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GITR intrinsically sustains early type 1 and late follicular helper CD4 T cell accumulation to control a chronic viral infection.

Clouthier DL, Zhou AC, Wortzman ME, Luft O, Levy GA, Watts TH - PLoS Pathog. (2015)

Bottom Line: While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection.GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i.GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during chronic infection remain incompletely defined. While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection. Here we show that during a persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13, mice lacking the glucocorticoid-induced tumor necrosis factor receptor related protein (GITR) exhibit defective CD8 T cell accumulation, increased T cell exhaustion and impaired viral control. Differences in CD8 T cells and viral control between GITR+/+ and GITR-/- mice were lost when CD4 T cells were depleted. Moreover, mixed bone marrow chimeric mice, as well as transfer of LCMV epitope-specific CD4 or CD8 T cells, demonstrated that these effects of GITR are largely CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IFNγ+IL-2+ Th1 cells, facilitating CD8 T cell expansion and early viral control. GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CD8 T cells to between days four and eight p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.

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GITR co-stimulation activates classical NF-κB and the Akt-mTORC1 signaling axis to regulate CD4 T cell accumulation post-priming.(A, B) C57BL/6 mice received a 1:1 mixture of GITR+/+ and GITR-/- SMARTA, and were infected the following day with LCMV cl 13. At day eight p.i., proportions of GITR+ and GITR- cells were evaluated, with gating strategy shown in B. (C) 106 or (D) 104 GITR+/+ and GITR-/- CFSE-labeled CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice one day prior to LCMV cl 13 infection. The total numbers of GITR+ and GITR- SMARTA cells in the spleen at different time points following LCMV cl 13 infection are shown. Each symbol in C shows mean ± SEM of at least two to three mice per group, representative of at least two experiments per time point. (E) Proportions of GITR+ and GITR- of: total, CD44hi, T-bet+ Th1, and Tfh SMARTA were evaluated in the spleen at day eight p.i. (F) The proportions of IFNγ+ or IFNγ+IL-2+, and the quantity of IFNγ produced per cell were evaluated in GITR+ and GITR- SMARTA CD4 T cells following five hours of GP61–80 peptide restimulation, with representative staining shown in G. (H) 106 GITR+/+ and GITR-/- CFSE-labeled CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice. At days three and five p.i., CFSE dilution was evaluated. (I) 106 GITR+/+ and GITR-/- CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice as in A. At day three post-infection, cells were stained directly ex vivo for (p) p65 NF-κB pSer529 and (p)S6 ribosomal protein pSer235/236. (J) Activated GITR+/+ SMARTA were expanded for two days and then serum starved for 12 hours prior to engaging with 10μg/mL anti-GITR (cone DTA-1) or Rat IgG, followed by evaluation of phosphorylation at Thr308 of Akt. MFI of pThr308 following stimulation is shown, representative of three independent experiments. Numbers adjacent to the representative histograms of I and J are median MFI values. Each symbol in D–G represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two experiments with a total of eight mice. Data in H show a single representative experiment with two-three mice per group, with at least two independent repeats. Data in I are pooled from two independent experiments with a total of seven mice.
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ppat.1004517.g007: GITR co-stimulation activates classical NF-κB and the Akt-mTORC1 signaling axis to regulate CD4 T cell accumulation post-priming.(A, B) C57BL/6 mice received a 1:1 mixture of GITR+/+ and GITR-/- SMARTA, and were infected the following day with LCMV cl 13. At day eight p.i., proportions of GITR+ and GITR- cells were evaluated, with gating strategy shown in B. (C) 106 or (D) 104 GITR+/+ and GITR-/- CFSE-labeled CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice one day prior to LCMV cl 13 infection. The total numbers of GITR+ and GITR- SMARTA cells in the spleen at different time points following LCMV cl 13 infection are shown. Each symbol in C shows mean ± SEM of at least two to three mice per group, representative of at least two experiments per time point. (E) Proportions of GITR+ and GITR- of: total, CD44hi, T-bet+ Th1, and Tfh SMARTA were evaluated in the spleen at day eight p.i. (F) The proportions of IFNγ+ or IFNγ+IL-2+, and the quantity of IFNγ produced per cell were evaluated in GITR+ and GITR- SMARTA CD4 T cells following five hours of GP61–80 peptide restimulation, with representative staining shown in G. (H) 106 GITR+/+ and GITR-/- CFSE-labeled CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice. At days three and five p.i., CFSE dilution was evaluated. (I) 106 GITR+/+ and GITR-/- CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice as in A. At day three post-infection, cells were stained directly ex vivo for (p) p65 NF-κB pSer529 and (p)S6 ribosomal protein pSer235/236. (J) Activated GITR+/+ SMARTA were expanded for two days and then serum starved for 12 hours prior to engaging with 10μg/mL anti-GITR (cone DTA-1) or Rat IgG, followed by evaluation of phosphorylation at Thr308 of Akt. MFI of pThr308 following stimulation is shown, representative of three independent experiments. Numbers adjacent to the representative histograms of I and J are median MFI values. Each symbol in D–G represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two experiments with a total of eight mice. Data in H show a single representative experiment with two-three mice per group, with at least two independent repeats. Data in I are pooled from two independent experiments with a total of seven mice.

