CD4+ CD25- FoxP3+ regulatory cells are the predominant responding regulatory T cells after human rotavirus infection or vaccination in gnotobiotic pigs

Abstract

The distribution and dynamic changes of CD4(+) CD25(+) FoxP3(+) and CD4(+) CD25(-) FoxP3(+) regulatory T (Treg) cells induced by human rotavirus (HRV) infection and vaccination were examined in neonatal gnotobiotic pigs infected with virulent HRV (VirHRV) or vaccinated with attenuated HRV (AttHRV). Subsets of gnotobiotic pigs in the AttHRV and control groups were challenged with VirHRV at post-inoculation day (PID) 28. We demonstrated that VirHRV infection or AttHRV vaccination reduced frequencies and numbers of tissue-residing Treg cells, and decreased the frequencies of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) producing CD4(+) CD25(-) Treg cells in ileum, spleen and blood at PID 28. The frequencies of IL-10 and TGF-β producing CD4(+) CD25(-) Treg cells in all sites at PID 28 were significantly inversely correlated with the protection rate against VirHRV-caused diarrhoea (r = -1, P < 0.0001). Hence, higher frequencies of functional CD4(+) CD25(-) Treg cells can be an indicator for poorer protective immunity against rotavirus. Our results highlighted the importance of CD4(+) CD25(-) Treg cells over CD4(+) CD25(+) Treg cells in rotavirus infection and immunity. AttHRV vaccination (induction of immune effector responses) reduced the expansion of CD4(+) CD25(-) Treg cells in ileum seen in the challenged naive pigs during the acute phase of VirHRV infection and preserved normal levels of intestinal TGF-β producing Treg cells post-challenge. The reduced suppressive effect of Treg cells in AttHRV-vaccinated pigs would unleash effector/memory T-cell activation upon challenge. Preserving TGF-β producing CD4(+) CD25(-) Treg cells is important in maintaining homeostasis. Based on our findings, a model is proposed to depict the dynamic equilibrium course of Treg and effector T-cell responses after primary rotavirus infection/vaccination and challenge.

Figure 1

Representative dot plots of frequencies of FoxP3⁺ regulatory T (Treg) cells among CD4⁺ T cells (a), CD4⁺ CD25⁺ and CD4⁺ CD25⁻ Treg cells (b), and transforming growth factor-β (TGF-β) producing CD4⁺ CD25⁻ Treg cells (c) in ileum of gnotobiotic (Gn) pigs from the five treatment groups. Mononuclear cells (MNC) were stained freshly without in vitro stimulation (same for MNC in all Treg-cell figures). In panels a and c, the numbers in the rectangles in dot plots are the frequencies of FoxP3⁺ Treg cells among CD4⁺ T cells and TGF-β⁺ cells among CD4⁺ CD25⁻ Treg cells, respectively. In panel b, the numbers at the upper left and right corners of dot plots are the frequencies of CD4⁺ CD25⁻ Treg cells (CD4⁺ CD25⁻ FoxP3⁺) and CD4⁺ CD25⁺ Treg cells (CD4⁺ CD25⁻ FoxP3⁺) respectively, among gated MNC.

Figure 2

Frequencies of FoxP3⁺ regulatory T (Treg) cells among CD4⁺ cells (a), CD4⁺ CD25⁺ and CD4⁺ CD25⁻ Treg cells among mononuclear cells (MNC) (b), and numbers of CD4⁺ CD25⁺ and CD4⁺ CD25⁻ Treg cells per tissue (c) in intestinal lymphoid tissues of gnotobiotic (Gn) pigs. The graph in panel a shows the frequencies of FoxP3⁺ cells among CD4⁺ cells. The two graphs in panel b show the frequencies of CD4⁺ CD25⁺ and CD4⁺ CD25⁻ Treg cells, respectively. The two graphs in panel c show the absolute numbers of CD4⁺ CD25⁺ and CD4⁺ CD25⁻ Treg cells per tissue, respectively. Data are presented as mean frequency or number ± SEM (n = 3 to n = 6). Different lowercase letters above bars indicate significant differences in frequencies compared among virulent human rotavirus (VirHRV) infection, attenuated human rotavirus (AttHRV) inoculation and control groups at PID 28 for the same cell type and tissue (Kruskal–Wallis test, P < 0·05); different capital letters above bars indicate significant differences in frequencies compared among Mock/VirHRV, AttHRV/VirHRV groups at post-challenge day (PCD) 7 and the control group at post-inoculation day (PID) 28 for the same cell type and tissue (Kruskal–Wallis test, P < 0·05), whereas shared letters indicate no significant difference.

Figure 3

Frequencies of FoxP3⁺ regulatory T (Treg) cells among CD4⁺ cells (a), CD4⁺ CD25⁺ and CD4⁺ CD25⁻ Treg cells among mononuclear cells (MNC) (b), and numbers of CD4⁺ CD25⁺ and CD4⁺ CD25⁻ Treg cells per tissue (c) in spleen and blood of gnotobiotic (Gn) pigs. See Fig. 2 legend for panel description and statistical analysis.

Figure 4

Frequencies of interleukin-10 (IL-10) (a) or transforming growth factor-β (TGF-β) (b) producing CD4⁺ CD25⁺ and CD4⁺ CD25⁻ regulatory T (Treg) cells in intestinal and systemic lymphoid tissues of gnotobiotic (Gn) pigs. The two graphs in panel a depict the frequencies of IL-10 producing CD4⁺ CD25⁺ and CD4⁺ CD25⁻ Treg cells and the two graphs in panel b depict the frequencies of TGF-β producing CD4⁺ CD25⁺ and CD4⁺ CD25⁻ Treg cells. Data are presented as mean frequency ± SEM (n = 3 to n = 6). See Fig. 2 legend for statistical analysis.

Figure 5

Human rotavirus (HRV)-specific interferon-γ (IFN-γ) producing CD4⁺ T-cell responses in intestinal and systemic lymphoid tissues of gnotobiotic (Gn) pigs. Mononuclear cells (MNC) were stimulated with semi-purified attenuated HRV (AttHRV) antigen in vitro for 17 hr. Brefeldin A was added for the last 5 hr to block secretion of cytokines produced by the T cells. IFN-γ production was detected by intracellular staining and flow cytometry and presented as IFN-γ⁺ CD4⁺ cells among CD3⁺ cells. Data are presented as mean frequency ± SEM (n = 3 to n = 6). See Fig. 2 legend for statistical analysis.

Figure 6

Model for the dynamics of CD4⁺ CD25⁻ regulatory T (Treg) cell responses after primary infection/vaccination and challenge. During the acute phase of rotavirus infection/vaccination [post-inoculation days (PID) 1–7], human rotavirus-specific interferon-γ (IFN-γ) producing effector T helper type 1 cell (Th1_eff) responses are induced in the intestinal lymphoid tissue; meanwhile there is an expansion of CD25⁻ Treg cells in the same site. At the convalescent phase after rotavirus infection/vaccination, the host resumes immune homeostasis and establishes the equilibrium between Th1_eff cells and Treg cells but at a different level, with increased numbers of Th1_eff cells and decreased numbers of Treg cells (a). After challenge, vaccinated and partially protected pigs develop anamnestic Th1_eff cell responses that are stronger and faster than those after primary infection but the Treg cell levels remain similar to pre-challenge levels (b). Fully protected pigs do not develop an anamnestic Th1_eff cell immune response.