expression in Tcons from a pair of 7-month-old mice. Scatter plots show mean ± SEM of GATA3+Bcl6+ and GATA3−Bcl6+ Tfh cells. F. B220 and CD93 staining of splenocytes and LN cells. Scatter plots show mean ± SEM of B220+CD93− mature B-cell percentages and numbers. G. GL7 and Fas expression in total B220+ and in IgM−IgD−B220+ cells. H. Scatter plots show mean ± SEM of GC-B cell percentages and numbers in 5–14-month-old mice. I. Overlaid histogram shows GL7 expression in GC-B cells. J. Intracellular IgG1 and IgG2b staining in GC-B cells. K. IgG1+ and IgG2b+ percentages in GC-B cells. L. Serum IgM, IgG1, IgG2b, IgG3, and IgE concentrations. M. CD23 expression in B cell populations. Data shown are representative of or pooled from at least four experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 determined by two-tail unpaired Student t test.

Development of autoimmune diseases and deregulated Tfh/Tfr cell and GC-responses in female Dgka-/-zf/f-Foxp3YFPCre/+ mice.

Three–nine-month-old female Dgka-/-zf/f-Foxp3YFPCre/+(DKO-Crehet) and WT-Foxp3YFPCre/+ (WT-Crehet) mice were analyzed. A. Body weights. B. Representative picture of spleen and mLNs, total cell numbers in the indicated organs. C. H&E staining of kidney thin sections. Bottom low shows higher magnification. D. Seral anti-dsDNA and ssDNA autoantibodies. E. Antinuclear antibodies. F. IgG deposition in the kidney. G. Total B cell percentages and numbers. H. Fas and GL7 staining in splenic B220+ cells. I. GC-B cell percentages in splenic B220+ and B220+IgM−IgD− (DN) B cells and GC-B cell numbers in B220+ B cells. J. Seral Ig levels. K. IgG1+ and IgG2b+ cells in GC-B cells. L. CD4+Foxp3− Tcon percentages and numbers. M. Naïve and effector cell percentages in Tcons. N. Percentages of T-bet+, GATA3+, RORγt+, and Bcl6+ cells in Tcons. O. Assessment of Tfh cells. Representative FACS plots show gating of CXCR5+PD-1+ Tfh and CXCR5−PD-1+ Tph cells in splenic Tcons. Scatter plots show Tfh percentages and numbers. P. Assessment of Tfr cells. Representative FACS plots show gating of CXCR5+PD-1+ Tfr cells in splenic YFP+ and YFP− Foxp3+ Tregs. Scatter plots show Tfr percentages and numbers. Q. Scatter plots show Tfr/Tfh cell ratios. Data shown are representative of or pooled from 5–10 experiments except four experiments for Figure 8K. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 determined by two-tail pair-wise (Figure 8A, data with lines connecting WT-Crehet and αζDKO-Crehet mice) or unpaired Student t test.

Treg-specific DGKαζ deficiency conferred CD28-independent Treg development/homeostasis and GC responses and accelerated Treg-to-exTreg/exTreg-Tfh conversion.

