Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees that recovered from hepatitis C

Abstract

Hepatitis C virus (HCV) poses a global health problem because it readily establishes persistent infection and a vaccine is not available. CD4(+)CD25(+) T cells have been implicated in HCV persistence because their frequency is increased in the blood of HCV-infected patients and their in vitro depletion results in increased IFN-gamma production by HCV-specific T cells. Studying a well-characterized cohort of 16 chimpanzees, the sole animal model for HCV infection, we here demonstrate that the frequency of Foxp3(+)CD4(+)CD25(+) regulatory T cells (T(Regs)) and the extent of suppression was as high in spontaneously HCV-recovered chimpanzees as in persistently HCV-infected chimpanzees. Foxp3(+)CD4(+)CD25(+) T(Regs) suppressed IFN-gamma production, expansion, and activation-induced cell death of HCV-specific T cells after recovery from HCV infection and in persistent HCV infection. Thus, T(Reg) cells control HCV-specific T cells not only in persistent infection but also after recovery, where they may regulate memory T-cell responses by controlling their activation and preventing apoptosis. However, Foxp3(+)CD4(+)CD25(+) T(Reg) cells of both HCV-recovered and HCV-infected chimpanzees differed from Foxp3(+)CD4(+)CD25(+)T(Reg) cells of HCV-naive chimpanzees in increased IL-2 responsiveness and lower T-cell receptor excision circle content, implying a history of in vivo proliferation. This result suggests that HCV infection alters the population of Foxp3(+)CD4(+)CD25(+) T(Reg) cells.

Figure 1.

Frequency, phenotype, and function of CD4⁺CD25⁺ T cells of HCV-naive chimpanzees. (A) CD4⁺CD25⁺ T cells (bold histogram line) of a representative HCV-naive chimpanzee (Ch6497) displayed a nonactivated (CD69^–), undifferentiated (CD27⁺CD28⁺) central memory (CD45RO⁺CCR7⁺CD62L⁺) phenotype with higher levels of CD45RO and intracellular CTLA-4 expression than CD4⁺CD25^– T cells (dotted histogram line). The thin histogram line to the left in each graph indicates the negative control. (B) CD4⁺CD25⁺ but not CD4⁺CD25^– T cells express Foxp3. Numbers in the left graph indicate the percentage of events in the respective gate. (C) Purified CD4⁺CD25⁺ T cells do not proliferate in response to plate-bound anti-CD3, but suppress the proliferation of CD4⁺CD25^– T cells (left panel). Moreover, CD4⁺CD25^– T cells proliferate more vigorously in response to plate-bound anti-CD3 than total CD4⁺ T cells (right panel). Mean and SD are indicated.

Figure 2.

Expression of FoxP3. CD4⁺CD25⁺ T cells of HCV-naive, HCV-recovered, and persistently HCV-infected chimpanzees express FoxP3 at the RNA (A) and protein (B) levels. The data are representative of 12 experiments, performed with T cells from 2 HCV-naive, 4 HCV-recovered, and 3 persistently HCV-infected chimpanzees. Functional analyses of the same T-cell subpopulations are shown in Figure 3.

Figure 3.

CD4⁺CD25⁺ T_Regs of HCV-naive, HCV-recovered, and persistently HCV-infected chimpanzees all exert suppressive function but differ in their response to exogenous IL-2. (A) Purified CD4⁺CD25⁺ T cells (2.5 × 10⁴/well) of HCV-naive, HCV-recovered, and persistently HCV-infected chimpanzees did not proliferate in response to plate-bound anti-CD3, but suppressed the proliferation of CD4⁺CD25^– T cells. The data are representative of 15 experiments, performed with T cells from 2 HCV-naive, 4 HCV-recovered, and 3 persistently HCV-infected chimpanzees. CD4⁺CD25⁺ and CD4⁺CD25^– T-cell populations are identical to those in Figure 2. (B) IL-2 responsiveness differed between CD4⁺CD25⁺ T cells of HCV-naive chimpanzees and those of chimpanzees that had experienced HCV infection (here shown for Ch1605, Ch6412). The results are representative for 2 HCV-naive and 4 HCV-recovered chimpanzees. Error bars indicate SD. n.s. indicates not significant.

