Anergy in vivo: down-regulation of antigen-specific CD4+ Th1 but not Th2 cytokine responses.

Abstract

Efficient immunologic tolerance, defined as antigen-specific unresponsiveness, can be peripherally induced by the i.v. injection of syngeneic splenocytes coupled with antigen using ethylene carbodiimide (ECDI). We have previously reported that unresponsiveness induced via i.v. injection of syngeneic splenocytes coupled with intact, UV-inactivated Theiler's murine encephalomyelitis virus (TMEV-SP) resulted in 'split tolerance'. Both virus-specific delayed-type hypersensitivity and IgG2a levels were inhibited, whereas IgG1 levels were increased when compared with sham tolerized controls. In the present report we demonstrate that tolerance induced by i.v. injection of TMEV-coupled splenocytes resulted in antigen-specific inhibition of T cell proliferation, as well as IL-2 and IFN-gamma production in response to both whole TMEV and the immunodominant viral epitope. Additionally, tolerance induction resulted in abrogation of Th1-derived [IL-2, IFN-gamma and LT/tumor necrosis factor-beta (TNF-beta)] cytokine mRNA expression in response to in vitro stimulation with UV-inactivated TMEV as determined by reverse transcriptase polymerase chain reaction. In contrast, expression of Th2-derived (IL-4, IL-6 and IL-10) cytokine mRNA was not affected in tolerized mice. Tolerance functioned directly at the level of CD4+ Th1 cells at both the induction and effector limbs as depletion of CD8+ T cells both prior to in vivo tolerization or in vitro culture had no effect on inhibition of Th1-specific responses. The mechanism of in vivo tolerance induction appeared to be anergy of CD4+ Th1 cells since IL-2, IFN-gamma and LT/TNF-beta mRNA expression as well as virus-specific proliferative responses could be restored by addition of rIL-2 to in vitro cultures of tolerant, CD4+ Th1 populations. These results suggest that in vivo 'split tolerance' induced by i.v. injection of ECDI-fixed, antigen-coupled splenocytes involves anergy of TMEV-specific, CD4+ Th1 lymphocytes and concomitant priming of Th2 cells. The induction of antigen-specific, in vivo anergy has important implications in the design of therapeutic strategies for immunopathologic diseases mediated by Th1 lymphocytes, especially T cell-mediated autoimmune disorders.