Foxp3+ regulatory T cells impede the priming of protective CD8+ T cells

Abstract

T cell activation is controlled by incompletely defined opposing stimulation and suppression signals that together sustain the balance between optimal host defense against infection and peripheral tolerance. In this article, we explore the impacts of Foxp3(+) regulatory T cell (Treg) suppression in priming Ag-specific T cell activation under conditions of noninfection and infection. We find the transient ablation of Foxp3(+) Tregs unleashes the robust expansion and activation of peptide-stimulated CD8(+) T cells that provide protection against Listeria monocytogenes infection in an Ag-specific fashion. By contrast, Treg ablation had nonsignificant impacts on the CD8(+) T cell response primed by infection with recombinant L. monocytogenes. Similarly, nonrecombinant L. monocytogenes administered with peptide stimulated the expansion and activation of CD8(+) T cells that paralleled the response primed by Treg ablation. Interestingly, these adjuvant properties of L. monocytogenes did not require CD8(+) T cell stimulation by IL-12 produced in response to infection, but instead were associated with sharp reductions in Foxp3(+) Treg suppressive potency. Therefore, Foxp3(+) Tregs impose critical barriers that, when overcome naturally during infection or artificially with ablation, allow the priming of protective Ag-specific CD8(+) T cells.

Figure 1

Foxp3⁺ Treg-ablation primes the expansion and activation of peptide stimulated T cells. A. Percent and number of OVA-specific CD90.1⁺ CD8⁺ T cells among splenocytes day 5 after stimulation with cognate peptide in the presence (Foxp3-WT) or absence of Tregs (Foxp3-DTR). Each group of mice received DT treatment one day before and on the day of peptide inoculation. B. CFSE expression among OVA-specific CD90.1⁺ CD8⁺ T cells 60 hrs (day 2.5) and 120 hrs (day 5) after stimulation with cognate peptide in Treg-sufficient (Foxp3-WT) or Treg-ablated mice (Foxp3-DTR) (black line histogram), compared with unstimulated cells (filled histogram) or unlabelled cells (gray line histogram). C. Expression of CD25 and IFN-γ production by CD90.1⁺ (line histogram) or bulk CD8⁺ T cells (filled histogram) day 5 after peptide stimulation in Treg-sufficient (Foxp3-WT) or mice ablated of Tregs on day before and on the day of peptide inoculation (Foxp3-DTR). D. Percent and number of CD90.1⁺ CD8⁺ cells among splenocytes at the indicated time points after peptide stimulation. These data are representative of three experiments containing 10–12 mice per group. Bar, one standard error.

Figure 2

Peptide-stimulated CD8⁺ T cells primed in the absence of Tregs protect against Lm infection. Bacterial CFUs in the spleen (top) or liver (bottom) day 3 after infection with either Lm-OVA or Lm-10403s for the indicated mice stimulated with OVA_257–264 peptide (and treated with DT one day prior and on the day of peptide inoculation) 30 days prior to infection. These data are representative of three experiments containing 8–12 mice per group.

Figure 3

Secondary expansion and cytokine production for peptide-stimulated CD8⁺ T cells primed initially in Treg-ablated or Treg-sufficient mice. A. Percent and number of CD90.1⁺ CD8⁺ cells among splenocytes prior to (D30), or days 3 (D30 + 3) and 5 (D30 + 5) after Lm-OVA infection in mice treated with peptide 30 days prior. B. Representative FACS plots demonstrating percent IFN-γ, IL-2, and TNF-α producing CD90.1⁺ CD8⁺ T cells day 5 after secondary Lm-OVA infection (D30 + 5 after peptide stimulation) directly ex-vivo (no stim) or after peptide stimulation. These data are representative of three experiments containing 8–12 mice per group. Bar, one standard error.