Figure 4.

CD4⁺CD25⁺ T_Regs of HCV-recovered chimpanzees and CD4⁺CD25⁺ T_Regs of persistently HCV-infected chimpanzees differ from CD4⁺CD25⁺ T_Regs of HCV-naive chimpanzees in their TREC content. The TREC copy number of CD4⁺CD25⁺ cells was divided by that of CD4⁺CD25^– cells for normalization. The dotted line indicates an equal TREC copy number in CD4⁺CD25⁺ and CD4⁺CD25^– populations. The TREC copy number was assessed in the same cell populations as in the suppression assays (Figure 3).

Figure 5.

CD4⁺CD25⁺ T_Regs suppress HCV-specific CD8⁺ T cells. (A) Frequency of CD25⁺ T cells in the blood of HCV-naive, HCV-recovered, and persistently HCV-infected chimpanzees. (B) Depletion of CD25⁺ T cells from PBMCs increased HCV-specific IFN-γ responses of HCV-recovered chimpanzees, especially if they had recovered from repeated HCV rechallenges. The data were calculated from duplicate cultures and are representative of 16 of 18 experiments, the remaining 2 of 18 experiments showed no increase in IFN-γ responses. In contrast, the increase of IFN-γ responses was weaker or absent in persistently HCV-infected chimpanzees (P < .01 when the increase in IFN-γ responses was compared between the recovered/rechallenged and the persistently HCV-infected chimpanzees). The data are representative of 12 experiments in persistently HCV-infected chimpanzees. Horizontal bold lines indicate the mean response. (C) Increase of HCV-specific IFN-γ responses after depletion of CD25⁺ T cells from PBMCs. The depleted T-cell populations exerted suppressor function when added back to the culture (Figure S3). (D) CD4⁺CD25⁺ T_Regs of an HCV-recovered chimpanzees inhibited IFN-γ secretion of autologous NS5B peptide-specific CD8⁺ T cells, did not produce IFN-γ and did not require CD4⁺CD25^– cells to exert suppressive function (for additional experiments, see Figure S3). APC indicates antigen-presenting cells (irradiated, CD4- and CD8-depleted PBMCs).

Figure 6.

Effect of CD4⁺CD25⁺ cells on expansion of HCV-specific, peptide-stimulated CD8 T-cell populations. Depletion of CD25⁺ T cells from PBMCs of HCV-recovered chimpanzees (A) and persistently HCV-infected chimpanzees (B) resulted in increased expansion of tetramer-positive, HCV-specific CD8⁺ T cells on stimulation with cognate peptide. Seven representative experiments of a total of 15 are shown.

Figure 7.

CD4⁺CD25⁺ T cells protect CD8⁺ T cells from AICD. (A) Apoptosis of anti-CD3–stimulated CD8⁺ T cells was diminished in the presence of CD4⁺CD25⁺ T cells, compared with the presence of CD4⁺CD25^– T cells. Anti-CD3 stimulation resulted in a 57% increase of apoptosis over baseline, stimulation with PMA/ionomycin yielded similar results (not shown). (B-C) Apoptosis of HCV peptide-stimulated T-cell lines of an HCV-recovered (Ch1605) and a persistently HCV-infected chimpanzee (Ch6412) was diminished in the presence of CD4⁺CD25⁺ T_Regs, compared with the presence of CD4⁺CD25^– T cells. The results are representative for 6 experiments, using HCVcore_41^-, HCV-NS3_1444^-, and HCV-NS3_1357^-stimulated T-cell lines of recovered and persistently infected chimpanzees.