Production of IL-10 by CD4+ regulatory T cells during the resolution of infection promotes the maturation of memory CD8+ T cells

Journal name:
Nature Immunology
Volume:
16,
Pages:
871–879
Year published:
DOI:
doi:10.1038/ni.3224
Received
Accepted
Published online

Abstract

Memory CD8+ T cells are critical for host defense upon reexposure to intracellular pathogens. We found that interleukin 10 (IL-10) derived from CD4+ regulatory T cells (Treg cells) was necessary for the maturation of memory CD8+ T cells following acute infection with lymphocytic choriomeningitis virus (LCMV). Treg cell–derived IL-10 was most important during the resolution phase, calming inflammation and the activation state of dendritic cells. Adoptive transfer of IL-10-sufficient Treg cells during the resolution phase 'restored' the maturation of memory CD8+ T cells in IL-10-deficient mice. Our data indicate that Treg cell–derived IL-10 is needed to insulate CD8+ T cells from inflammatory signals, and reveal that the resolution phase of infection is a critical period that influences the quality and function of developing memory CD8+ T cells.

At a glance

Figures

  1. IL-10 is required during the resolution phase of infection to allow optimal maturation of memory CD8+ T cells.
    Figure 1: IL-10 is required during the resolution phase of infection to allow optimal maturation of memory CD8+ T cells.

    (a) Flow cytometry of cells from the spleen of wild-type (WT) and Il10−/− mice 60 d after infection with LCMV (Armstrong strain), assessing the response of T cells positive for the epitope gp(33–41) (gp33). Numbers in plots indicate percent gp33+ T cells (far left) or percent KLRG1CD127+ cells (top left quadrant) or KLRG1+CD127 cells (bottom right quadrant) in the gp33+ T cell population (middle) or KLRG1CD62L+ cells in the gp33+ T cell population (far right). Below, frequency of CD127+KLRG1 cells (left), CD127KLRG1+ cells (middle) or CD62L+KLRG1 cells (right) among gp33+ T cells (assessed on days 45–60). (b) Expression of CD127, KLRG1, CXCR3, CD27, TCF-1 and granzyme B (GzmB) in wild-type and Il10−/− gp33+ T cells from mice as in a. Numbers in plots indicate mean fluorescence intensity (MFI) of marker (horizontal axis) in wild-type cells (top number) or Il10−/− cells (number below). (c) Frequency of CD127+KLRG1, CD127KLRG1+ and CD62L+KLRG1 cells among gp33+ T cells from mice infected with LCMV (Armstrong strain) (day 0) and given mock injection of PBS or injection of antibody to IL-10 (α-IL-10) on days (D) 0–30, 0–8, 8–30 or 15–30 (horizontal axis), assessed at day 30. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (unpaired two-tailed Student's t-test). Data are from one experiment representative of five experiments with at least four mice per group (a,b) or three experiments (c; mean and s.e.m.).

  2. IL-10 is important in insulating CD8+ T cells from inflammatory signals following viral infection.
    Figure 2: IL-10 is important in insulating CD8+ T cells from inflammatory signals following viral infection.

    Flow cytometry of cells from wild-type and Il10−/− mice infected with LCMV (Armstrong strain) and given mock injection of PBS or injection of CpG B at day 8 (D8) or day 15 (D15) after infection, assessed 60 d after infection (numbers in plots (top) as in Fig. 1a, top). Below, summary of results above (gp33+ cells among CD8+ T cells (far left) or as in Fig. 1a, bottom). *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired two-tailed Student's t-test). Data are from one experiment representative of three experiments with at least four mice per group (mean and s.e.m.).

  3. CD4+ Treg cell-derived IL-10 is critical for the maturation of memory CD8+ T cells.
    Figure 3: CD4+ Treg cell–derived IL-10 is critical for the maturation of memory CD8+ T cells.

