IFNγ-induction of TH1-like regulatory T cells controls antiviral responses

Abstract

Regulatory T (T_reg) cells are an immunosuppressive population that are required to maintain peripheral tolerance and prevent tissue damage from immunopathology, via anti-inflammatory cytokines, inhibitor receptors and metabolic disruption. Here we show that T_reg cells acquire an effector-like state, yet remain stable and functional, when exposed to interferon gamma (IFNγ) during infection with lymphocytic choriomeningitis and influenza A virus. T_reg cell-restricted deletion of the IFNγ receptor (encoded by Ifngr1), but not the interleukin 12 (IL12) receptor (encoded by Il12rb2), prevented T_H1-like polarization (decreased expression of T-bet, CXC motif chemokine receptor 3 and IFNγ) and promoted T_H2-like polarization (increased expression of GATA-3, CCR4 and IL4). T_H1-like T_reg cells limited CD8⁺ T cell effector function, proliferation and memory formation during acute and chronic infection. These findings provide fundamental insights into how T_reg cells sense inflammatory cues from the environment (such as IFNγ) during viral infection to provide guidance to the effector immune response. This regulatory circuit prevents prolonged immunoinflammatory responses and shapes the quality and quantity of the memory T cell response.

Extended Data Fig. 1 ∣. IFNγ and T-bet expression in T_reg cells during acute and chronic infection.

a, Gating strategy for all flow cytometric experiments. b-f, Foxp3^Cre-YFP mice remained uninfected (n = 5) or were infected (n = 6) with Arm. or Cl. 13. b, c, Expression of Ifng (b) and Tbx21 (c) mRNA by purified splenic T_reg cells were determined by qPCR after ex vivo stimulation with anti-CD3/anti-CD28 (Arm.) or PMA and Ionomycin (Cl. 13). c, Linear regression of Tbx21 and Ifng co-expression. d, Foxp3^Cre-YFP mice (n = 14) were infected with Arm. and stimulated ex vivo with anti-CD3 and expression of T-bet and IFNγ was measured by flow cytometry. Linear regression of T-bet and IFNγ co-expression. e, f, Foxp3^Cre-YFP mice remained uninfected or were infected with Arm. or Cl. 13. Expression of Ifng (e) (n = 15, 5 independent experiments, Uninfected; n = 12, Arm.; n = 17 Cl. 13) and Tbx21 (f) (n = 16, 5 independent experiments, Uninfected; n = 12, Arm.; n = 14, Cl. 13) mRNA by sorted splenic CD4⁺ T_conv cells and T_reg cells, without stimulation, were determined by qPCR. b-f, Data are presented as mean values and represent biologically independent mice and (b-d and as indicated) or 3 (e, f) or 5 (as indicated) independent experiments. Statistical significance was determined by multiple unpaired Student’s t-test relative to Uninfected (b, c), or by simple linear regression (d) or by a two-way ANOVA with multiple comparisons and multiple unpaired t-tests relative to CD4⁺ T_conv cells (e, f) (P values indicated when significant); NS, not significant.

Extended Data Fig. 2 ∣. T_H1 signatures in T_reg cells and CD4⁺T_conv cells during acute and chronic infection.

a, Diagram of Il12rb2.Thy1.1^L/LhNGR targeted mouse. b, c, Il12rb2.Thy1.1^L/LhNGR mice (n = 11) were infected with Arm. and splenic Thy1.1⁻ and Thy1.1⁺ CD4⁺ T_conv. cells purified and expression of Il12rb2 by Thy1.1⁻ and Thy1.1⁺ CD4⁺ T_conv. cells was determined by qPCR (b). Thy1.1⁻ and Thy1.1⁺ CD4⁺ T_conv. cells were serum starved and treated with PBS or IL-12, and induction of pSTAT4 was measured by flow cytometry (c). d, e, Foxp3^Cre-YFP mice (n = 16, 5 independent experiments, Uninfected; n = 12, Arm.; n = 14, Cl. 13) remained uninfected or were infected with Arm. or Cl. 13, expression of Il12rb2 mRNA (d) and Ifngr1 mRNA (e) by sorted splenic CD4⁺ T_conv cells and T_reg cells, without stimulation, were measured by qPCR. f-i, Expression of IFNGR1 (f) (n = 7, 2 independent experiments, Uninfected; n = 9, 2 independent experiments, Arm.; n = 15, Cl. 13), T-bet (g) (n = 7, Uninfected; n = 11, Arm.; n = 8, Cl. 13), CXCR3 (h) (n = 11, Uninfected; n = 9, 2 independent experiments, Arm.; n = 15, Cl. 13), T-bet (n = 7, 2 independent experiments, Uninfected; n = 11, 2 independent experiments, Arm.; n = 16, Cl. 13), GATA-3 (n = 6, 2 independent experiments, Uninfected; n = 9, 2 independent experiments, Arm.; n = 17, Cl. 13), and RORγt (n = 3, 2 independent experiments, Uninfected; n = 11, 2 independent experiments, Arm.; n = 8, 2 independent experiments, Cl. 13), (i) by splenic CD4⁺ T_conv cells and T_reg cells were determined flow cytometry. b-i, Data are presented as mean values and represent biologically independent mice and 2 (g, and as indicated) or 3 (b-f, h, i) or 5 (as indicated) independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test relative to Thy1.1⁻ (b) or CD4⁺ T_conv cells (f-h), or by a two-way AVOVA with multiple comparisons (c), or by a two-way ANOVA with multiple comparisons (d-e) and multiple unpaired t-tests relative to CD4⁺ T_conv cells (d, e, i) (P values indicated when significant); NS, not significant.

