Molecular mechanism of immune suppression by negative-feedback regulation

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To understand negative regulation of T cell activation, we have analyzed three molecules with inhibitory function, an inhibitory adaptor, Gab2,an inhibitory co-stimulation receptor, CTLA-4,and a newly ITAM^+ molecule, NFAM-1.Gab2 is phosphorylated by ZAP-70 upon TCR stimulation and mediated negative signals through recruitment of a phosphatase SHP-2. Furthermore, we found Gab2 associates with Grb2/Gads by competing with SLP-76,which is another inhibitory mechanism to mediate inhibition. Gab2-Tg mice show decreased T cell activation and block negative selection of thymocytes. These findings indicate that Gab2 function as an inhibitory adaptor for activation and development of T cells.We analyze the function of CTLA-4 in Treg function by establishing CTLA-4-Tg mice since Treg shows constitutive expression of CTLA-4 and could be involved in Treg-mediated suppression. In Tg mice, the number of Treg was reduced, but their suppressive function was not altered. In addition, by isolating Trig from CTLA-4-deficient thymocytes, we found that CTLA-4-/-Treg exhibited similar suppressive function. We concluded that CTLA-4 is not critical for suppressive function of Treg.We have developed a functional cloning system (NACS) using a reporter T cells expressing NFAT-GFP and a CD8-chimeric cDNA library, and cloned a new ITAM-bearing molecule, NFAM-1. When expressed in T or B cells, NFAM-1-ITAM was tyrosine-phosphorylated and recruited ZAP-70 or Syk upon stimulation. NFAM-1-Tg mice revealed strong blockade of development of B cells but not T cells. However, the NFAM-1-KO mice showed no significant defects of the development of both T and B cell development, suggesting that there is a redundancy with other molecules in vivo. NFAM-1 did not have any charged amino acid residues within the transmembrane region, it may represent a new family of ITAM^+ molecules.