Investigation into Cardiac Myhc-α 334–352-Specific TCR Transgenic Mice Reveals a Role for Cytotoxic CD4 T Cells in the Development of Cardiac Autoimmunity

Figures

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  Figure 1. Tg mice express Myhc-α 334–352-specific TCRs on both CD4+ and CD8+ T cells. Thymocytes and splenocytes were prepared from NTLM and Tg mice, and the cells were stained with antibodies for CD3, CD4, CD8, and TCR vβ4. Their corresponding cell populations were analyzed by flow cytometry, as shown in left and right panels, respectively. While panels (a–c) respectively represent the flow cytometric plots of the subsets of cells positive for anti-CD3, CD4, and CD8, and TCR vβ4 in CD4+ and CD8+ T cells, panel (d) indicates the comparative analysis of the above populations. Mean ± SEM values obtained from three to four mice in each group are shown. *** p ≤ 0.001 and **** p ≤ 0.0001.

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  Figure 2. Both CD4+ and CD8+ T cells from naïve Tg mice respond to Myhc-α 334–352 and their responses are comparable. (a) Proliferative response. Lymphocytes prepared from naïve Tg mice were stimulated with Myhc-α 334–352 or RNase 43–56 (control) for two days, and after pulsing with [3H] thymidine for 16 h, proliferative responses were measured as CPM. (b) CFSE staining. Lymphocytes were stained with CFSE prior to stimulation with or without Myhc-α 334–352 or RNase 43–56 (50 µg/mL). IL-2 medium was added on day 2, and cells harvested on day 4 were stained with anti-CD4, anti-CD8, and 7-AAD. The top, middle, and bottom panels, respectively, represent the flow cytometric plots of cells positive for CD4 and CD8 in relation to CFSE; histogram plots of overlays between medium, RNase 43–56, and Myhc-α 334–352 treatments; and bar diagrams of whole CD4+ or CD8+ T cells or their low- and high-expressing cells. Mean ± SEM values are obtained from three mice in each group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

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  Figure 3. Naïve Tg T cells produce cytokines representing various T helper subsets. (a) Cytokine bead array analysis. Lymphocytes from naïve Tg mice were stimulated with or without Myhc-α 334–352 or RNase 43–56 (control) (50 µg/mL) for two days, and supernatants were analyzed for the indicated cytokines using the LEGENDplex Murine Th Cytokine Panel (12-plex; BioLegend), as described in the Methods section. (b) Intracellular staining. Lymphocytes were stimulated as above for two days, and IL-2 medium was then added. Cells harvested on day 4 were stained with anti-CD4, anti-CD8, and 7-AAD; after fixing and permeabilizing, cells were stained with cytokine antibodies, and cytokine-positive cells within CD4 or CD8 subsets were analyzed by flow cytometry. Mean ± SEM values are obtained from three mice in each group. * p ≤ 0.05 and **** p ≤ 0.0001.

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  Figure 4. CD4+ T cells in Tg mice express markers of cytotoxicity similar to CD8+ CTLs. (a) Markers of CD8+ CTLs and their expression in CD4+ T cells. Lymphocytes obtained from naïve Tg mice were stimulated with or without Myhc-α 334–352 or RNase 43–56 (control) (50 µg/mL) for two days, and IL-2 medium was then added. On day 4 post-stimulation with peptides, surface expression of CD107a was analyzed with respect to CD8+ and CD4+ T cells. Additionally, intracellular staining was performed to analyze the expression of IFN-γ and GrB corresponding to CD8+ and CD4+ T cells. (b) Other markers of cytotoxic T cells. Cells were stained with antibodies for the indicated markers, and CD4+ or CD8+ T cells expressing CD107a were analyzed in relation to NKG2A, NKG2D, and CRTAM expression by flow cytometry. Intranuclear EOMES expression was evaluated after staining with antibodies for CD4 or CD8 and CD107a, as described in the Methods section. Mean ± SEM values are obtained from three mice in each group. * p ≤ 0.01, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

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  Figure 5. Tg mice develop myocarditis in response to Myhc-α 334–352 immunization. Naïve mice: Hearts collected from naïve Tg mice were evaluated for histological changes by H and E staining. Panels (a,b) indicate normal endocardium and minimal myocardial inflammation (arrow), respectively. Immunized mice: Tg mice were immunized with Myhc-α 334–352. On day 21, hearts were collected for histological assessment, as described above. Panels (c,d) indicate marked subendocardial myocarditis and myocardial mononuclear cell inflammatory infiltrates, respectively (arrows). Original magnification, ×20 (bar = 100 µm, applies to all panels). Representative images are shown from eight naïve and four immunized Tg mice.

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  Figure 6. Proliferative responses of CD4+ and CD8+ T cells obtained from Tg mice immunized with Myhc-α 334–352 were comparable. (a) Proliferative response. Lymphocytes obtained from Tg mice immunized with Myhc-α 334–352 were stimulated with Myhc-α 334–352 or RNase 43–56 (control). After two days, cells were pulsed with [3H] thymidine, and 16 h later, proliferative responses were measured as CPM. (b) CFSE staining. Cells were stained with CFSE before stimulating with or without Myhc-α 334–352 or RNase 43–56 (50 µg/mL); IL-2 medium was added on day 2. On day 4, cells were stained with anti-CD4, anti-CD8, and 7-AAD. The top, middle, and bottom panels represent the flow cytometric plots of cells positive for CD4 and CD8 vs. CFSE; histogram plots of overlays between medium, RNase 43–56, and Myhc-α 334–352 stimulations; and bar diagrams of the whole CD4+ or CD8+ T cells and their respective low- or high-expressing cells. Mean ± SEM values are obtained from three mice in each group. * p ≤ 0.05, *** p ≤ 0.001, and **** p ≤ 0.0001.

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  Figure 7. Tg T cells obtained from mice immunized with Myhc-α 334–352 produce mainly inflammatory cytokines. (a) Cytokine bead array analysis. Lymphocytes obtained from Tg mice immunized with Myhc-α 334–352 were stimulated with or without Myhc-α 334–352 or RNase 43–56 (control) (50 µg/mL); supernatants harvested on day 2 were analyzed for the indicated cytokines using the LEGENDplex Murine Th Cytokine Panel (12-plex; BioLegend), as described in the Methods section. (b) Intracellular staining. Cells were stimulated as above, and after two days, IL-2 medium was added. On day 4 post-stimulation, cells were stained with anti-CD4, anti-CD8, and 7-AAD. After fixation and permeabilization, cells were stained with antibodies for the indicated cytokines, and cells positive for each cytokine were analyzed in the CD4 or CD8 subsets by flow cytometry. Mean ± SEM values are obtained from three mice in each group. ** p ≤ 0.01 and **** p ≤ 0.0001.