Mentions: To distinguish between the effects of GITR on CD4 T cell accumulation versus differentiation, we transferred a 1:1 mixture of GITR+/+:GITR-/- TCR transgenic SMARTA cells one day prior to LCMV cl 13 infection (Fig. 7A). We used the congenic marker CD45.1 and GITR expression to identify GITR+/+ and GITR-/- SMARTA cells within the same mouse (Fig. 7B). Differences in GITR+/+ and GITR-/- SMARTA accumulation showed a trend starting at day five p.i., with significant differences by day eight p.i., with a 3.2:1 ratio (Fig. 7, C and D). The ratio of GITR+/+:GITR-/- CD44hi, Tfh, and Th1 SMARTA cells was uniformly 3:1, indicating that GITR is not skewing the differentiation of particular CD4 effector T cell sub-populations, rather it contributes to the accumulation of all CD4 T cells (Fig. 7E). While there was a lower frequency of GITR- CD4 T cells producing IFNγ after GP61–80 peptide restimulation, GITR+ and GITR- IFNγ+ SMARTA produced a similar amount of IFNγ per cell. There was also a consistent trend toward fewer GITR- IL-2+ IFNγ+ SMARTA cells relative to the GITR+ SMARTA (Fig. 7, F and G).


GITR intrinsically sustains early type 1 and late follicular helper CD4 T cell accumulation to control a chronic viral infection.

Clouthier DL, Zhou AC, Wortzman ME, Luft O, Levy GA, Watts TH - PLoS Pathog. (2015)

GITR co-stimulation activates classical NF-κB and the Akt-mTORC1 signaling axis to regulate CD4 T cell accumulation post-priming.(A, B) C57BL/6 mice received a 1:1 mixture of GITR+/+ and GITR-/- SMARTA, and were infected the following day with LCMV cl 13. At day eight p.i., proportions of GITR+ and GITR- cells were evaluated, with gating strategy shown in B. (C) 106 or (D) 104 GITR+/+ and GITR-/- CFSE-labeled CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice one day prior to LCMV cl 13 infection. The total numbers of GITR+ and GITR- SMARTA cells in the spleen at different time points following LCMV cl 13 infection are shown. Each symbol in C shows mean ± SEM of at least two to three mice per group, representative of at least two experiments per time point. (E) Proportions of GITR+ and GITR- of: total, CD44hi, T-bet+ Th1, and Tfh SMARTA were evaluated in the spleen at day eight p.i. (F) The proportions of IFNγ+ or IFNγ+IL-2+, and the quantity of IFNγ produced per cell were evaluated in GITR+ and GITR- SMARTA CD4 T cells following five hours of GP61–80 peptide restimulation, with representative staining shown in G. (H) 106 GITR+/+ and GITR-/- CFSE-labeled CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice. At days three and five p.i., CFSE dilution was evaluated. (I) 106 GITR+/+ and GITR-/- CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice as in A. At day three post-infection, cells were stained directly ex vivo for (p) p65 NF-κB pSer529 and (p)S6 ribosomal protein pSer235/236. (J) Activated GITR+/+ SMARTA were expanded for two days and then serum starved for 12 hours prior to engaging with 10μg/mL anti-GITR (cone DTA-1) or Rat IgG, followed by evaluation of phosphorylation at Thr308 of Akt. MFI of pThr308 following stimulation is shown, representative of three independent experiments. Numbers adjacent to the representative histograms of I and J are median MFI values. Each symbol in D–G represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two experiments with a total of eight mice. Data in H show a single representative experiment with two-three mice per group, with at least two independent repeats. Data in I are pooled from two independent experiments with a total of seven mice.
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Related In: Results  -  Collection