A–H. Analyses of WT-Foxp3YFPCre/YFPCre, Dgka-/-zf/f-Foxp3YFPCre/YFPCre (DKO), Dgka-/-zf/f-Foxp3YFPCre/YFPCre-CD28-/- (TKO), and Foxp3YFPCre/YFPCre-CD28-/- (CD28KO). A. Treg percentages and numbers (mean ± SEM) in the thymus, spleen, and mLNs. B. Mean ± SEM of cTreg and eTreg percentages. C. Tfh-cell percentages. D. Tfr-cell percentages. E. Representative FACS plots showing Fas and GL7 staining in live gated splenic B220+IgM−IgD− (DN) B cells. Scatter plots show mean ± SEM of GC-B cell percentages. F. Seral antibody levels. G. Seral anti-ssDNA and dsDNA autoantibody levels. H. Seral antinuclear antibodies in CD28-/-and TKO mice. I–K. Analyses of mixed BM chimeric mice reconstituted with a mixture of BM cells of CD45.1+ WT BM cells with either CD45.2+ WT-Foxp3YFPCre/YFPCre, Dgka-/-zf/f-Foxp3YFPCre/YFPCre(DKO), Dgka-/-zf/f-Foxp3YFPCre/YFPCre-CD28-/-(TKO), or Foxp3YFPCre/YFPCre-CD28-/- (28KO) BM cells. I. Ratios of CD45.2+ test Treg percentages in CD4+ T cells/CD45.1+ WT Treg percentage in CD4+ T cells in individual chimeric mice. J. Representative FACS plots showing PD-1 and CXCR5 staining in live gated mLN CD45.1+ control and CD45.2+ test CD4+Foxp3− Tcons and CD4+Foxp3+ Tregs. K. Percentages of Tfh and Tfr cells as well as Tfr/Tfh ratios of CD45.1+ and CD45.2+ origins in individual chimeric mice.

Data shown are representative of or pooled from 6–11 experiments for A–H and 3–5 experiments for I–K. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by two-tailed unpaired Student t-test.

Enhanced conversion to exTreg/exTreg-Tfh cells and pathogenicity of αζDKO Tregs.

A–C. Comparison of DEGs in both Treg-αζDKO Tregs and Tcons compared with their corresponding WT controls. A. DEGs that were shared or not shared in αζDKO Tregs and Tcons.

B. Concordant and discordant expression of DEGs in Treg-αζDKO Tregs and Tcons. C. Expression of indicated Treg-associated genes in Treg-αζDKO Tregs and Tcons.

D -M. Adoptive transfer experiments. 5 × 105 double-sorted CD45.2+ Tregs from WT-Foxp3YFPCre/YFPCre or CD45.1+CD45.2+ Tregs from DGKα-/-ζf/f-Foxp3YFPCre/YFPCre mice were co-injected i.v. with 1 × 106 WT CD45.1+ CD4+ T cells into TCRβ-/- mice. Serum were collected and mice were euthanized for the experiment 10 weeks after transfer. D. Experimental scheme. 5 × 105 double-sorted CD45.2+ Tregs from WT-Foxp3YFPCre/YFPCre or CD45.1+CD45.2+ Tregs from Dgka-/-zf/f-Foxp3YFPCre/YFPCre mice were co-injected i.v. with 1 × 106 WT CD45.1+ CD4+ T cells into TCRβ-/- mice. Serum were collected and mice were euthanized for the experiment 10 weeks after transfer. E. Spleen sizes and total cell numbers (mean ± SEM). F. Representative FACS plot showing YFP levels in live gated CD45.2+ or CD45.1+CD45.2+CD4+TCRβ+ cells. G. Scatter plot showing means ± SEM of CD4+YFP− exTreg percentages. H. Representative FACS plots showing PD-1 and CXCR5 staining in YFP+ 45.2+/CD45.1+CD45.2+CD4+TCRβ+ Tregs and YFP− CD45.2+ or CD45.1+CD45.2+CD4+TCRβ+ exTregs. I. Mean ± SEM of Tfr and ExTreg-Tfh cell percentages. J. FACS plots showing Fas and GL7 staining in B220+ IgM−IgD− B cells. Scatter plot showing mean ± SEM of GC-B cell percentages. K. IgG1+ and IgG2b+ cells in GC-B cells in the spleen and mLNs. L. Seral anti-ssDNA and -dsDNA antibody titers. M. IgG deposition in the kidney. Representative immunofluorescence of anti-IgG staining of kidney sections is shown. Scatter plot shows mean ± SEM of IgG+ glomerulus numbers per 10 × 10 field.

Figure 10L is from one experiment and represents two experiments. Other data are representative of or pooled from 4–8 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by two-tailed unpaired Student t-test.