Non-cytotoxic functions of lymphoycte granule exocytosis

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T lymphocytes function by secretion of mediators which influence other cells. Secretion occurs via two pathways: the "constitutive" pathway, in which newly synthesized proteins pass through the Golgi and are immediately released by exocytosis of small vesicles; and the "regulated" pathway in which mediators are stored in larger granules until TcR engagement signals their exocytosis. Although in lymphocytes the regulated pathway has been exclusively associated with cytotoxicity and the secretion of perforin and granzymes, we have investigated whether other mediators are secreted via this pathway, and if it operates in both CD4+ and CD8+ T cells. We have evaluated the importance of granule exocytosis to chemokine secretion by both CD8+ and CD4+ T cell blasts. Purified subpopulations of both CD4+ and CD8+ human T cell blasts were found to rapidly secrete the granule enzyme b-hexosaminidase in response to plate-bound anti-CD3, suggesting that CD4+ T cells have a functional granule-like compartment. We measured the secretion of chemokines and g-interferon under these conditions, using resistance to cycloheximide (which blocks protein synthesis) and Brefeldin A (which blocks Golgi export) to assess the contribution of granules. In both CD4+ and CD8+ T cells, TcR-induced secretion of RANTES and MIP-1a at 2-4 hours was resistant to these drugs, while TcR-induced g-interferon was abolished. At later times, the constitutive pathway dominates chemokine secretion. Microscopy and flow cytometry show that RANTES is present in granules in perforin-negative CD8+ and CD4+ T cell blasts. These results demonstrate that granules rapidly deliver non-cytotoxic mediators in both CD8+ and CD4+ effector T cells. Further evidence for operation of the regulated pathway in CD4+ T cells comes from flow cytometry experiments showing that the lysosomal membrane markers LAMP-1 and LAMP-2 are undetectable on the surface of resting T blasts, but are expressed on the surface of both CD4+ and CD8+ blasts within an hour of TcR engagement.