T cells expressing the gamma delta T cell receptor induce apoptosis in cardiac myocytes.
OBJECTIVE
Enterovirus infections are major etiological factors in myocarditis and dilated cardiomyopathy. Using an experimental murine model of this disease, previous studies have shown that myocarditis susceptibility depends upon activation of T lymphocytes expressing the gamma delta T cell receptor (TcR), and that only mouse strains which accumulate gamma delta T cells in the myocardium show apoptosis of myocytes or evidence of dilated cardiomyopathy-like disease. The objective of the present studies is to demonstrate that gamma delta T cells directly induce greater Fas-dependent apoptosis of cultured myocytes than T cells expressing the alpha beta TcR.


METHODS
Bl.Tg.E alpha mice were infected for 7 days with coxsackievirus B3 (CVB3). Hearts were removed and were either formalin-fixed, sectioned and stained with hematoxylin and eosin for inflammation, and using TdT-TUNEL for apoptosis, or were minced and collagenase digested for isolation of gamma delta+ and alpha beta+ T cells using immunomagnetic bead separation. Neonatal cultures of cardiac myocytes were isolated from mice less than 2 days old by collagenase and pancreatin digestion, and were either untreated or infected with virus. Levels of Fas (CD95) were measured using FITC-conjugated hamster anti-mouse Fas monoclonal antibody and flow cytometry. Susceptibility of myocytes to Fas-dependent killing was measured by 51Cr-release by labeled myocytes incubated for 4 h on either 3T3-mock or 3T3-FasL transfected cell monolayers. Killing by T cells was also measured in a 4 h 51Cr-release assay. Fas-dependent and perforin-dependent cytotoxicity was determined by specific blocking using either Fas-Fc or concanamycin A.


RESULTS
Virally infected myocyte cultures showed significantly enhanced Fas expression compared to uninfected cells, with maximal upregulation of Fas occurring 18-24 h after virus infection. Both infected and uninfected myocytes were selectively killed by FasL-transfected 3T3 cells but not by mock control cells. Approximately 38% of CD3+ lymphocytes isolated from the heart express the gamma delta TcR with the remainder expressing the alpha beta TcR. Both gamma delta+ and alpha beta+ T cells lysed myocyte targets. Blocking studies indicate that gamma delta+ T cells induced predominantly Fas-mediated killing, while alpha beta+ cell produced more perforin-mediated death, although these effectors were capable of Fas-dependent killing as well.


CONCLUSIONS
These studies demonstrate that T cells expressing the gamma delta TcR are more effective mediators of myocyte apoptosis than alpha beta+ T cells in vitro and suggests that these effectors may be primarily responsible for myocardial injury associated with dilated cardiomyopathy-like signs during coxsackievirus B3-induced myocarditis.
Huber
S A
SA
Department of Pathology, University of Vermont, Colchester 05446, USA. shuber@salus.uvm.edu
D000818:Animals; D017209:Apoptosis; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002450:Cell Communication; D002478:Cells, Cultured; D018920:Coculture Techniques; D003601:Cytotoxicity Tests, Immunologic; D005434:Flow Cytometry; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009205:Myocarditis / Q000276:immunology* / Q000503:physiopathology; D009206:Myocardium / Q000276:immunology*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics / Q000378:metabolism*; D019014:fas Receptor / Q000378:metabolism
D016692:Receptors, Antigen, T-Cell, gamma-delta; D019014:fas Receptor