Here, we have studied the activity of a novel proteintyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colonystimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.

Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.

When challenged with extracellular ATP, leukocytes respond and activate processes attributed to the P2X7 receptor (P2X7R), an unusual ligand-gated ion channel. To prove P2X7R involvement, blood samples from P2X7R-deficient mice were characterized. Monocytes and lymphocytes associated with wild-type blood responded to ATP and underwent volume/shape changes and shed L-selectin. In contrast, leukocytes from P2X7R-deficient animals demonstrated no change in physical properties or L-selectin expression following ATP challenge. Blood stimulated with LPS or ATP individually generated minimal quantities of the leaderless polypeptide IL-1β, but sequential treatment of wild-type, but not P2X7R-deficient, blood with LPS and ATP yielded large amounts of cell-free cytokine. Based on these differences, wild-type and P2X7R-deficient animals were compared following induction of monoclonal anti-collagen-induced arthritis. Ab-treated wild-type animals subsequently challenged with LPS developed inflamed, swollen paws; their joint cartilage demonstrated lesions, loss of proteoglycan content, and the presence of collagen degradation products. P2X7R-deficient animals subjected to the same challenge were markedly less affected; both the incidence and severity of disease were reduced. These data indicate that ATP does act via the P2X7R to affect leukocyte function and that the P2X7R can serve as an important component of an in vivo inflammatory response.

Cathepsins have been implicated in the degradation of proteins destined for the MHC class II processing pathway and in the proteolytic removal of invariant chain (Ii), a critical regulator of MHC class II function. Mice lacking the lysosomal cysteine proteinase cathepsin S (catS) demonstrated a profound inhibition of Ii degradation in professional APC in vivo. A marked variation in the generation of MHC class II-bound Ii fragments and presentation of exogenous proteins was observed between B cells, dendritic cells, and macrophages lacking catS. CatS-deficient mice showed diminished susceptibility to collagen-induced arthritis, suggesting a potential therapeutic target for regulation of immune responsiveness.