Branched chain α‐ketoacid dehydrogenase kinase 111–130, a T cell epitope that induces both autoimmune myocarditis and hepatitis in A/J mice

Abstract

IntroductionOrgan‐specific autoimmune diseases are believed to result from immune responses generated against self‐antigens specific to each organ. However, when such responses target antigens expressed promiscuously in multiple tissues, then the immune‐mediated damage may be wide spread.MethodsIn this report, we describe a mitochondrial protein, branched chain α‐ketoacid dehydrogenase kinase (BCKDk) that can act as a target autoantigen in the development of autoimmune inflammatory reactions in both heart and liver.ResultsWe demonstrate that BCKDk protein contains at least nine immunodominant epitopes, three of which, BCKDk 71–90, BCKDk 111–130 and BCKDk 141–160, were found to induce varying degrees of myocarditis in immunized mice. One of these, BCKDk 111–130, could also induce hepatitis without affecting lungs, kidneys, skeletal muscles, and brain. In immunogenicity testing, all three peptides induced antigen‐specific T cell responses, as verified by proliferation assay and/or major histocompatibility complex class II/IAk dextramer staining. Finally, the disease‐inducing abilities of BCKDk peptides were correlated with the production of interferon‐γ, and the activated T cells could transfer disease to naive recipients.ConclusionsThe disease induced by BCKDk peptides could serve as a useful model to study the autoimmune events of inflammatory heart and liver diseases.

β1-Adrenergic Receptor Contains Multiple IAk and IEk Binding Epitopes That Induce T Cell Responses with Varying Degrees of Autoimmune Myocarditis in A/J Mice

“…Analysis was performed by a board-certified pathologist blinded to treatment. After ascertaining the inflammatory changes, total number of inflammatory foci were determined by sections with the largest number of foci or by adding non-overlapping foci across sections as reported previously ( 26 , 28 , 30 , 31 ).…”

“…To detect T cells in hearts, formalin-fixed paraffin-embedded tissue sections were stained with rabbit anti-mouse CD3 (clone SP7, 1:100, Abcam, Cambridge, MA, USA), rabbit anti-mouse CD4 (polyclonal, 1:100, Novus Biologicals, Littleton, CO, USA), and rabbit anti-mouse CD8 (clone EP1150Y, 1:100, Novus Biologicals) or their isotype controls. For non-T cells namely, neutrophils, macrophages, and B cells, rat anti-mouse Ly6G (clone 1A8, 1:250, Leinco Technologies, Fenton, MO, USA), rabbit anti-mouse CD11b (clone EPR1344, 1:3,500, Abcam) ( 30 ), and rat anti-mouse CD19 (clone 6OMP31, 1:1000, Thermo Fisher Scientific, San Diego, CA, USA) and their isotype controls were used, respectively. In brief, after deparaffinization, rehydration, and blockade of endogenous peroxidase activity, antigen retrieval was performed by treating the sections with 10 mM sodium citrate buffer (pH 6.0) in a water bath at 98°C for 15–40 min or using a pressure cooker.…”

Identification of Immunogenic Epitopes That Permit the Detection of Antigen-Specific T Cell Responses in Multiple Serotypes of Group B Coxsackievirus Infections

“…Lymph node cells (LNCs) and splenocytes obtained from animals on day 10 post-immunization or infected animals at termination were used to assess their proliferative responses based on tritiated 3 [H] thymidine-incorporation assay [20][21][22]. After stimulating the cells with indicated peptides for 2 days, and adding 3 [H] thymidine (1 µci/well) during the next 16 h, proliferative responses were measured as counts per minute (cpm).…”

“…Europium-labeled streptavidin (SA) was then added to a concentration of 1 µg/mL in dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) buffer followed by DELFIA-enhancement solution (Perkin Elmer, Waltham, MA, USA). After measuring the fluorescence intensities at excitation/emission wavelengths of 340/615 nm, the half-maximal inhibitory concentration (IC 50 ) values were then calculated as described [20][21][22].…”

“…All sections were examined by a board-certified pathologist blinded to treatment. The total number of inflammatory cell foci was determined as reported previously [20,21].…”