Extended Data Fig. 3 ∣. Transcriptomic analysis of Ifngr1-deficient T_reg cells verses control, during chronic infection.

a, Expression of the deleted region of Ifngr1, normalized to the intact region of Ifngr1, from uninfected Ifngr1^L/LFoxp3^Cre-YFP (n = 5) compared to Foxp3^Cre-YFP mice (n = 6). Thy1.2⁻ cells, CD8⁺ T cells, CD4⁺ T_conv cells and T_reg cells were sorted, gDNA was isolated and qPCR was performed. b, Expression of IFNGR1 in T_reg cells from uninfected Foxp3^Cre-YFP (n = 7) and Ifngr1^L/LFoxp3^Cre-YFP (n = 5) mice was measured by flow cytometry. c, d, Foxp3^Cre-YFP mice remained uninfected (n = 15, 5 independent experiments, control; n = 10, 4 independent experiments, Ifngr1-deficient) or were infected with Arm. (n = 9, 2 independent experiments, control; n = 8, 2 independent experiments, Ifngr1-deficient) or Cl. 13 (n = 15, control; n = 14 (c), n = 15 (d), Ifngr1-deficient) and percentage of Foxp3+of CD4⁺ T cells (c), number of T_reg cells (d, upper) and CD4⁺ T_conv cells (d, lower) per spleen, were determined by flow cytometry. e-g, scRNA-seq of splenic T_reg cells from uninfected (n = 3) or Cl. 13 infected (n = 5) (d16) Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice. e, Distribution of cells amongst clusters. f, Top 50 ranked significant differentially expressed genes from T_reg cells in Foxp3^Cre-YFP and Ifngr1^L/LFoxp3^Cre-YFP mice infected with Cl. 13. g, GSEA overview illustrating pathways upregulated in the control T_reg cell gene set compared to Ifngr1-deficient T_reg cells during Cl. 13 infection. h, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice remained uninfected and the expression of T-bet (n = 7), GATA-3 (n = 6, control; n = 5, Ifngr1-deficient) and RORγt (n = 3, control; n = 4, Ifngr1-deficient) by splenic T_reg cells were measured by flow cytometry. a-h, Data are presented as mean values and represents biologically independent mice and 2 (a, b, e-h and as indicated) or 3 (c, d) or 5 (as indicated) independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test (b), or by multiple unpaired t-tests (a, c, d, h), relative to Foxp3^Cre-YFP mice. Adjusted P values were determined by one-way ANOVA relative to Foxp3^Cre-YFP mice (e) or by Wilcoxon rank-sum test (f) or Kolmogorov-Smirnov test (g) relative to infected Foxp3^Cre-YFP mice (P values indicated when significant); NS, not significant.

Extended Data Fig. 4 ∣. Intrinsic effects of Ifngr1 deletion from T_reg cells using Ifngr1^L/LFoxp3^Cre-YFPFoxp3^Cre-ERT2-GFP heterozygous mice.

a-e, Heterozygous Foxp3^Cre-YFP.Foxp3^Cre-ERT2-GFP (Cre Het) (n = 5, Uninfected; n = 12, Cl. 13) and Ifngr1^L/LFoxp3^Cre-YFP.Foxp3^Cre-ERT2-GFP (L/L Het) (n = 10, Uninfected; n = 19, Cl. 13) female mice remained uninfected (2 independent experiments) or were infected with Cl. 13 (3 independent experiments). a, Percent expression of GFP⁺ and YFP⁺ by splenic CD4⁺ T cells were determined by flow cytometry. b-e, Expression of the CXCR3 (b), CCR4 (c), CD127 (d) and KLRG1 (e) in splenic T_reg cells from indicated mice were measured by flow cytometry. f-h, Heterozygous Foxp3^Cre-YFP.Foxp3^Cre-ERT2-GFP (Cre Het) and Ifngr1^L/LFoxp3^Cre-YFP.Foxp3^Cre-ERT2-GFP (L/L Het) female mice were infected with Cl. 13. f, Gating strategy for IFNGR1⁻ and IFNGR1⁺ splenic T_reg cells (g, h) from indicated mice. g, h, Expression of T-bet (g) (n = 10, Cre HET; n = 15, L/L HET) and GATA-3 (h) (n = 10, Cre HET; n = 12, L/L HET) by IFNGR1⁻ and IFNGR1⁺ splenic T_reg cells from indicated mice, were measured by flow cytometry. a-h, Data are presented as mean values and represent biologically independent mice and 2 (as indicated) or 3 (a-e, g, h and as indicated) independent experiments. Statistical significance was determined by a two-way AVOVA with multiple comparisons (a, g, h) or by one-way AVOVA with multiple comparisons (b-e) (P values indicated when significant); NS, not significant.