    (a) Frequency of CD127+KLRG1, CD127KLRG1+ and CD62L+KLRG1 cells in the gp33+ T cell population from Il10f/f, Il10f/fCd4-Cre, Il10f/fLyz2-Cre, and Il10f/fCd11c-Cre mice 60 d after infection with LCMV (Armstrong strain). (b) Flow cytometry (top) of T cells from the spleen of 10BiT IL-10 reporter mice 15 d after infection with LCMV (Armstrong strain). Numbers in quadrants (middle) indicate percent CD62L+CD69 cells (top left), CD62LCD69+ cells (bottom right) or CD62LCD69+ cells (bottom left) among CD25+CD4+ cells gated at left (outlined area); number adjacent to outlined area (right) indicates percent KLRG1+CD69+ cells among CD62LCD69+ cells gated at left (green outlined area). Right, frequency of CD62L+CD69, CD62LCD69 or CD62LCD69+ cells among CD25+CD4+ cells (colors match middle plot at left), or KLRG1+ cells among CD62LCD69+ cells, at days 0, 8 and 15 after infection as above. (c) Expression of IL-10 (assessed as Thy-1.1) by subsets (above plots) of cells from mice as in b (top). Right, frequency of Thy-1.1+ cells (left) and MFI of Thy-1.1 (right) among the Thy-1.1+ cells in those subsets (as in b) at days 0, 8 and 15 after infection as above. (d) Flow cytometry (left) of cells from Il10f/f and Il10f/fFoxp3-Cre mice at 60 d after infection with LCMV (Armstrong strain) (numbers in plots as in Fig. 1a, top). Right, summary of results at left (as in Fig. 1a, bottom). (e) Expression of CD127, KLRG1, CXCR3, CD27, TCF-1 and granzyme B in Il10f/f and Il10f/fFoxp3-Cre gp33+ T cells. Numbers in plots indicate MFI of marker (horizontal axis) in Il10f/f cells (top number) or Il10f/fFoxp3-Cre cells (number below). *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired two-tailed Student's t-test). Data are from one experiment representative of three experiments with three to six mice per group (mean and s.e.m. in ad).

  4. CD4+ Treg cell-derived IL-10 is necessary for the formation of protective memory CD8+ T cells.
    Figure 4: CD4+ Treg cell–derived IL-10 is necessary for the formation of protective memory CD8+ T cells.

    Flow cytometry (left) of splenic T cells from host mice (Ly5.1+) given no cells (None) or 1.5 × 105 gp33+ CD8+ T cells pooled from congenically mismatched (Ly5.2+) Il10f/f and Il10f/fFoxp3-Cre mice 45 d after infection of donor mice with LCMV (Armstrong strain), followed by challenge of the recipient mice with recombinant L. monocytogenes expressing the gp(33–41) epitope 1 d after cell transfer and analysis 4 d after challenge. Numbers adjacent to outlined areas (far left) indicate percent Ly5.2+ (donor) CD44+ cells. Middle, frequency (middle left) and abundance (middle right) of donor CD8+ T cells in the spleen of host mice. Far right, bacterial burden in the liver of host mice, presented as colony-forming units (CFU) per gram of liver. *P < 0.001 and **P < 0.0001 (unpaired two-tailed Student's t-test). Data are from one experiment representative of two experiments with at least four mice per group (mean and s.e.m.).

  5. Virus-specific CD8+ T cells from mice lacking CD4+ Treg cell-derived IL-10 display a robust inflammatory gene signature.
    Figure 5: Virus-specific CD8+ T cells from mice lacking CD4+ Treg cell–derived IL-10 display a robust inflammatory gene signature.