Figure 4

Foxp3⁺ Treg-ablation does not impact the expansion and activation of antigen-specific CD8⁺ T cells primed by recombinant Lm-OVA infection. A. Percent and number of OVA-specific CD90.1⁺ CD8⁺ T cells among splenocytes day 5 after Lm-OVA infection in Foxp3-WT or Foxp3-DTR mice each treated with DT one-day prior and on the day of infection. B. Expression of CD25 and IFN-γ production by CD90.1⁺ (line histogram) or bulk CD8⁺ T cells (filled histogram) day 5 after Lm-OVA infection in Treg-ablated (Foxp3-DTR) or Treg-sufficient (Foxp3-WT) controls. These data are representative of three experiments containing 10–12 mice per group.

Figure 5

Non-recombinant Lm-10403s primes the expansion and activation of peptide stimulated T cells. A. Percent and number of CD90.1⁺ CD8⁺ T cells among splenocytes day 5 after stimulation with OVA peptide alone (no infection) or peptide stimulation plus Lm infection (Lm-10403s). B. Expression of CD25 and IFN-γ production by CD90.1⁺ (line histogram) or bulk CD8⁺ T cells (filled histogram) recovered from mice day 5 after peptide stimulation alone (no infection) or peptide stimulation plus Lm infection (Lm-10403s). C. Percent OVA-specific CD90.1⁺ among CD8⁺ T cells day 5 after stimulation with OVA peptide plus Lm-10403s in mice treated with anti-IL-12 neutralizing or isotype (rat IgG2a) antibodies (top). Percent IL-12 receptor deficient OT-1 or WT OT-1 (each CD45.2⁺) among CD8⁺ T cells after adoptive transfer into CD45.1⁺ mice, and day 5 after stimulation with OVA peptide and Lm-10403s (bottom). Percent IFN-γ production for each group of OT-1 cells (line histogram) or bulk CD8⁺ T cells (filled histogram) after peptide stimulation. These data are representative of three experiments containing 8–12 mice per group.

Figure 6

Reduced Treg suppressive potency after Lm infection. A. Purification of Foxp3⁺ Tregs as GFP⁺ CD4⁺ cells in Foxp3^GFP reporter mice after Lm infection. Percent GFP or Foxp3 expression among CD4⁺ splenocytes at each time point after infection before sorting (Pre-sort) and among all cells after sorting (Post-sort) for GFP⁺ CD4⁺ cells. B. CFSE dilution among CD8⁺ CD45.1⁺ responder cells after co-culture with GFP⁺ Tregs at the indicated ratio and stimulation with anti-CD3 antibody (line histogram), no stimulation controls (dark filled histogram), or stimulation without Tregs (light filled histogram). C. Percent CD8⁺ CD45.1⁺ responder cells in each CFSE generation after co-culture with GFP⁺ Tregs from mice at the indicated time point after infection at a 1:8 ratio. These data are representative of three experiments containing Tregs from 6–8 mice per infection time point. Bar, one standard error.

Figure 7

Antigen-pulsed dendritic cells do not override the impacts of Treg-suppression. A. Relative expression of MHC-I (H-2K^b) or CD80 on CD11c⁺ cells isolated directly ex vivo from naïve Treg sufficient mice (gray filled histogram and symbols), or mice day 5 after Lm infection (red line histogram and symbols) or ablation of Foxp3⁺ Tregs (blue line histogram and symbols) compared with JAWS II cells maintained in vitro (black line histogram and symbols). B. Percent and number of OVA-specific CD90.1⁺ CD8⁺ T cells among splenocytes day 5 after stimulation with peptide pulsed or unpulsed JAWS II dendritic cells in the presence (Foxp3-WT) or absence of Tregs (Foxp3-DTR). Expression of CD25 and IFN-γ production by CD90.1⁺ (line histogram) or bulk CD8⁺ T cells (filled histogram) day 5 after peptide stimulation in Treg-sufficient (Foxp3-WT) or mice ablated of Tregs one day before and on the day of peptide inoculation (Foxp3-DTR). These data are representative of three experiments containing 8–12 mice per group. Bar, one standard error.