Project 1: In recent years, evidence has been presented which posits a key role for the IL-12R beta2 chain in Th1/Th2 T cell differentiation. In particular, it has been suggested that down-regulation of this receptor and consequent cessation of IL-12 signaling is a prerequisite of Th2 T cell development. In the present study we examined this proposition by determining the effect of IL-12R beta2 chain signaling on beta2-chain transfected Th2 T cell lines and on Th2 T cells derived from mice bearing an IL-12R beta2 chain transgene. Using the first approach, we showed that D10.G4.1 T cells, a well-established Th2 T cell line, can be transfected with a vector containing an IL-12R beta2 chain cDNA under the control of a CD2 promotor and locus control region. Such T cells express an IL-12R composed of the beta1 and beta2 chains and transduce a STAT4 signal. However, when stimulated by antigen in the presence of IL-12, they do not produce significant amounts of IFN-gamma or manifest reduced IL-4 production. Using the second approach, we showed initially that double transgenic mice bearing both a transgene for the OVA TCR and a gene for the IL-12R beta2 chain do in fact overexpress IL-2R beta2 chains upon stimulation with OVA. We then showed that Th2 T cell lines derived from such mice and expressing the IL-12R beta2 chain produce minimal amounts of IFN- gamma upon restimulation with IL-12 and do not manifest reduced production of IL-4. Taken together, these studies show that Th2 T cells, cannot be made to revert to a Th1 phenotype even when re- stimulated through a competent IL-12 receptor. It is thus evident that down-regulation of the IL-12R beta2 chain is not the controlling factor in the switch to Th2 T cell development. Project 2: The role of TGF- beta1 in the regulation of T cell responses continues to be perplexing, possibly because its role is influenced by the type of T cell being regulated and the presence or absence of modifying cytokines in the microenvironment. In the present study, we attempted to overcome such problems by assessing TGF-beta1 effects under highly defined conditions. In initial studies we showed that TGF-beta1 has a profound inhibitory effect on na?ve CD4+ T cell responses whether the cells are stimulated under neutral, Th1 and Th2 priming conditions. In addition, if naive T cells are primed in the presence of TGF-beta1 they exhibit secondary anti-CD3/anti-CD28-induced and antigen-specific immune responses even in the absence of TGF-beta during secondary stimulation; thus, TGF-beta1 has an imprinting on ?carry-over? inhibitory effect. The impaired secondary immune response was not reversed by the addition of IL-2 and is not due to reduced expression of co-stimulatory molecules such as CD25, CD28, or CD80. A different pattern of TGF- beta1-mediated immune regulation was observed in relation to fully differentiated antigen-specific memory CD4+ T cells. Here we observed that antigen-specific activation of memory Th1 T cells is inhibited by TGF-beta1, but required higher levels of TGF-beta1 than observed for inhibition of na?ve T cells. This inhibition was associated with partial inhibition of STAT4 phosphorylation. In contrast, memory Th2 T cells are not subjected to TGF-beta1-mediated suppression, and STAT6 activation is not affected by TGF-beta1. These studies reveal that TGF- beta1 is a powerful negative regulatory of the primary immune response of CD4+ T cells, but only Th1 T cells are subject to such regulation after the memory stage of T cell differentiation has been reached. Thus, these studies define the potential regulatory role of TGF- beta1 in Th1 and Th2 T cell-mediated host defense and autoimmunity. - IL-12, IL-12R, Th1/Th2 differentiation, IFN-gamma, IL-4, TGF-beta1, T cell priming, STAT4, STAT6. - Human Subjects
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