    (a) RNA-seq analysis of selected biologically relevant genes among mRNA isolated from gp33+ CD8+ T cells and CD8+ T cells positive for the epitope nucleoprotein residues 396–404, pooled from Il10f/f and Il10f/fFoxp3-Cre mice 15 d after infection with LCMV (Armstrong strain), presented as expression (log2) in Il10f/fFoxp3-Cre cells relative to that in Il10f/f cells (key below; columns indicate paired replicates); left margin, grouping of genes by signature (key, bottom right), as determined by published gene sets8, 35, 36, 37, 38, 50. (b) GSEA of gene sets from the Molecular Signatures Database of the Broad Institute, showing gene sets with significant enrichment (false-discovery rate, <10−5) and their enrichment score (where a positive score indicates 'enrichment' (higher expression) in the Il10f/fFoxp3-Cre sample relative to that in the Il10f/f sample), with members of the gene set presented in the ranked list of genes ('bar code' below) and the signal-to-noise ranking metric (bar at bottom), assessing effector signatures versus (vs) memory signatures in CD8+ T cells, based on published gene sets35, 36. (c,d) GSEA of genes upregulated (Up) or downregulated (Down) after stimulation with IFN-α and IFN-β (collectively called 'IFN-α/β')) (c) or IL-12 (d), based on published gene sets37; presented as in b. Data are from three independent experiments with three mice per group pooled for each sample.

  6. Enhanced maturation of DCs and inflammation in the absence of Treg cell-derived IL-10.
    Figure 6: Enhanced maturation of DCs and inflammation in the absence of Treg cell–derived IL-10.

    (a) Flow cytometry (top) analyzing the expression of CD86, CD80, PD-L1 and PD-L2 in CD45+CD11chiMHCII+ DCs from Il10f/f and Il10f/fFoxp3-Cre mice 15 d after infection with LCMV (Armstrong strain). Bottom, summary of results above. (b) Immunoassay of IL-6, IL-1β, TNF and IL-12p70 in the serum of Il10f/f and Il10f/fFoxp3-Cre mice left uninfected (day 0) or at day 15 after infection with LCMV. *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired two-tailed Student's t-test). Data are from one experiment representative of three experiments with at three to five mice per group (mean and s.e.m.).

  7. Transfer of IL-10-competent CD4+ Treg cells during the resolution phase of LCMV infection is sufficient to 'rescue' the defect in the maturation of memory CD8+ T cells in Il10-/- mice.
    Figure 7: Transfer of IL-10-competent CD4+ Treg cells during the resolution phase of LCMV infection is sufficient to 'rescue' the defect in the maturation of memory CD8+ T cells in Il10−/− mice.

    (a) Frequency of CD127+KLRG1, CD127KLRG1+ and CD62L+KLRG1 cells in the gp33+ T cell population of Foxp3GFP-DTR mice infected with LCMV (Armstrong strain) and given mock injection of PBS or injection of diphtheria toxin (DT) on day 0 (D0), day 8 (D8) or day 15 (D15) after infection, followed by analysis 60 d after infection. (b) Expression of CD86, CD80, PD-L1 and PD-L2 in DCs from wild-type and Il10−/− host mice infected with LCMV (Armstrong strain) and, 8 d later, given no cells (None) or 3 × 105 Foxp3+ CD4+ Treg cells isolated from 'infection-matched' Foxp3GFP-DTR donor mice (+Treg), followed by analysis 15 d after infection of host mice. (c) Flow cytometry (left) of cells from Il10−/− mice as in b, analyzed 60 d after infection of host mice. Numbers adjacent to outlined areas indicate percent gp33+ T cells (far left); numbers in quadrants (near left) indicate percent KLRG1CD127+ cells (bottom right) or KLRG1+CD127 cells (top left) in the gp33+ T cell population. Right, frequency of CD127+KLRG1cells or CD127KLRG1+ cells among gp33+ T cells. (d) Expression of CD127, KLRG1, TCF-1 and granzyme B in gp33+ T cells from Il10−/− mice as in b. Numbers in plots indicate MFI of marker (horizontal axis) in cells from Il10−/− mice given no cells (top number) or given CD4+ Treg cells (number below). *P < 0.05; **P < 0.01 and ***P < 0.001 (unpaired two-tailed Student's t-test). Data are from one experiment representative of three experiments (a) or two experiments (bd) with three to seven mice per group (mean and s.e.m.).

  8. IL-10 is required for optimal maturation of memory CD8+ NP396+ T cells.
    Supplementary Fig. 1: IL-10 is required for optimal maturation of memory CD8+ NP396+ T cells.

