Heart non-specific effector CD4+ T cells protect from postinflammatory fibrosis and cardiac dysfunction in experimental autoimmune myocarditis

Figures

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  Fig. 1. Teff phenotype of heart-reactive CD4+ T cells in α-MyHC/CFA-induced EAM. BALB/c mice received subcutaneous injection of α-MyHC/CFA into the right inguinal region as shown in panel a. CD4+ T cells obtained from the right iLN 7 days after immunization were sorted into four fractions, labelled with CFSE and cultured for 3 days on APCs with or without α-MyHC. Panel b shows the gating strategy for CD4+ T cell subpopulations in viable CD4+ CD45+ population isolated from iLN: Teff effector, Tcm central memory, Tn naïve and Tun undefined T cells. Percentage of proliferating CFSE-labelled CD4+ T cell subpopulations is quantified in panel c. Data are representative of three independent experiments, *p < 0.05 (unpaired Student’s t test). Panel d shows representative phenotype analyses of viable CD4+CD45+ cells and frequencies of CD4+CD44hiCD62Llow cells (Teff) in indicated organs at day 17 (active myocarditis). Teff from respective organs were sorted, labelled with CFSE and stimulated with α-MyHC peptide in presence of APCs for 5 days. Proliferation is shown as dilution of CFSE dye in viable CD3+CD4+ population. Supernatant levels of indicated cytokines secreted by Teff isolated from the…

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  Fig. 2. Cells with Teff signature effectively infiltrate cardiac tissue. Indicated Teff (3–5 × 106) were adoptively transferred into sub-lethally irradiated BALB/c recipients. Top panel a shows representative heart histology of the respective recipient 10 days after adoptive transfer. Bottom panel a shows immunohistochemistry for CD45.1 and CD90.1 in hearts of indicated recipients. Scale bars = 100 μm. Panel b shows analysis of donor CD4+CD45.1+ cells in blood (top) and heart (bottom) 10 days after adoptive transfer of CD45.1+ Teff. Analysis of donor CD45.1+ and DO11.10+ Teff in indicated organs 10 days after adoptive transfer is shown in panel c and quantification of total heart-inflammatory CD3+CD4+ DO11.10+ T cells is presented in panel d. Arrows show gating strategies and numbers indicate percentage of cells in the adjacent gate. Data are representative of six mice, p value calculated with unpaired Student’s t-test. CD45.1+ Teff—CD4+ cells from α-MyHC-stimulated (72 h) splenocytes/iLN cells of α-MyHC/CFA immunized CD45.1-tg mice (EAM d17); TCR-M Teff—CD4+ cells from α-MyHC-stimulated (72 h) splenocytes of TCR-M-tg (CD90.1+) mice; DO11.10+ Teff—CD4+ cells from OVA-stimulated (72 h)…

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  Fig. 3. Differential kinetic of antigen-dependent and antigen-independent expansion of Teff during myocarditis. CD45.1+ Teff (4 × 106), TCR-M Teff (4.5 × 106) and DO11.10+ Teff (4.5 × 106) cells were co-delivered into sub-lethally irradiated BALB/c recipients. Panel a shows analysis of donor CD4+ T cell frequency in indicated organs 4 days after adoptive transfer. Arrows show gating strategies and numbers indicate percentage of cells in the adjacent gate. Data are representative of four mice per time point. Panel b presents frequencies of indicated donor Teff among heart-infiltrating CD4+ T cells at days 4 and 10 after adoptive transfer. Donor CD45.1+, TCR-M and DO11.10+ Teff were generated as described in Fig. 2