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Show All Figures
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ppat.1004517.g007: GITR co-stimulation activates classical NF-κB and the Akt-mTORC1 signaling axis to regulate CD4 T cell accumulation post-priming.(A, B) C57BL/6 mice received a 1:1 mixture of GITR+/+ and GITR-/- SMARTA, and were infected the following day with LCMV cl 13. At day eight p.i., proportions of GITR+ and GITR- cells were evaluated, with gating strategy shown in B. (C) 106 or (D) 104 GITR+/+ and GITR-/- CFSE-labeled CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice one day prior to LCMV cl 13 infection. The total numbers of GITR+ and GITR- SMARTA cells in the spleen at different time points following LCMV cl 13 infection are shown. Each symbol in C shows mean ± SEM of at least two to three mice per group, representative of at least two experiments per time point. (E) Proportions of GITR+ and GITR- of: total, CD44hi, T-bet+ Th1, and Tfh SMARTA were evaluated in the spleen at day eight p.i. (F) The proportions of IFNγ+ or IFNγ+IL-2+, and the quantity of IFNγ produced per cell were evaluated in GITR+ and GITR- SMARTA CD4 T cells following five hours of GP61–80 peptide restimulation, with representative staining shown in G. (H) 106 GITR+/+ and GITR-/- CFSE-labeled CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice. At days three and five p.i., CFSE dilution was evaluated. (I) 106 GITR+/+ and GITR-/- CD45.1+ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice as in A. At day three post-infection, cells were stained directly ex vivo for (p) p65 NF-κB pSer529 and (p)S6 ribosomal protein pSer235/236. (J) Activated GITR+/+ SMARTA were expanded for two days and then serum starved for 12 hours prior to engaging with 10μg/mL anti-GITR (cone DTA-1) or Rat IgG, followed by evaluation of phosphorylation at Thr308 of Akt. MFI of pThr308 following stimulation is shown, representative of three independent experiments. Numbers adjacent to the representative histograms of I and J are median MFI values. Each symbol in D–G represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two experiments with a total of eight mice. Data in H show a single representative experiment with two-three mice per group, with at least two independent repeats. Data in I are pooled from two independent experiments with a total of seven mice.
Mentions: To distinguish between the effects of GITR on CD4 T cell accumulation versus differentiation, we transferred a 1:1 mixture of GITR+/+:GITR-/- TCR transgenic SMARTA cells one day prior to LCMV cl 13 infection (Fig. 7A). We used the congenic marker CD45.1 and GITR expression to identify GITR+/+ and GITR-/- SMARTA cells within the same mouse (Fig. 7B). Differences in GITR+/+ and GITR-/- SMARTA accumulation showed a trend starting at day five p.i., with significant differences by day eight p.i., with a 3.2:1 ratio (Fig. 7, C and D). The ratio of GITR+/+:GITR-/- CD44hi, Tfh, and Th1 SMARTA cells was uniformly 3:1, indicating that GITR is not skewing the differentiation of particular CD4 effector T cell sub-populations, rather it contributes to the accumulation of all CD4 T cells (Fig. 7E). While there was a lower frequency of GITR- CD4 T cells producing IFNγ after GP61–80 peptide restimulation, GITR+ and GITR- IFNγ+ SMARTA produced a similar amount of IFNγ per cell. There was also a consistent trend toward fewer GITR- IL-2+ IFNγ+ SMARTA cells relative to the GITR+ SMARTA (Fig. 7, F and G).

Bottom Line: While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection.GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i.GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during chronic infection remain incompletely defined. While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection. Here we show that during a persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13, mice lacking the glucocorticoid-induced tumor necrosis factor receptor related protein (GITR) exhibit defective CD8 T cell accumulation, increased T cell exhaustion and impaired viral control. Differences in CD8 T cells and viral control between GITR+/+ and GITR-/- mice were lost when CD4 T cells were depleted. Moreover, mixed bone marrow chimeric mice, as well as transfer of LCMV epitope-specific CD4 or CD8 T cells, demonstrated that these effects of GITR are largely CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IFNγ+IL-2+ Th1 cells, facilitating CD8 T cell expansion and early viral control. GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CD8 T cells to between days four and eight p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.

Show MeSH
Related in: MedlinePlus