    (a) Analysis of the virus-specific CD8+ T cell response 60 days post acute LCMV Armstrong infection. The percentages and numbers of GP33+ T cells along with the (b) representative plots and analysis of the NP396+ T cell response in the spleen of wild type and Il10–/– mice are shown. Percentages and numbers of NP396+ T cells, along with the percentages of CD127+KLRG1-, CD127-KLRG1+, and CD62L+KLRG1- cells in the NP396+ T cell population are shown. (c) Mice were infected with acute LCMV and treated with αIL-10 between days 0-30, 0-8, 8-30, 15-30 or mock injected with PBS. Mice were sacrificed at day 30 and the percentage and numbers of GP33+ T cells determined. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. (*, p < 0.01; **, p < 0.001). Data are from one experiment representative of 5 experiments (a, b) or 3 experiments (c) with at least 4 mice per group carried out 45-60 days (a, b) or 30 days (c) following LCMV Armstrong infection (mean and s.e.m).

  9. IL-10 is required in a CD8+ T cell-extrinsic and CD8+ T cell-intrinsic manner to allow the maturation and survival of memory CD8+ T cells.
    Supplementary Fig. 2: IL-10 is required in a CD8+ T cell–extrinsic and CD8+ T cell–intrinsic manner to allow the maturation and survival of memory CD8+ T cells.

    50,000 Il10raf/f or Il10raf/fCd4-Cre P14 GP33+ cells were transferred into congenically-mismatched mice one day prior to infection with acute LCMV infection. Analysis of the P14 GP33+ T cell response was carried out 30 days p.i. (a) Representative plots and analysis of the P14 GP33+ T cell response. Percentages and numbers of P14 GP33+ T cells, along with the percentages of CD127+KLRG1-, CD127-KLRG1+, and CD62L+KLRG1- cells in the P14 GP33+ T cell population are shown. (b) Representative histograms of GzmB and Tcf1 expression in Il10raf/f (black) or Il10raf/fCd4-Cre (gray filled) P14 GP33+ cells. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. (*, p < 0.05; **, p < 0.01). Data are from one experiment representative of 2 experiments with 4 mice per group carried out 30 days following LCMV Armstrong infection (mean and s.e.m).

  10. CD4+ Treg cells continue to produce IL-10 during the resolution phase of infection.
    Supplementary Fig. 3: CD4+ Treg cells continue to produce IL-10 during the resolution phase of infection.

    (a) Analysis of the GP33+ T cell response 60 days post acute LCMV infection in Il10f/f, Il10f/fCd4-Cre, Il10f/fLyz2-Cre, and Il10f/fCd11c-Cre mice. Percentages and numbers of GP33+ T cells were determined. (b) Analysis of IL-10 production by T cells following acute LCMV infection in IL-10 reporter (10BiT Thy1.1 mice). Percentages and numbers of IL-10-Thy1.1 reporter-positive cells at multiple time points post LCMV infection are shown. (c) Intravenously (i.v.) administered anti-CD4 antibody was used to distinguish circulating (red pulp localized) versus resident (white pulp localized) CD4+ T cells. (d) Analysis of the GP33+ T cell response 60 days post acute LCMV Armstrong infection. Percentages and numbers of GP33+ T cells in Il10f/f and Il10f/f Foxp3-Cre mice are shown. Data are from one experiment representative of 3 experiments (a, b, c, d) with 3-6 mice per group carried out 45-60 days (a, d), or 0, 8, and 15 days (b, c) following LCMV Armstrong infection (mean and s.e.m).

  11. Validation of RNA-seq results.
    Supplementary Fig. 4: Validation of RNA-seq results.

    (a) Expression of Zeb2, Ccr7, Cx3cr1, and Pim1 was determined by qPCR analysis of cDNA isolated from pooled GP33+ and NP396+ CD8+ T cells at 15 days post acute LCMV infection from Il10f/f and Il10f/f Foxp3-Cre mice. Expression of TCF-1 and GzmB in GP33+ in T cells at 15 days post acute LCMV infection from Il10f/f and Il10f/f Foxp3-Cre mice was determined by flow cytometric analysis. (b) Representative plots of the GP33+ T cell response in Il10f/f and Il10f/f Foxp3Cre mice at day 15 post LCMV infection. Data are from one experiment representative of 3 experiments with 3-5 mice per group carried out 15 days following LCMV Armstrong infection (mean and s.e.m).