Extended Data Fig. 5 ∣. T_reg cells maintain stability during chronic infection.

a, DE of genes induced (upper), or repressed (lower), by Foxp3 in splenic T_reg cells of Foxp3^Cre-YFP mice compared to Ifngr1^L/LFoxp3^Cre-YFP mice, that remained uninfected (n = 3) or were infected with Cl. 13 (n = 5) was measured by scRNA-seq. b, c Foxp3^Cre-ERT2-GFPRosa26^LSL.tdTom mice were treated with tamoxifen for 7d and then remained uninfected or were infected with Cl. 13. b, Experimental schema. c, Representative flow cytometry plot of tdTom and GFP co-expression in splenic CD4⁺ T cells at steady state (n = 7) or at 16 (n = 5, 2 independent experiments) and 35 (n = 11, 2 independent experiments) dpi with Cl. 13. d-f, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with Cl. 13 and levels of Foxp3 (d) (n = 9, control; n = 10, Ifngr1-deficient) and percentage of Nrp1 (e) (n = 9, control; n = 8, Ifngr1-deficient) CD25 (f, left) (n = 11) and level of CD25 (f, right) (n = 11) by splenic T_reg cells were determined by flow cytometry. a, c-f, Data are presented as mean values and represent biologically independent mice and 2 (a, d-f and as indicated) or 3 (c) independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test relative to Foxp3^Cre-YFP mice (d-f). Adjusted P value was determined by Wilcoxon rank-sum test (a) (P value indicated when significant); NS, not significant.

Extended Data Fig. 6 ∣. IFNγ promotes T_H1-like polarization of T_reg cells during Influenza A viral infection.

a-d, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with IAV. a, b, Percentage of initial body weight over time (a) (numbers of mice used indicated in parentheses) and at day 6 (b) (n = 16, control; n = 17, Ifngr1-deficient). c, d, Expression of T-bet (c) (n = 9, control; n = 7, Ifngr1-deficient) and GATA-3 (d) (n = 10, control; n = 8, Ifngr1-deficient) by lung T_reg cells were measured by flow cytometry. a-d. Data are presented as mean values and represent biologically independent mice and 2 (a, c, d) or 3 (b) independent experiments. Statistical significance was determined by two-way ANOVA with multiple comparisons (a) or by an unpaired two-tailed Student’s t-test (b-d), all relative to Foxp3^Cre-YFP mice (P values indicated when significant); NS, not significant.

Extended Data Fig. 7 ∣. Ifngr1 deletion from T_reg cells does not affect viral load nor markers associated with CD8⁺ T cell exhaustion during chronic infection.

