Anergy in vivo: Down-regulation of antigen-specific CD4⁺ T_h1 but not T_h2 cytokine responses

Efficient immunologic tolerance, defined as antlgen-speclflc unresponslveness, can be peripherally induced by the l.v. Injection of syngenelc splenocytes coupled with antigen using ethylene carbodilmlde (ECDI). We have previously reported that unresponslveness induced via l.v. Injection of syngenelc splenocytes coupled with intact, UV-lnactlvated Theiler's murine encephalomyelitis virus (TMEV-SP) resulted in 'split tolerance'. Both vtrus-speclflc delayed-type hypersensltlvlty and lgG2a levels were inhibited, whereas lgG1 levels were increased when compared with sham tolerized controls. In the present report we demonstrate that tolerance induced by l.v. Injection of TMEV-coupled splenocytes resulted in antigen-specific inhibition of T cell proliferation, as well as IL-2 and IFN-γ production in response to both whole TMEV and the immunodomlnant viral epitope. Additionally, tolerance induction resulted in abrogation of T_h1 -derived [IL-2, IFN-γ and LT/tumor necrosis factor-β (TNF-β)] cytokine mRNA expression in response to In vitro stimulation with UV-inactlvated TMEV as determined by reverse transcrlptase polymerase chain reaction. In contrast, expression of T_h2-derived (IL-4, IL-6 and IL-10) cytokine mRNA was not affected in tolerized mice. Tolerance functioned directly at the level of CD4⁺ T_h1 cells at both the induction and effector limbs as depletion of CD8⁺ T cells both prior to in vivo tolerizatlon or in vitro culture had no effect on inhibition of T_h1-specific responses. The mechanism of In vivo tolerance induction appeared to be anergy of CD4⁺ T_h1 cells since IL-2, IFN-γ and LT/TNF-β mRNA expression as well as virus-specific prollferatlve responses could be restored by addition of rlL-2 to In vitro cultures of tolerant, CD4⁺ T_h1 populations. These results suggest that in vivo 'split tolerance' Induced by l.v. Injection of ECDI-flxed, antigen-coupled splenocytes involves anergy of TMEV-speclflc, CD4⁺ T_h1 lymphocytes and concomitant priming of T_h2 cells. The induction of antlgen-speclflc, in vivo anergy has important implications in the design of therapeutic strategies for immunopathologic diseases mediated by T_h1 lymphocytes, especially T cell-mediated autoimmune disorders.