Cross-regulation of T regulatory-cell response after coxsackievirus B3 infection by NKT and γδ T cells in the mouse

Abstract

Coxsackievirus B3 (CVB3) variants H3 and H310A1 differ by a single nonconserved amino acid in the VP2 capsid region. C57Bl/6 mice infected with the H3 virus develop myocarditis correlating with activation of T cells expressing the Vγ4 T cell receptor chain. Infecting mice with H310A1 activates natural killer T (NKT; mCD1d-tetramer(+) TCRβ(+)) cells, but not Vγ4 T cells, and fails to induce myocarditis. H310A1 infection preferentially activates M2 alternatively activated macrophage and CD4(+)FoxP3 (T regulatory) cells, whereas CD4(+)Th1 (IFN-γ(+)) cells are suppressed. By contrast, H3 virus infection activates M1 proinflammatory and CD4(+)Th1 cells, but not T regulatory cells. The M1 macrophage show significantly increased CD1d expression compared to M2 macrophage. The ability of NKT cells to suppress myocarditis was shown by adoptive transfer of purified NKT cells into H3-infected NKT knockout (Jα18 knockout) mice, which inhibited cardiac inflammation and increased T regulatory cell response. Cardiac virus titers were equivalent in all mouse strains indicating that neither Vγ4 nor NKT cells participate in control of virus infection. These data show that NKT and Vγ4 cells cross-regulate T regulatory cell responses during CVB3 infections and are the primary factor determining viral pathogenesis in this mouse model.

Figure 1

C57Bl/6 male mice infected with either H3 or H310A1 virus. Percentage myocardium inflammation and cardiac virus titer in mice infected with either H3 or H310A1 virus (A and B). Lymphoid cells labeled with antibodies to Vγ4 TCR chain and CD69 early activation marker (C) or with mCD1d-tetramer and antibody to TCRβ for NKT cells (D). Values are given as data from individual mice with means ± SEM indicated for each group. ^∗∗P < 0.01 H310A1-infected mice versus H3-infected mice.

Figure 2

Histology of C57Bl/6, NKT KO, or γδ KO 5- to 8-week-old male mice either uninfected or infected 7 days earlier with either 10² PFU H3 or 10⁴ PFU H310A1 virus i.p. Hearts were removed, perfused with PBS, formalin fixed, paraffin embedded, sectioned, and then stained with H&E. Representative examples of histology from each group are provided from the animals reported in Figure 3A.

Figure 3

C57Bl/6 (B6), NKT KO, and γδ KO mice were infected i.p. with either 10² PFU H3 (A and C) or 10⁴ PFU H310A1 (B and D) virus and sacrificed 7 days later. Hearts were evaluated for myocarditis by image analysis (A and B) and cardiac virus titers by the plaque forming assay (C and D). Values represent individual mice with means ± SEM of each group indicated. ^∗∗P < 0.01 infected NKT KO or γδ KO mice versus B6 mice.

Figure 4

Characterization of infiltrating lymphoid cells in H3- and H310A1-infected mice. Lymphocytes were isolated from hearts of C57Bl/6, NKT KO, or γδ KO mice infected i.p. 7 days earlier with either 10² PFU H3 or 10⁴ PFU H310A1. A: The total infiltrating lymphoid cells isolated from the heart of each individual mouse were determined by trypan blue exclusion. Lymphoid cells from the hearts (10⁵ cells) were stimulated with ionomycin, PMA, and BFA for 4 hours, labeled with either anti-CD4 or anti-CD8, fixed, permeabilized, and then labeled with anti–IFN-γ to demonstrate Th1 cells (B); labeled with anti-CD4, fixed, permeabilized, and labeled with anti-FoxP3 to demonstrate T regulatory cells (C); labeled with anti-Vγ4 and anti-CD69 (D); or labeled with mCD1d-tetramer and anti-TCRβ to demonstrate NKT cells (E). F: Lymphocytes were obtained from hearts of C57Bl/6 mice infected 7 days earlier with either 10² PFU H3 (for Vγ4 cell isolation) or 10⁴ PFU H310A1 (for NKT cell isolation) and labeled with either CD1d-tetramer and antibody to TCRβ (for NKT cells), or with antibody to Vγ4 and CD69 (for Vγ4 cells), and were then subjected to sterile sorting. The indicated cytokine evaluation was performed using commercial kits according to the manufacturer's directions. Values represent the number of the specific cells in the heart-derived lymphoid cell population and were determined by multiplying the percentage of positive cells as determined by flow cytometry multiplied by the number of lymphoid cells isolated from each heart. Values represent means ± SEM of 3 to 8 mice/group. ^∗∗P < 0.01 H3-infected NKT KO or γδ KO mice versus H3-infected B6 mice. ^†P < 0.05, ^††P < 0.01 H310A1-infected NKT KO or γδ KO mice versus H310A1-infected B6 mice. ^‡‡P < 0.01 cytokine values for Vγ4 cells versus NKT cells.