a, b, Foxp3^Cre-YFP, Ifngr1^L/L or Ifngr1^L/LFoxp3^Cre-YFP mice remained uninfected or were infected with Cl. 13. a, Liver histological score from uninfected mice (n = 2) or mice infected with Cl. 13 8 dpi (n = 4, Foxp3^Cre-YFP; n = 3, Ifngr1^L/L; n = 3, Ifngr1^L/LFoxp3^Cre-YFP). Score determined as: 1= Triaditis, rare sinusoidal lymphocytes; 2= Triaditis, prominent sinusoidal lymphocytes; 3= Triaditis, prominent sinusoidal lymphocytes, single cell apoptosis. b, Serum AST activity was determined in uninfected mice (n = 9, Foxp3^Cre-YFP; n = 2, 1 independent experiment, Ifngr1^L/L; n = 8, Ifngr1^L/LFoxp3^Cre-YFP) or mice infected with Cl. 13, 8 dpi (n = 13, Foxp3^Cre-YFP; n = 14, Ifngr1^L/LFoxp3^Cre-YFP) or 16 dpi (n = 10, Foxp3^Cre-YFP; n = 2, 1 independent experiment, Ifngr1^L/L; n = 9, Ifngr1^L/LFoxp3^Cre-YFP). c, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with Cl. 13 and the viral load in serum d16 (n = 12, control; n = 11, Ifngr1-deficient; 2 independent experiments) and d30 (n = 11, control; n = 10, Ifngr1-deficient; 2 independent experiments), kidney (n = 7, control; n = 6, Ifngr1-deficient; 1 independent experiment) and liver (n = 5, control; n = 3, Ifngr1-deficient; 1 independent experiment) were determined. d, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFPmice were infected with Cl. 13 and treated with 200 μg isotype control (n = 3, control; n = 4, Ifngr1-deficient) or anti-PDL1 (n = 5, control; n = 2, Ifngr1-deficient) every 3 d from 26-38 dpi and the viral load in kidney was determined. e, f, Foxp3^Cre-YFP (n = 19) or Ifngr1^L/LFoxp3^Cre-YFP (n = 16) mice were infected with Cl. 13 and IR co-expression (indicated on top) with PD1 (e), and SPICE plots visualizing multiple combinations of IR co-expression (f), by pooled Tetramer⁺ (GP₃₃, GP₂₇₆, NP₃₉₆) splenic CD8⁺ T cells were determined by flow cytometry. g-j, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with Cl. 13 and treated with isotype control (n = 7) or anti-PDL1 (n = 6, control; n = 8, Ifngr1-deficient) as in (d). g, h, Expression of T-bet by bulk splenic CD8⁺ T cells (g) and PD1 by pooled Tetramer⁺ (GP₃₃, GP₂₇₆, NP₃₉₆) splenic CD8⁺ T cells (h) were determined by flow cytometry. i, Percentage of pT_ex (Tcf1⁺TIM3⁻) and tT_ex (TCF1⁻TIM3⁺) splenic CD8⁺ T cells were determined by flow cytometry. j, Percentage of tT_ex splenic GP33⁺CD8⁺ T cells was determined by flow cytometry. a-j, Data are presented as mean values and represent biologically independent mice and 1 (a, d, and as indicated), 2 (c, g-j) or 3 (b, e, f) independent experiments. a-e, g-j, Statistical significance was determined by a two-way ANOVA with multiple comparisons (a, b) or by an unpaired two-tailed Student’s t-test relative to Foxp3^Cre-YFP mice (c, e) or my multiple unpaired Student’s t-tests (d, g-j) (P values indicated when significant); NS, not significant.

Extended Data Fig. 8 ∣. Ifngr1-deficient T_reg cells from infected mice maintain suppressive function of naïve CD4⁺T_conv cells in vitro and do not impact T_H polarization of CD4⁺T_conv cells in vivo.

a,b, in vitro microsuppression assay of splenic uninfected CD4⁺ T_conv (T_responders) by splenic T_reg cells from Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice infected with LCMV Arm. (a) or Cl. 13 (b) (numbers of mice used indicated in parentheses). c, in vitro microsuppression assay of splenic CD44^hiCD62L^lo CD4⁺ T_conv (T_eff) from Arm. infected Foxp3^Cre-YFP mice by splenic T_reg cells from Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice infected with LCMV Arm. (numbers of mice used indicated in parentheses). d-g, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice infected with Cl. 13 and expression of T-bet, GATA-3 and RORγt (d) (n = 8), T-bet and GATA-3 co-expression (e) (n = 8), CXCR3 (f) (n = 8) and CCR4 (g) (n = 8, control; n = 9, Ifngr1-deficient) by splenic CD4⁺ T_conv cells were measured by flow cytometry. h, Expression of the deleted region of Ifng, normalized to the intact region of Ifng, from Ifng^L/LFoxp3^Cre-YFP mice (n = 4) compared to Foxp3^Cre-YFP mice (n = 3). Thy1.2⁻ cells, CD8⁺ T cells, CD4⁺ T_conv cells and T_reg cells were sorted, gDNA was isolated and qPCR was performed. a-h Data are presented as mean values and represent biologically independent mice and 1 (h), 2 (b-g) or 3 (a) independent experiments. Statistical significance was determined by two-way ANOVA (a-c), or by multiple unpaired Student t-tests (d, e, h), or by an unpaired two-tailed Student’s t-test (f, g), relative to Foxp3^Cre-YFP mice (P values indicated when significant); NS, not significant.

Extended Data Fig. 9 ∣. Transcriptomic analysis reveals that TH1-like T_reg cells inhibit CD8⁺ T cell memory during LCMV infection.