  12. Heat map of differentially expressed genes based on RNA-seq results.
    Supplementary Fig. 5: Heat map of differentially expressed genes based on RNA-seq results.

    Genes with a p-adjusted value < 0.2 (Benjami-Hochberg) and the corresponding log2 fold-change in mRNA isolated from pooled GP33+ and NP396+ CD8+ T cells at 15 days post acute LCMV infection from Il10f/f and Il10f/f Foxp3-Cre mice.

  13. Virus-specific CD8+ T cells from mice lacking CD4+ Treg cell-derived IL-10 display a robust inflammatory and effector gene signature.
    Supplementary Fig. 6: Virus-specific CD8+ T cells from mice lacking CD4+ Treg cell–derived IL-10 display a robust inflammatory and effector gene signature.

    mRNA was isolated from pooled GP33+ and NP396+ CD8+ T cells at 15 days post acute LCMV infection from Il10f/f and Il10f/f Foxp3-Cre mice and compared by RNA-seq. (a) Gene set plots showing individual log2 fold-changes of with corresponding standard error based on published effector vs memory gene set. Gene Set Enrichment Analysis (GSEA) was performed using gene sets from the Broad MSigDB collection; select significantly enriched gene sets (FDR < 1e-5) are shown with their running Enrichment Score (ES) (line), where members of the gene set appear in the ranked list of genes (barcode), and the signal to noise ranking metric (bar). A positive ES signifies enrichment in the Il10f/f Foxp3-Cre sample relative to the WT condition of a given gene set; i.e., more highly expressed. (b) GSEA results of CpG (c) and poly:IC stimulated genes (bottom) were visualized. (d) Normalized enrichment scores for Gene Set Enrichment Analysis. Normalized enrichment scores (NES) was calculated for select significantly enriched gene sets (FDR < 1e-5). Gene set name, figure GSEA plots shown in, and NES are shown in table.

  14. Transfer of IL-10-competent CD4+ Treg cells during the resolution phase of LCMV infection is sufficient to /`rescue/' the maturation defect of memory CD8+ T cells in Il10-/- mice.
    Supplementary Fig. 7: Transfer of IL-10-competent CD4+ Treg cells during the resolution phase of LCMV infection is sufficient to ‘rescue’ the maturation defect of memory CD8+ T cells in Il10–/– mice.

    Analysis of the GP33+ T cell response 60 days p.i. in Foxp3GFP-DTR mice treated with diphtheria toxin at day -1, day 8, or day 15 p.i. or mock injected with PBS. (a) Percentage and numbers of GP33+ T cells are shown. Representative of 3 independent experiments with 3-6 mice per group carried out 45-60 days following LCMV Armstrong infection. (b) Analysis of the GP33+ T cell response 60 days post acute LCMV Armstrong infection in Il10–/– mice and Il10–/– mice that were administered 3x105 Foxp3+ CD4+ T cells isolated from coinfected Foxp3GFP-DTR mice at day 8 p.i. Percentage and numbers of GP33+ T cells are shown. Representative of 2 independent experiments with 3-7 mice per group carried out 60 days following LCMV Armstrong infection. Data are from one experiment representative of 3 experiments (a) or 2 experiments (b) with 3-7 mice per group carried out 45-60 days following LCMV Armstrong infection (mean and s.e.m).

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Author information

  1. Present address: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin, USA.

    • Weiguo Cui
  2. These authors jointly directed this work.

    • Joe Craft &
    • Susan M Kaech

Affiliations

  1. Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut, USA.

    • Brian J Laidlaw,
    • Weiguo Cui,
    • Robert A Amezquita,
    • Simon M Gray,
    • Tianxia Guan,
    • Yisi Lu,
    • Yasushi Kobayashi,
    • Richard A Flavell,
    • Steven H Kleinstein,
    • Joe Craft &
    • Susan M Kaech
  2. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.

