Impaired thymic tolerance to α-myosin directs autoimmunity to the heart in mice and humans

Abstract

Autoimmunity has long been linked to myocarditis and its sequela, dilated cardiomyopathy, the leading causes of heart failure in young patients. However, the underlying mechanisms are poorly defined, with most clinical investigations focused on humoral autoimmunity as the target for intervention. Here, we show that the α-isoform of myosin heavy chain (α-MyHC, which is encoded by the gene Myh6) is the pathogenic autoantigen for CD4+ T cells in a spontaneous mouse model of myocarditis. Further, we found that Myh6 transcripts were absent in mouse medullary thymic epithelial cells (mTECs) and peripheral lymphoid stromal cells, which have been implicated in mediating central and peripheral T cell tolerance, respectively. Transgenic expression of α-MyHC in thymic epithelium conferred tolerance to cardiac myosin and prevented myocarditis, demonstrating that α-MyHC is a primary autoantigen in this disease process. Remarkably, we found that humans also lacked α-MyHC in mTECs and had high frequencies of α-MyHC-specific T cells in peripheral blood, with markedly augmented T cell responses to α-MyHC in patients with myocarditis. Since α-MyHC constitutes a small fraction of MyHC in human heart, these findings challenge the longstanding notion that autoimmune targeting of MyHC is due to its cardiac abundance and instead suggest that it is targeted as a result of impaired T cell tolerance mechanisms. These results thus support a role for T cell-specific therapies for myocarditis.

Figure 1. Intra- and intermolecular spreading of humoral autoimmunity from regions unique to cardiac-specific α-MyHC in longitudinal serum samples from individual DQ8⁺NOD mice.

(A) Schematic of the percent amino acid sequence identity between the different MyHC isoforms (dashed and solid lines) and their predominant site of expression (solid arrows) in mice and humans. (B) Western blots with 0.25 μg/lane each of purified mouse cardiac myosin (CM) and soleus myosin (SM), 1 μg total mouse heart myofibrillar extracts (He), and 0.25 μg purified mouse cardiac troponin I and cardiac troponin T (cTnI/cTnT) were probed with serial sera from the same DQ8⁺NOD mouse at ages 4, 6, and 10 weeks. The positions of MyHC and cTnT are indicated. Shown is a representative result of independent experiments from 4 different mice. (C) Western blots with 0.25 μg/lane each of purified huCM-A, huCM-V, huSM, mouse cardiac, and soleus myosin were probed with sera from a DQ8⁺NOD mouse at ages 4 and 8 weeks.

Figure 2. Mortality from myocarditis in DQ8⁺NOD mice (WT) in the absence of B cells or T cells.

(A) Kaplan-Meier survival curves of female DQ8⁺NOD μMT^+/–, WT, μMT^–/–, and Cα^–/– mice followed through 37 weeks of age. The mortality from myocarditis did not differ statistically between DQ8⁺NOD WT and μMT^+/– mice but was significant (P < 0.001) in all other comparisons. (B) Heart histology shows the absence of infiltrates in Cα^–/–DQ8⁺NOD mice and severe lymphocytic infiltrates and cardiac thrombosis (T) in μMT^–/–DQ8⁺NOD mice. Original magnification, ×37.5.

Figure 3. The generation and characterization of CD4⁺ T cell clones isolated directly from myocarditis lesions of DQ8⁺NOD mice.

(A) The schema shows the protocol of establishing CD4⁺ T cell clones from the myocarditic hearts. (B) Proliferation assay indicates that both clone E and clone 2 recognize cardiac myosin but not soleus myosin. Data are presented as mean ± SD. (C) ELISPOT assay shows that clone E produces IFN-γ but not IL-4 in response to cardiac myosin, with no responses to soleus myosin. (D) Clone E also recognizes huCM-A in proliferation assays. SI, stimulation index. (E) Clone E responds to cardiac myosin only in the context of DQ8⁺ APCs and not NOD WT (I–A^g7) APCs. (F) Comparison of IFN-γ ELISA responses of splenic T cells to cardiac and soleus myosin in individual DQ8⁺NOD mice (n = 7) with antibody titers ranging from 1:100 to 1:6,400. Shown is the mean value of duplicate wells for each analysis (**P < 0.01).

Figure 4. CD4⁺ T cell clones specific for α-MyHC induce severe myocarditis when transferred into immunodeficient DQ8⁺Rag^–/–NOD mice.