a-c, scRNA-seq of splenic pooled Tetramer⁺ (GP₃₃, GP₂₇₆, NP₃₉₆) CD8⁺ T cells from Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice (n = 3), 16 dpi with Cl. 13. a, UMAP embedding Tetramer⁺ CD8⁺ T cells into a two-dimensional space to generate 8 distinct clusters, with categories shown. b, Distribution of cells amongst clusters from individual mice. c, Percentage of cells in the memory cluster. d, e, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with Arm. and the expression of effector CD8⁺ cells (d) (n = 18, control; n = 16, Ifngr1-deficient) and the ratio of MPECs to SLECs within splenic pooled CD44^hiCD62L^lo Tetramer⁺ CD8⁺ T cells (e) (n = 8) were determined by flow cytometry. f, g, Expression of Ifngr1 within the bulk population (f) and within the memory cluster (g) of the splenic pooled Tetramer⁺ CD8⁺ T cells from the scRNA-seq dataset in a-c (n = 3). h-j, Rag1^−/− mice were reconstituted with Ifngr1^−/− (n = 12) or control (WT) (n = 13) T_reg cells and infected with Arm. h, i, Frequency of splenic CD44^hiCD62L^lo (h) and CD44^hiCD62L^hi (i, left), and number of CD44^hiCD62L^hi (i, right) pooled Tetramer⁺ CD8⁺ T cells were determined by flow cytometry. j, Frequency of (left) and total number of (right) MPECs (upper) and SLECs (lower), and the ratio of MPECs to SLECs, splenic pooled Tetramer⁺ CD8⁺ T cells were determined by flow cytometry. k, heatmap of DE of the IL16/CD4/CCR5 signaling axis in T_reg cells from the scRNA-seq dataset in a-c. l-n, Foxp3^Cre-YFP and Ifngr1^L/LFoxp3^Cre-YFP mice were infected with Arm. and transcripts of the IL16/CD4/CCR5 signaling axis in T_reg cells (l) (n = 11) or Il16 (m) (n = 11) and Ccl5 (n) (n = 6, control; n = 9, Ifngr1-deficient) expression in T_reg cells and CD44^hiCD62L^lo CD4⁺and CD8⁺ T cells, were determined by qPCR. a-n, Data are presented as mean values and represent biologically independent mice and 1 (a-c, f, g), 2 (e, k, n) or 3 (d, h-j, l, m) independent experiments. Statistical significance and P values were determined by an unpaired two-tailed Student’s t-test relative to the Foxp3^Cre-YFP mice (c-g) or Rag1^−/− + WT T_reg cells (h-j), or by multiple unpaired Student t-tests relative to Foxp3^Cre-YFP mice (l) or by two-way ANOVA with multiple comparisons. (m,n), Adjusted P values were determined by one-way ANOVA (b) or Wilcoxon rank-sum test (k) relative to infected Foxp3^Cre-YFP mice (P values indicated when significant); NS, not significant.

Extended Data Fig. 10 ∣. T_H1-like T_reg cells do not impact tissue resident memory CD8⁺ T during IAV infection rechallenge.

a-g, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with PR8 only (n = 9, control; n = 8, Ifngr1-deficient) or X31 and challenged with PR8 (n = 10, control; n = 9, Ifngr1-deficient). a, Experimental schema. b, Expression of LAP/TGFβ1 by lung T_reg cells by percent (upper) and level of expression within LAP/TGFβ1⁺ T_reg cells (lower) were determined by flow cytometry. c, Percentage (upper), and number (lower), of lung IAV pooled Tetramer⁺ (PA₂₂₄ and NP₃₆₆) CD8⁺ T were determined by flow cytometry. d, Expression of IFNγ by lung CD8⁺ T cells was measured, after in vitro stimulation with pooled IAV peptides (PA_224-233 and NP_366-372), by flow cytometry. e, Expression of CXCR3 by lung pooled Tetramer⁺ CD8⁺ T cells was measured by flow cytometry. f, Percentage (left), and number (lower), of CD103⁺CD69⁺ lung pooled Tetramer⁺ CD8⁺ T cells were determined by flow cytometry. g, Blinded histology scores (areas indicated) of paraffin-embedded hematoxylin and eosin stained, lung tissue sections. b-g, Data are presented as mean values and represent biologically independent mice and 3 independent experiments. Statistical significance was determined by multiple unpaired Student’s t-tests relative to Foxp3^Cre-YFP mice (P values indicated when significant); NS, not significant.

Fig. 1 ∣. T_reg cells acquire a T_H1-like program during acute and chronic infection.

