CD8+ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL. TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route. These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.
Abstract. To investigate the question of whether lyric granules share a common biogenesis with lysosomes, cloned cytolytic T cell lines were derived from a patient with I-cell disease. The targeting of two soluble lyric granule components, granzymes A and B, was studied in these cells which lack a functional mannose-6-phosphate (Man-6-P) receptor-mediated pathway to lysosomes. Using antibodies and enzymatic substrates to detect the lytic proteins, I-cells were found to constitutively secrete granzymes A and B in contrast to normal cells in which these proteins were stored for regulated secretion. These results suggest that granzymes A and B are normally targeted to the lytie granules of activated lymphocytes by the Man-6-P receptor.In normal cells, the grartzymes bear Man-6-P residues, since the oligosaccharide side chains of granzymes A and B, as well as radioactive phosphate on granzyme A from labeled cells, were removed by endoglycosidase H (Endo H). However, in I-ceils, granzymes cannot bear Man-6-P and granzyme B acquires complex glycans, becoming Endo H resistant.Although the levels of granzymes A and B in cytolytic I-cell lymphocytes are <30% of the normal levels, immunolocalization and cell fractionation of granzyme A demonstrated that this reduced amount is correctly localized in the lytic granules. Therefore, a Man-6-P receptor~'ndependent pathway to the lytic granules must also exist. Cathepsin B colocalizes with granzyme A in both normal and I-cells indicating that lysosomal proteins can also use the Man-6-P receptor-independent pathway in these cells. The complete overlap of these lysosomal and lytic markers implies that the lytic granules perform both lysosomal and secretory roles in cytolytic lymphocytes. The secretory role of lytic granules formed by the Man-6-P receptor-independent pathway is intact as assessed by the ability of I-cell lymphocytes to lyse target cells by regulated secretion.
SummaryTransgenic mice carrying and expressing the human CD3e gene incorporate the corresponding protein product into T cell receptor (TCR)/CD3 complexes on thymocyte and T cell surfaces. The chimeric antigen receptors allow normal T cell development and selection of repertoires in vivo and are able to transduce activation signals in vitro. We have exploited the ability to distinguish mouse (m) and human (h)CD3e chains to analyze the stoichiometry of CD3e in transgenic mouse TCRs. Immunoprecipitation and fluorescence resonance energy transfer experiments demonstrate that such TCRs can contain both h-and mCD3e chains, implying that more than one CD3e subunit occurs per TCR. Antigen comodulation studies are consistent with a stochastic use ofh-or mCD3e during receptor assembly, and further suggest a structure for the TCR/CD3 complex with two CD3e chains. The determination ofCD3e subunit stoichiometry, together with existing biochemical data, allows the generation of a minimal model for the structure of the TCR and illustrates the potential value of the transgenic approach to the analysis of complex receptors.
CTLs from patients with Chediak-Higashi syndrome (CHS) are unable to destroy target cells recognized via the TCR. To determine the mechanism responsible for the loss of cytotoxicity, CD8+ CTL clones have been derived from a patient with CHS. Individual CTL clones show poor killing that can be increased in longer assays. However, in the presence of cycloheximide, the small amount of killing observed is abolished, indicating killing arises from newly synthesized proteins, rather than from proteins stored in granules. In this study, we show that the CHS CTL clones express normal levels of the lytic proteins granzyme A, granzyme B, and perforin, which are processed properly during biosynthesis and targeted correctly to giant lytic granules. Despite the difference in size, CHS and normal lytic granules are similar, in that both contain the lysosomal enzyme cathepsin D and the lytic protein granzyme A, and lack the mannose-6-phosphate receptor (MPR). However, unlike normal CTL clones, the CHS CTL clones are unable to secrete their giant granules in which the lytic proteins are stored. After cross-linking the TCR, CHS CTL clones fail to secrete granzyme A, as assayed by both enzyme release and confocal microscopy. We suggest that the defect in CHS lies in a protein that is involved in membrane fusion and is essential for the secretion of lysosomal compartments in certain hemopoietic cells.
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