Figure 5

Representative flow histograms of NKT cells as indicated by labeling with mCD1d-tetramer and anti-TCRβ and Vγ4⁺CD69⁺ cells in B6, NKT KO, and γδ KO mice infected with H3 virus 7 days earlier.

Figure 6

Macrophage phenotype and innate effector cells. A: C57Bl/6, NKT KO, and γδ KO mice were infected i.p. with 10² PFU H3 virus 7 days earlier, and the lymphoid cells infiltrating the hearts of individual mice were retrieved and evaluated for M1 or M2 macrophage phenotype. The cells were labeled with antibody to CD11b and Ly6C, fixed, permeabilized, and then labeled with antibodies to IL-10 (M2) or IL-12 (M1). ^∗P < 0.05, ^∗∗P < 0.01 IL-10⁺ macrophage versus IL-12⁺ macrophage. B: C57Bl/6 and NKT KO mice were infected with 10⁴ PFU H310A1 i.p. Groups of infected NKT KO mice also received 0.5 × 10⁴ purified NKT cells i.v. through the tail vein. The NKT cells were isolated by flow sorting of doubly labeled mCD1d-tetramer/anti-TCRβ⁺ lymphoid cells infiltrating the hearts of C57Bl/6 mice infected 7 days earlier with 10⁴ PFU H310A1 as described in Figure 4 and in Materials and Methods. One group of NKT KO mice given NKT cells also was injected i.p. with 200 μg of monoclonal anti–IL-4. NKT cells and anti–IL-4 were injected on the same day as virus. All mice were sacrificed 7 days later, and lymphoid cells infiltrating the hearts were retrieved and evaluated for CD11b, Ly6C^hi, and IL-10 (M2 macrophage) or IL-12 (M1 macrophage, C57Bl/6 only). ^∗P < 0.05, ^∗∗P < 0.01 versus C57Bl/6 IL-10⁺ cells. Replicate groups of H310A1-infected mice with or without NKT cells and anti–IL-4 as represented in B were evaluated for percent myocardium inflamed (C), cardiac virus titers (D), and number of CD4⁺FoxP3⁺ cells/heart (E). ^∗P < 0.05, ^∗∗P < 0.01 versus C57Bl/6 mice. ^†P < 0.05 versus NKT KO and NKT KO + NKT cells + anti–IL-4 groups. Values represent number of cells in the lymphoid cells from individual mice with the means ± SEM for each group indicated.

Figure 7

CD1d expression on CD11b⁺ cells isolated from the heart. C57Bl/6 mice were infected 7 days earlier with either H3 or H310A1 virus. Lymphoid cells infiltrating the heart were labeled with antibodies to CD3, CD11b, and CD1d. A: Representative histograms of flow analysis of CD3-CD11b⁺ cells for CD1d expression. B: Percent CD11b⁺ cells that are also CD1d⁺. C: Mean fluorescence intensity of CD1d on CD11b⁺ cells. Data represent means ± SEM of 4 to 6 mice/group. ^∗P < 0.05, ^∗∗P < 0.01 H310A1-infected (white bars) mice versus H3-infected (black bars) mice.