Regulatory T cell suppressive potency dictates the balance between bacterial proliferation and clearance during persistent Salmonella infection

Abstract

The pathogenesis of persistent infection is dictated by the balance between opposing immune activation and suppression signals. Herein, virulent Salmonella was used to explore the role and potential importance of Foxp3-expressing regulatory T cells in dictating the natural progression of persistent bacterial infection. Two distinct phases of persistent Salmonella infection are identified. In the first 3-4 weeks after infection, progressively increasing bacterial burden was associated with delayed effector T cell activation. Reciprocally, at later time points after infection, reductions in bacterial burden were associated with robust effector T cell activation. Using Foxp3(GFP) reporter mice for ex vivo isolation of regulatory T cells, we demonstrate that the dichotomy in infection tempo between early and late time points is directly paralleled by drastic changes in Foxp3(+) Treg suppressive potency. In complementary experiments using Foxp3(DTR) mice, the significance of these shifts in Treg suppressive potency on infection outcome was verified by enumerating the relative impacts of regulatory T cell ablation on bacterial burden and effector T cell activation at early and late time points during persistent Salmonella infection. Moreover, Treg expression of CTLA-4 directly paralleled changes in suppressive potency, and the relative effects of Treg ablation could be largely recapitulated by CTLA-4 in vivo blockade. Together, these results demonstrate that dynamic regulation of Treg suppressive potency dictates the course of persistent bacterial infection.

Figure 1. Tempo of persistent Salmonella infection in F1 129SvJ X C57BL/6 mice.

A. Recoverable CFUs at the indicated time points after infection from the spleen (top) and liver (bottom) after infection with 10⁴ S. enterica serotype Typhimurium (strain SL1344) in F1 129SvJ X C57BL/6 (left) or 10² in C57BL/6 (right) mice. †, all mice died or were moribund. B. Spleen size in F1 129SvJ X C57BL/6 mice at the indicated time points after infection. Absolute number of splenocyte cells (C) and percent CD4⁺ and CD8⁺ cells (D) in F1 129SvJ X C57BL/6 mice at the indicated time points after infection. These data reflect eight to ten mice per time point representative of three independent experiments. Bar, standard error.

Figure 2. T cell activation kinetics during persistent Salmonella infection.

A. Percent CD44^hiCD62L^lo cells among CD4⁺ and CD8⁺ T cells at the indicated time points after infection with 10⁴ S. enterica serotype Typhimurium in F1 129SvJ X C57BL/6 mice. B. Percent IFN-γ producing CD4⁺ and CD8⁺ T cells after Salmonella infection and ex vivo stimulation with anti-CD3/CD28 antibody (black histogram) or no stimulation control (shaded histogram) at the indicated time points after infection. C. Expression levels of CD25 (top) and CD69 (bottom) by CD4⁺ and CD8⁺ T cells at the indicated time points post-infection (black histograms) compared to naïve F1 control mice (shaded histograms). These data reflect eight to ten mice per time point representative of three independent experiments.

Figure 3. CD4⁺ T cells are required for reductions in Salmonella pathogen burden during persistent infection.

A. Percent CD4⁺ and CD8⁺ T cells 14 days after treatment with each indicated antibody in mice beginning day 31 after Salmonella infection. B. Recoverable Salmonella CFUs in the spleen (left) and liver (right) for mice treated with each antibody for six days (31+6) or 14 days (31+14). These data reflect six to twelve mice per time point representative of three independent experiments each with similar results. Bar, standard error. *, p<0.05; **, p<0.001.

Figure 4. Parallel expansion of Foxp3⁺ and Foxp3-negative CD4⁺ T cells during persistent Salmonella infection.

Representative FACS plots (A) and composite data (B) indicating percent Foxp3⁺ cells among CD4⁺ T cells at the indicated time points after infection with 10⁴ Salmonella in F1 129SvJ X C57BL/6 mice. C. Total numbers of Foxp3⁺CD4⁺ Tregs and Foxp3-negative non-Treg CD4⁺ T cells among splenocytes during persistent infection. These data reflect six to eight mice per time point representative of three independent experiments each with similar results. Bar, standard error.

Figure 5. Dynamic regulation of Treg suppressive potency during persistent Salmonella infection.