    • Robert A Amezquita,
    • Richard A Flavell &
    • Susan M Kaech
  3. Department of Pathology, Yale School of Medicine, New Haven, Connecticut, USA.

    • Steven H Kleinstein
  4. Interdepartmental Program in Computational Biology and Bioinformatics, Yale School of Medicine, New Haven, Connecticut, USA.

    • Steven H Kleinstein
  5. Department of Internal Medicine (Rheumatology), Yale School of Medicine, New Haven, Connecticut, USA.

    • Joe Craft

Contributions

B.J.L., J.C., S.M.K. conceived of and designed the experiments, analyzed the data and wrote the manuscript; and B.J.L., W.C., R.A.A., S.M.G., T.G., Y.L., Y.K., S.H.K. and R.A.F. performed the experiments.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: IL-10 is required for optimal maturation of memory CD8+ NP396+ T cells. (77 KB)

    (a) Analysis of the virus-specific CD8+ T cell response 60 days post acute LCMV Armstrong infection. The percentages and numbers of GP33+ T cells along with the (b) representative plots and analysis of the NP396+ T cell response in the spleen of wild type and Il10–/– mice are shown. Percentages and numbers of NP396+ T cells, along with the percentages of CD127+KLRG1-, CD127-KLRG1+, and CD62L+KLRG1- cells in the NP396+ T cell population are shown. (c) Mice were infected with acute LCMV and treated with αIL-10 between days 0-30, 0-8, 8-30, 15-30 or mock injected with PBS. Mice were sacrificed at day 30 and the percentage and numbers of GP33+ T cells determined. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. (*, p < 0.01; **, p < 0.001). Data are from one experiment representative of 5 experiments (a, b) or 3 experiments (c) with at least 4 mice per group carried out 45-60 days (a, b) or 30 days (c) following LCMV Armstrong infection (mean and s.e.m).

  2. Supplementary Figure 2: IL-10 is required in a CD8+ T cell–extrinsic and CD8+ T cell–intrinsic manner to allow the maturation and survival of memory CD8+ T cells. (69 KB)

    50,000 Il10raf/f or Il10raf/fCd4-Cre P14 GP33+ cells were transferred into congenically-mismatched mice one day prior to infection with acute LCMV infection. Analysis of the P14 GP33+ T cell response was carried out 30 days p.i. (a) Representative plots and analysis of the P14 GP33+ T cell response. Percentages and numbers of P14 GP33+ T cells, along with the percentages of CD127+KLRG1-, CD127-KLRG1+, and CD62L+KLRG1- cells in the P14 GP33+ T cell population are shown. (b) Representative histograms of GzmB and Tcf1 expression in Il10raf/f (black) or Il10raf/fCd4-Cre (gray filled) P14 GP33+ cells. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. (*, p < 0.05; **, p < 0.01). Data are from one experiment representative of 2 experiments with 4 mice per group carried out 30 days following LCMV Armstrong infection (mean and s.e.m).

  3. Supplementary Figure 3: CD4+ Treg cells continue to produce IL-10 during the resolution phase of infection. (49 KB)

    (a) Analysis of the GP33+ T cell response 60 days post acute LCMV infection in Il10f/f, Il10f/fCd4-Cre, Il10f/fLyz2-Cre, and Il10f/fCd11c-Cre mice. Percentages and numbers of GP33+ T cells were determined. (b) Analysis of IL-10 production by T cells following acute LCMV infection in IL-10 reporter (10BiT Thy1.1 mice). Percentages and numbers of IL-10-Thy1.1 reporter-positive cells at multiple time points post LCMV infection are shown. (c) Intravenously (i.v.) administered anti-CD4 antibody was used to distinguish circulating (red pulp localized) versus resident (white pulp localized) CD4+ T cells. (d) Analysis of the GP33+ T cell response 60 days post acute LCMV Armstrong infection. Percentages and numbers of GP33+ T cells in Il10f/f and Il10f/f Foxp3-Cre mice are shown. Data are from one experiment representative of 3 experiments (a, b, c, d) with 3-6 mice per group carried out 45-60 days (a, d), or 0, 8, and 15 days (b, c) following LCMV Armstrong infection (mean and s.e.m).