(A) The host received 2.2 × 10⁷ clone E cells and was sacrificed 21 days later. H&E staining revealed extensive lymphocytic infiltration of the heart with cardiac myocyte destruction (insets). In contrast, skeletal muscle (B) was devoid of infiltrates. Infiltrates were also observed around the α-MyHC–expressing cardiac muscle layer surrounding the pulmonary veins (D, arrow). Heart (C) and lungs (lower panel of D) of saline control DQ8⁺Rag^–/–NOD mice that did not receive clone E are shown for comparison. The data are representative of 2 separate clone E transfers into DQ8⁺Rag^–/–NOD recipients. Scale bars: 200 μm in left panel of A, B, and C; 100 μm in right panels of A; and 300 μm in D.

Figure 5. Lack of expression of α-MyHC in thymic and LNSC subsets is associated with reduced T cell tolerance to cardiac myosin.

(A) Expression of control and tissue-restricted genes in whole thymus, purified mTECs, cTECs, DCs, and macrophages (Mϕ) from B6, NOD, and DQ8⁺NOD mice was determined by RT-PCR. Values represent microarray data (see Supplemental Table 1). Actb, β-actin; Ctsl, cathepsin L, Ctss, cathepsin S; Ins2, insulin II; Myh6, α-MyHC (red); Myh7, β-MyHC; Tnnt2, cardiac troponin T; Tnni3, cardiac troponin I; Chrna1, cholinergic receptor, nicotinic, α polypeptide 1; Co, plasmid DNA as positive control. (B) Relative mRNA expression levels of Myh6, Myh7, and GAD67 were assessed by quantitative RT-PCR in purified mouse mTECs and cTECs and control tissues. Data were normalized to Actb. (C) Analysis of Myh6 and Myh7 expression in purified LNSC subsets: FRCs, LECs, BECs, and DN cells. Mlana, tyrosinase, retinal S antigen (Ag), and Aire are positive control genes for FRC, LEC, BEC, and DN subsets, respectively. (D) T cell tolerance to cardiac and soleus myosin as assessed by in vitro recall proliferative responses of representative B6 and NOD mice 10 days after immunization with cardiac myosin (filled diamonds), and soleus myosin (open circles). Data represent mean ± SD of responses measured in triplicate. (E) Responses to indicated myosins in B6 and NOD mice as assessed by SI. Immunization and recall proliferation responses were performed as in D, and SI at 25 μg/ml antigen concentration was calculated as (counts – background counts/background counts) (*P < 0.05). The solid lines represent the mean values for each group.

Figure 6. Expression of α-MyHC in thymus prevents the development of myocarditis.

(A) Schema of the transgenic strategy to target expression of α-MyHC into thymic epithelium. (B) Western blot analysis of expression of α-MyHC protein in thymic, splenic, and lymph node lysates from NOD WT and the indicated samples probed with a monoclonal antibody to MyHC. (C) TaqMan RT-PCR analysis of expression of transgenic α-MyHC transcripts in thymus (Thy), spleen (Sp), lymph node, heart (He), kidney (Kid), liver (Liv), and lung (Lu). Shown are the representative results of 3 TOM⁺TA⁺ mice; values are expressed relative to TOM⁺TA⁺ thymus. n.d., not detected. (D) Prevalence of cardiac myosin autoantibodies in the indicated mice at 10 weeks of age (***P < 0.0001). (E) Comparison of IFN-γ responses of splenic T cells to stimulation with cardiac myosin in 22- and 12-week-old TOM⁺TA⁺DQ8⁺ mice and a 12-week-old female littermate. Shown is the mean ± SD of triplicate wells for each analysis. (F) H&E staining of heart sections from a 19-week-old female TOM⁺TA⁺DQ8⁺ and a littermate. Original magnification, ×10 and ×40 (insets). Scale bars: 400 μm and 100 μm (insets).

Figure 7. Lack of thymic expression of α-MyHC (MYH6) is associated with impaired T cell tolerance to cardiac myosin in humans.

(A) Relative expression levels of MYH6, MYH7, GAD65, and GAD67 were assessed by quantitative RT-PCR in purified human mTECs and cTECs and the respective control human tissues: heart atria, soleus muscle, and brain. Data from 3 human TEC samples are shown. Data were normalized to GAPDH, and relative expression was with respect to the control tissues. (B) IFN-γ ELISPOT analyses indicate that human peripheral blood MNCs respond to huCM-A but not to huSM. The panels show, from left to right, the mean ± SD of triplicate wells for each analysis on 3 patients with inflammatory heart disease (Pt 1: DR3, DR7; DQB1*0201,*0303, Pt 2: DR4, DR16; DQB1*0301,*0502, and Pt 3: DR3, DR4;DQB1*0201,*0302) and 3 subjects without clinical heart disease (C 1: DR1, DR3; DQB*0501, *0201; C 2: DQB1*0503, *0301, and C 3: DR3, DR7; DQB*0202, *0201). Representative IFN-γ responses of Pt 3 and C 3 are shown in the inset. Data in A and B are presented as mean ± SD.