a, Foxp3^Cre-YFP mice remained uninfected or were infected with Arm. or Cl. 13 and the expression of IFNγ by splenic CD8⁺ T cells, CD4⁺ T_conv cells and T_reg cells were measured by flow cytometry following ex vivo stimulation with anti-CD3 (Arm.) (n = 7, uninfected; n = 9, Arm.; two independent experiments) or PMA and Ionomycin (Cl. 13) (n = 8, uninfected; n = 15, Cl. 13). Uninfected mice were used as controls for the different stimulation conditions. b,c, Foxp3^Cre-YFP mice remained uninfected (n = 11) or were infected with Arm. (n = 16, (b); n = 9, (c)) or Cl. 13 (n = 10, two independent experiments) and the expression of T-bet (b) and CXCR3 (c) in splenic T_reg cells were measured by flow cytometry. d–g, Mice remained uninfected or were infected with Arm. or Cl. 13. d,e, Expression of Il12rb2 (Thy1.1) in Il12rb2.Thy1.1 ^L/L.hNGFR (d) (n = 4, uninfected; n = 7 Arm. and Cl. 13) and IFNGR1 in Foxp3^Cre-YFP mice (e) (n = 11, uninfected; n = 11, two independent experiments, Arm.; n = 15, Cl. 13), within splenic CD4⁺ T_conv cells and T_reg cells were measured by flow cytometry. f,g, Expression of Il12rb2 (Thy1.1) in Il12rb2. Thy1.1^L/L.hNGFR (f) (n = 4, uninfected; n = 7, Arm. and Cl. 13) and IFNGR1 in Foxp3^Cre-YFP mice (g) (n = 11, uninfected; n = 9, two independent experiments, Arm.; n = 10, two independent experiments Cl. 13) was measured in splenic T-bet⁺ CD4⁺ T_conv cells and T_reg cells by flow cytometry. a–g, Data are presented as mean values and represent biologically independent mice and two (d,f and as indicated) and three (a–c,e,g) independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test relative to uninfected mice (a) or CD4⁺ T_conv cells (d–g), or by a one-way ANOVA with multiple comparisons (b,c) (P values indicated when significant). NS, not significant.

Fig. 2 ∣. Transcriptomic analysis reveals IFNγ-induced T_H1 profiles in T_reg cells during chronic infection.

a–c, scRNA-seq of splenic T_reg cells from uninfected (n = 3) or Cl. 13-infected (d16, n = 5) Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice. a, Variable genes were identified, used for principal component analysis and subjected to UMAP to generate five distinct clusters. b, Galaxy plots displaying the distribution of T_reg cells from Foxp3^Cre-YFP and Ifngr1^L/LFoxp3^Cre-YFP mice among clusters from individual uninfected or Cl. 13-infected mice. c, Volcano plot of the significant differentially expressed genes up in control and Ifngr1-deficient T_reg cells. The top 10 upregulated genes and selected T_H1 and T_H2 genes are bolded. d–f, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with Arm. or Cl. 13 and expression by splenic T_reg cells of CD127 (left), number of CD127⁺T_reg cells per spleen (right) (n = 16, Arm.; n = 11, two independent experiments, Cl. 13), expression of Bcl-2 (e) (n = 13), expression of KLRG1 in (left; n = 19, Arm.; n = 15 control, n = 16 Ifngr1-deficient Cl. 13) and number of KLRG1⁺T_reg cells per spleen (right; n = 16, Arm.; n = 15, control, n = 16, Ifngr1-deficient, two independent experiments, Cl. 13) were measured by flow cytometry. a–f, Data are presented as mean values and represent biologically independent mice and two (a–c and as indicated) and three (d–f) independent experiments. c, Adjusted P values were determined by Wilcoxon rank-sum test relative to Foxp3^Cre-YFP mice and are <0.05. P values were determined by multiple unpaired two-tailed Student’s t-test (d–f) (P values indicated when significant).

Fig. 3 ∣. Ifngr1-deficient T_reg cells program to a T_H2 phenotype during viral infection.

a,b, scRNA-seq of splenic T_reg cells from uninfected (n = 3) or Cl. 13-infected (d16, n = 5) Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice. a, Heat map of T_H1 and T_H2-related gene expression in control and Ifngr1-deficient T_reg cells. b, GSEA results of C7 category curated pathways showing downregulated genes from overall gene expression of control compared to Ifngr1-deficient T_reg cells. c–i, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice remained uninfected or were infected with Arm. or Cl. 13 and expression of T-bet (n = 11 control, n = 12 Ifngr1-deficient, two independent experiments, Arm.; n = 18 control, n = 15 Ifngr1-deficient, four independent experiments, Cl. 13), GATA-3 (n = 9 control, n = 12 Ifngr1-deficient, two independent experiments, Arm.; n = 11 control, n = 9 Ifngr1-deficient, two independent experiments, Cl. 13), RORγt (n = 11 control, n = 9 Ifngr1-deficient, two independent experiments, Arm.; n = 12 control, n = 11 Ifngr1-deficient, Cl. 13) (c), CXCR3 (d) (n = 11 control, n = 8 Ifngr1-deficient, uninfected; n = 16, Arm.; n = 15 control, n = 13 Ifngr1-deficient, Cl. 13), IFNγ (e) (n = 13 control, n = 12 Ifngr1-deficient, Arm., n = 11 control, n = 10 Ifngr1-deficient, two independent experiments, Cl. 13), IFNγ and T-bet (f) (n = 8), IL4 (g) (n = 9 control, n = 8 Ifngr1-deficient, Arm.; n = 11 control, n = 9 Ifngr1-deficient, Cl.13), IL5 (upper) (n = 8, two independent experiments), IL13 (lower) (n = 12) (h) and CCR4 (i) (n = 8 control, n = 9 Ifngr1-deficient, two independent experiments) by splenic T_reg cells was measured by flow cytometry. e–h, Cytokine expression was measured after ex vivo stimulation with anti-CD3 (Arm.) or PMA and Ionomycin (Cl. 13). a–i, Data are presented as mean values and represent biologically independent mice and 2 (a,b,f,g,i and as indicated), three (c–e,h) or four (as indicated) independent experiments. Adjusted P values were determined by Wilcoxon rank-sum test (a) or by Kolmogorov–Smirnov test (b), relative to infected Foxp3^Cre-YFP mice. P values were determined by multiple unpaired Student t-tests (c) or by an unpaired two-tailed Student’s t-test (d–i), relative to Foxp3^Cre-YFP mice (P values indicated when significant).