  4. Supplementary Figure 4: Validation of RNA-seq results. (65 KB)

    (a) Expression of Zeb2, Ccr7, Cx3cr1, and Pim1 was determined by qPCR analysis of cDNA isolated from pooled GP33+ and NP396+ CD8+ T cells at 15 days post acute LCMV infection from Il10f/f and Il10f/f Foxp3-Cre mice. Expression of TCF-1 and GzmB in GP33+ in T cells at 15 days post acute LCMV infection from Il10f/f and Il10f/f Foxp3-Cre mice was determined by flow cytometric analysis. (b) Representative plots of the GP33+ T cell response in Il10f/f and Il10f/f Foxp3Cre mice at day 15 post LCMV infection. Data are from one experiment representative of 3 experiments with 3-5 mice per group carried out 15 days following LCMV Armstrong infection (mean and s.e.m).

  5. Supplementary Figure 5: Heat map of differentially expressed genes based on RNA-seq results. (105 KB)

    Genes with a p-adjusted value < 0.2 (Benjami-Hochberg) and the corresponding log2 fold-change in mRNA isolated from pooled GP33+ and NP396+ CD8+ T cells at 15 days post acute LCMV infection from Il10f/f and Il10f/f Foxp3-Cre mice.

  6. Supplementary Figure 6: Virus-specific CD8+ T cells from mice lacking CD4+ Treg cell–derived IL-10 display a robust inflammatory and effector gene signature. (154 KB)

    mRNA was isolated from pooled GP33+ and NP396+ CD8+ T cells at 15 days post acute LCMV infection from Il10f/f and Il10f/f Foxp3-Cre mice and compared by RNA-seq. (a) Gene set plots showing individual log2 fold-changes of with corresponding standard error based on published effector vs memory gene set. Gene Set Enrichment Analysis (GSEA) was performed using gene sets from the Broad MSigDB collection; select significantly enriched gene sets (FDR < 1e-5) are shown with their running Enrichment Score (ES) (line), where members of the gene set appear in the ranked list of genes (barcode), and the signal to noise ranking metric (bar). A positive ES signifies enrichment in the Il10f/f Foxp3-Cre sample relative to the WT condition of a given gene set; i.e., more highly expressed. (b) GSEA results of CpG (c) and poly:IC stimulated genes (bottom) were visualized. (d) Normalized enrichment scores for Gene Set Enrichment Analysis. Normalized enrichment scores (NES) was calculated for select significantly enriched gene sets (FDR < 1e-5). Gene set name, figure GSEA plots shown in, and NES are shown in table.

  7. Supplementary Figure 7: Transfer of IL-10-competent CD4+ Treg cells during the resolution phase of LCMV infection is sufficient to ‘rescue’ the maturation defect of memory CD8+ T cells in Il10–/– mice. (21 KB)

    Analysis of the GP33+ T cell response 60 days p.i. in Foxp3GFP-DTR mice treated with diphtheria toxin at day -1, day 8, or day 15 p.i. or mock injected with PBS. (a) Percentage and numbers of GP33+ T cells are shown. Representative of 3 independent experiments with 3-6 mice per group carried out 45-60 days following LCMV Armstrong infection. (b) Analysis of the GP33+ T cell response 60 days post acute LCMV Armstrong infection in Il10–/– mice and Il10–/– mice that were administered 3x105 Foxp3+ CD4+ T cells isolated from coinfected Foxp3GFP-DTR mice at day 8 p.i. Percentage and numbers of GP33+ T cells are shown. Representative of 2 independent experiments with 3-7 mice per group carried out 60 days following LCMV Armstrong infection. Data are from one experiment representative of 3 experiments (a) or 2 experiments (b) with 3-7 mice per group carried out 45-60 days following LCMV Armstrong infection (mean and s.e.m).

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    Supplementary Figures 1–7

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