A. Percent Foxp3⁺ (left) and GFP⁺ (right) cells among CD4⁺ T cells from F1 and F1 Foxp3 ^GFP/− mice. B. Expression of Foxp3⁺ after cell sorting for GFP⁺CD4⁺ cells from F1 Foxp3 ^GFP/− mice at the indicated time points after infection. C. Percent CFSE^lo cells among CD45.1⁺CD4⁺ responder T cells (Tresp) after co-culture with the indicated ratio of GFP⁺(Foxp3⁺) Tregs isolated from mice at each time point after infection and stimulation with anti-CD3/CD28 (line histogram) or no stimulation (shaded histogram) (top). Relative suppression of CFSE dilution in CD45.1⁺CD4⁺ responder T cells by GFP⁺(Foxp3⁺) Tregs isolated at each time point after infection normalized to the suppression conferred by Tregs from naïve mice co-cultured with responder T cells at a 1∶1 ratio (dotted line) (bottom). These data are representative of three independent experiments each with similar results.

Figure 6. Shifts in Treg-mediated in vivo suppression during persistent Salmonella infection.

A. Representative FACS plots demonstrating the efficiency whereby Foxp3⁺ Tregs are ablated with DT treatment in F1 Foxp3 ^DTR compared with F1 Foxp3 ^WT (F1) control mice. The numbers indicate the percent Foxp3-expressing among CD4⁺ T cells after DT treatment. B. Representative FACS plots demonstrating percent (top) CD90.1⁺ OT-1 T cells among CD8⁺ splenocytes and total number (bottom) of CD90.1⁺CD8⁺ splenocytes in Treg-sufficient (F1) or Treg-ablated (F1 Foxp3 ^DTR) mice day 5 after injection of OVA_257–264 peptide or no peptide controls. C. Representative FACS plots (top) demonstrating percent CD90.1⁺ OT-I T cells among CD8⁺ splenocytes and composite data (bottom) depicting percent and total number CD90.1⁺CD8⁺ splenocytes after adoptive transfer into mice at the indicated time points after Salmonella infection and peptide stimulation. These data represent six to ten mice per group combined from three independent experiments each with similar results. Bar, standard error. **, p<0.01.

Figure 7. Expression of Treg-associated effector molecules during persistent Salmonella infection.

A. The relative expression of CTLA-4 and GITR on Foxp3⁺ Tregs (line histogram) or Foxp3-negative CD4⁺ T cells (shaded histogram) at the indicated time points during persistent infection. B. Expansion of Salmonella FliC_431–439-specific CD4⁺ T cells, and Foxp3-expression among these cells after staining with FliC:I-A^b tetramer and magnetic bead enrichment. The numbers in each plot represent the average cell number and percent Foxp3⁺ cells from 12 mice per time point combined from four independent experiments. C. Expression of CTLA-4 or GITR on FliC_431–439-specific Foxp3⁺ Tregs (line histogram) and Foxp3-negative CD4⁺ T cells (shaded histogram) at early (day 5) and late (day 37) time points during persistent Salmonella infection. These data reflect six mice per time point representative of two independent experiments each with similar results. Bar, standard error.

Figure 8. Relative impacts after Treg ablation on the tempo of persistent Salmonella infection.

A. Number of recoverable Salmonella CFUs from spleen (top) and liver (bottom) in Treg-ablated F1 Foxp3 ^DTR compared with Treg-sufficient F1 control mice when DT treatment was initiated at either early (day 5) or late (day 37) time points during persistent infection. B. Percent CD44^hiCD62L^lo among CD4⁺ T cells in Treg-ablated F1 Foxp3 ^DTR compared with Treg-sufficient F1 control mice when DT treatment was initiated on either day 5 or day 37 during persistent infection. C. Number of recoverable Salmonella CFUs from spleen (top) and liver (bottom) following CTLA-4 blockade beginning at either early (day 5) or late (day 37) time points post-infection in F1 mice. D. Percent CD44^hiCD62L^lo among CD4⁺ T cells when CTLA-4 blockade was initiated at either early (day 5) or late (day 37) time points post-infection. E. Number of recoverable Salmonella CFUs from spleen (top) and liver (bottom) following treatment with GITR-stimulating antibody beginning at either early (day 5) or late (day 37) time points post-infection in F1 mice. F. Percent CD44^hiCD62L^lo among CD4⁺ T cells when GITR stimulation was initiated at either early (day 5) or late (day 37) time points post-infection. These data reflect six to ten mice per group combined from two to three independent experiments each with similar results. Bar, standard error. *, p<0.05.