Fig. 4 ∣. IL12 does not induce T_H1-like nor suppress T_H2-like programming during chronic infection.

a, Expression of the deleted region of Il12rb2, normalized to the intact region of Il12rb2, from uninfected Il12rb2^L/LFoxp3^Cre-YFP mice (n = 4) compared to Foxp3^Cre-YFP mice (n = 3). Thy1.2⁻ cells, CD8⁺ T cells, CD4⁺ T_conv cells and T_reg cells were sorted, gDNA was isolated and qPCR was performed. b–f, Foxp3^Cre-YFP (n = 10) or Il12rb2^L/LFoxp3^Cre-YFP (n = 9) mice were infected with Cl. 13 and expression of T-bet, GATA-3 and RORγt (b), CXCR3 (c), IFNγ (d), CCR4 (e) and T_H2 cytokines (IL4, IL5 and IL13) (f) in splenic T_reg cells were measured by flow cytometry. a–f, Data are presented as mean values and represent biologically independent mice and one (a) or two (b–f) independent experiments. Statistical significance was determined by multiple unpaired Student t-tests (a,b) or by an unpaired two-tailed Student’s t-test (c–f), relative to Foxp3^Cre-YFP mice (P values indicated when significant).

Fig. 5 ∣. T_H1-like T_reg cells limit T cell effector function during chronic infection.

a, Percentage of initial body weight of uninfected or Cl. 13-infected Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice over time. The number of mice used is indicated in parentheses. Error bars represent standard error. b–h, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with Cl. 13 or remained uninfected as indicated. ALT activity (b) (n = 12 control, n = 14 Ifngr1-deficient, d8; n = 10 control, n = 9 Ifngr1-deficient, d16) and levels of IFNγ in serum (c) (n = 13 control, n = 11 Ifngr1-deficient, d8; n = 14 control, n = 12 Ifngr1-deficient, d16; n = 14, d30) were determined. d,e, Expression of IFNγ by splenic CD4⁺ T_conv cells (d) (n = 7 control, n = 10 Ifngr1-deficient, d16, two independent experiments; n = 19 control, n = 16 Ifngr1-deficient, d30) and CD8⁺ T cells (e) (n = 8 control, n = 9 Ifngr1-deficient, d16, two independent experiments; n = 19 control, n = 16 Ifngr1-deficient, d30) was measured by flow cytometry after in vitro stimulation with pooled LCMV peptides (GP_33–41, GP_276–286, NP_396–404 and GP_61–80). f, Percentage of splenic LCMV Tetramer⁺ CD8⁺ T was determined (n = 19, control; n = 16, Ifngr1-deficient). g, Number of CD4⁺ T_conv and CD8⁺ T cells per spleen determined by flow cytometry (n = 7). h, Heat map of cytokines, cytokine receptors, migratory and suppressive genes in T_reg cells (n = 3, uninfected; n = 5 Cl. 13). a–h, Data are presented as mean values and represent biologically independent mice and two (g,h, and as indicated) or three (a–f) independent experiments. Adjusted P values were determined by a Wilcoxon rank-sum test (h) relative to infected Foxp3^Cre-YFP mice. P values were determined by a chi-squared test across all time points (a), multiple unpaired Student t-tests (a–c) or unpaired two-tailed Student’s t-test (d–g), relative to Foxp3^Cre-YFP mice. (P values indicated when significant).

Fig. 6 ∣. IFNγ from CD8⁺T cells but not T_reg cells contributes to T_H1 polarization of T_reg cells during chronic infection.