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  Fig. 4. Transcriptomic analysis of heart-specific and heart non-specific Teff cells. CD45.1+ Teff (3 × 106) and DO11.10+ Teff (3 × 106) cells were co-delivered into sub-lethally irradiated BALB/c recipients. 10 days later, the whole splenocytes were isolated, stimulated with α-MyHC (or kept unstimulated for controls) for 24 h and CD45.1+ and DO11.10+ CD4+ cells were sorted for the whole transcriptome sequencing (RNAseq). Panel a illustrates the experimental setup. The number and the overlap of all differentially expressed genes (p < 0.05, log2ratio > 0.5 or < −0.5) in response to α-MyHC in CD45.1+ Teff and in DO11.10+ Teff is shown in panel b. Panel c shows heatmap of genes of the cytokine activity GO:0005125 category up-regulated (p < 0.05, log2ratio > 0.75) in CD45.1+ Teff and/or DO11.10+ Teff in response to α-MyHC treatment. Panel d shows heatmap of genes of the extracellular space GO:0005615 category differentially expressed (p < 0.05, log2ratio > 0.75 or < − 0.75) between CD45.1+ Teff and DO11.10+ Teff under stimulatory (+α-MyHC) condition. Cells obtained from 3 independent mice were used for RNAseq. Donor CD45.1+ and DO11.10+ Teff were generated as described in Fig. 2

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  Fig. 5. Heart non-specific Teff do not affect myocarditis severity. Myocarditis-inducing CD45.1+ Teff (4–6 × 106) were injected with or without DO11.10+ Teff (4–6 × 106) into sub-lethally irradiated BALB/c recipients. Representative histology (top) and immunohistochemistry for CD45 (middle) and CD3 (bottom) in hearts of indicated recipients 10 days after adoptive transfer are shown in panel a. Scale bar = 100 μm. Myocarditis severity scores and quantification of heart-infiltrating CD3+ and CD45+ cells are presented in panel b. p values calculated with unpaired Student’s t test. Donor CD45.1+ and DO11.10+ Teff were generated as described in Fig. 2

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  Fig. 6. Heart non-specific Teff prevents postinflammatory fibrosis and cardiac dysfunction. α-MyHC/CFA immunized BALB/c recipients received control solution (PBS) or DO11.10+ Teff (4–5 × 106) at days 17 and 20 of EAM and were analyzed for fibrotic and functional changes at day 40. Panel a shows representative Masson’s Trichrome staining and immunohistochemistry stainings for periostin, vimentin and αSMA in heart sections. Scale bar = 200 μm. Quantifications of the fibrotic and immunopositive areas are shown in panel b. p values calculated with the Mann–Whitney test. Panel c shows hydroxyproline content at day 40. Echocardiography measurements were performed in mice before immunization (d0) and at d40 of EAM. Panel d shows difference for d40 and d0 (Δ = d40–d0) for selected parameters. EF ejection fraction, FS fractional shortening, CO cardiac output. p values calculated with the Student’s t test. All echocardiographic parameters at d0 and d40 are available in the Suppl. Table 2. Analysis of heart weights at d40 is shown in panel e. HW/TL heart weight to tibial length. p values calculated with the Student’s t test. Donor DO11.10+ Teff were generated as described in Fig. 2

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  Fig. 7. Human Teff stimulated with γc-cytokines suppress myofibroblast signature of human cardiac fibroblasts. Human cardiac fibroblasts were co-cultured with sorted human Tn and Teff in the presence of IL-2, IL-7, IL-5 and IL-21 (γc-cytokines) for 7 days (human Tn and Teff were isolated as shown in Supp. Fig. 7). Panel a shows gene expression levels (normalized to GAPDH) in cardiac fibroblasts at day 7 at indicated conditions. Data are representative of three independent experiments, p values calculated with one-way ANOVA, * p < 0.05 the Dunnett post hoc test compared to fibroblasts with γc-cytokines. Panel b shows representative immunofluorescence for αSMA (green) and F-actin (phalloidin staining, red) and quantification of αSMA-positive cardiac fibroblasts cultured for 7 days in presence of γc-cytokines with or without human Teff. Data are representative of three independent experiments, p value calculated with unpaired Student’s t-test. Scale bar = 50 μm. Panel c shows relative intracellular levels of αSMA and collagen I in cardiac fibroblasts in presence of γc-cytokines and Teff measured with flow cytometry. Data are shown as fold change from three independent experiments, p values…