a,b, WT or Ifng^−/− mice were infected with Cl. 13 and expression of T-bet and CXCR3 (a) (n = 8) and GATA-3 (n = 8, two independent experiments), CCR4 (n = 8, two independent experiments), IL4 (n = 15, control; n = 14, Ifng-deficient), IL5 (n = 15, control; n = 14, Ifng-deficient) and IL13 (n = 15, control; n = 14, Ifng-deficient) (b) by splenic T_reg cells were measured by flow cytometry. c−e, Ifng^YFPFoxp3^RFP mice were infected with Cl. 13 for 8 d (n = 8) and 16 d (n = 13) and the percentage of IFNγ⁺ Thy1.2⁺ (c) and CD8⁺ T cells, CD4⁺ T_conv and T_reg cells within the Ifng⁺Thy1.2⁺ cells by percent (d) and number (e) were determined by flow cytometry. f, Expression of the T_H1 markers T-bet (n = 12, control; n = 11, Ifng-deficient) and CXCR3 (n = 8, two independent experiments) in T_reg cells from indicated mice were measured by flow cytometry. g, Expression of GATA-3, CCR4, IL4, IL5 and IL13 by splenic T_reg cells from indicated mice (n = 8) was measured by flow cytometry. h, Expression of the T_H1 markers T-bet (n = 9, control; n = 10, Ifng-deficient; two independent experiments), CXCR3 (n = 12, control; n = 13, Ifng-deficient) and IFNγ (n = 9, control; n = 10, Ifng-deficient; two independent experiments) in T_reg cells from indicated mice was measured by flow cytometry. i, Expression of GATA-3, CCR4, IL5, IL13 (n = 12, control; n = 13, Ifng-deficient) and IL4 (n = 7, two independent experiments) by splenic T_reg cells from indicated mice was measured by flow cytometry. a–i, Data are presented as mean values and represent biologically independent mice and two (a,c–e,g and as indicated) or three (b,f,h,i) independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test relative to WT mice (a,b), d8 (c,d) or Cre control mice (f–i) or by multiple unpaired Student t-tests relative to d8 and two-way ANOVA with multiple comparisons (e) (P values indicated when significant). NS, not significant.

Fig. 7 ∣. T_H1-like T_reg cells limit CD8⁺T memory formation and response to vaccination during acute and chronic infection.

a,b, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with Arm. Percentage of splenic SLECs and MPECS within effector CD8⁺ T cells at 8 dpi (a) (n = 18, control; n = 16, Ifngr1-deficient), and number of splenic pooled tetramer⁺ (GP₃₃, GP₂₇₆ and NP₃₉₆) CD8⁺ T cells (left; n = 18, control; n = 15, Ifngr1-deficient) and T_reg cells (right; n = 11, control; n = 10, Ifngr1-deficient; two independent experiments) at 100 dpi (b) were determined by flow cytometry. c–h, Rag1^−/− mice were reconstituted with Ifngr1^−/− or control (WT) T_reg cells and infected with Arm. c, Experimental schema. d, The frequency and number of T_CM (upper), T_EM (lower) and T_CM-to-T_EM ratio (n = 13, control; n = 12, Ifngr1-deficient), within splenic pooled tetramer⁺ CD8⁺ T cells were determined by flow cytometry. e, Coexpression of Eomes (upper) (n = 13, control; n = 12, Ifngr1-deficient) or T-bet (lower) (n = 10, control; n = 9, Ifngr1-deficient; two independent experiments) with CD27, within splenic pooled tetramer⁺ CD8⁺ T cells, was determined by flow cytometry. f–h, Frequency of CD69⁺ (f), CXCR6⁺CXCR3⁺ (g) and level of Blimp1 expression (h) in pooled tetramer⁺ CD8⁺ T cells (n = 10, control; n = 9, Ifngr1-deficient) from the liver were determined by flow cytometry. i–l, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were infected with LCMV Arm. and rechallenged with LM-GP33 on 35 dpi. i, Experimental schema. j,k, Total number of CD8⁺ T cells (j) and GP33⁺effector CD8⁺ T cells (k), per spleen (n = 16, control; n = 15, Ifngr1-deficient) were determined by flow cytometry. l, Expression of IFNγ by splenic CD8⁺ T cells (n = 13, control; n = 10, Ifngr1-deficient) was measured after in vitro stimulation with GP₃₃ peptide. m–o, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice were vaccinated with LM-GP33 and challenged with Cl. 13 on d34. m, Experimental schema. n, Frequency splenic GP33⁺ effector CD8⁺ T cells (n = 4, control LM-OVA; n = 7, control LM-GP33; n = 5, Ifngr1-deficient LM-OVA; n = 9, Ifngr1-deficient LM-GP33) was determined by flow cytometry. o, Expression of IFNγ by splenic CD8⁺ T cells (n = 4, control LM-OVA; n = 7, control LM-GP33; n = 5 Ifngr1-deficient, LM-OVA; n = 9, Ifngr1-deficient, LM-GP33) was measured after in vitro stimulation with GP₃₃ peptide. a,b,d–h,j–l,n,o, Data are presented as mean values and represent biologically independent mice and two (f–h,l,n,o and as indicated) or three (a,b,d,e,j,k) independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test relative to Foxp3^Cre-YFP mice (a,b) or Rag1^−/− + WT T_reg cells (d–h) or by multiple unpaired Student t-tests relative to Foxp3^Cre-YFP mice (j–l,n,o) (P values indicated when significant). NS, not significant.