We established long-term cell lines of cytotoxic T lymphocytes (CTL) specific for human T cell leukemia virus type I (HTLV-I) from peripheral blood lymphocytes (PBL) of a patient with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), an HTLV-I-carrier with Sjögren syndrome, and an asymptomatic HTLV-I-carrier, by repeated stimulation with autologous HTLV-I-infected T cells in vitro. CTL derived from the patient with HAM/TSP expressed CD8 antigen, and their function was restricted by HLA-A2. They showed cytotoxic effects predominantly against the target cells expressing HTLV-I p40tax among the autologous B cell lines infected with vaccinia recombinants containing various HTLV-I genes which served as targets. These data are consistent with the previously reported findings that fresh PBL of HAM/TSP patients contain p40tax-specific CTL activity. Furthermore, CTL derived from the patient with Sjögren syndrome without neurological involvement also demonstrated cytotoxicity predominantly to p40tax. The cytotoxicity to the target cells experimentally expressing p40tax was blocked by unlabeled HTLV-I-infected cells possessing HLA-A2. HTLV-I-specific cytotoxicity was also inhibited by unlabeled B cells bearing p40tax. Thus, HTLV-I p40tax-specific cytotoxicity is mediated by the major CTL population activated by native HTLV-I antigens in patients with HAM/TSP or Sjögren syndrome. In contrast to the CTL of these patients, CTL similarly induced from the asymptomatic HTLV-I-carrier, which were highly cytotoxic to autologous HTLV-I-infected T cells, did not show significant levels of cytotoxicity to autologous B cells expressing p40tax.(ABSTRACT TRUNCATED AT 250 WORDS)

Doc2 has two C2 domains that interact with Ca2؉ and phospholipid. Munc13 has two C2 domains and one C1 domain that interacts with phorbol ester or diacylglycerol (DAG) and phospholipid. Both Doc2 and Munc13 are implicated in Ca 2؉ -dependent neurotransmitter release, but their modes of action still remain unclear. We show here that Doc2 interacts with Munc13 both in a cell-free system and in intact PC12 cells during the high K We have isolated Doc2 as a novel protein having two C2 domains that interact with Ca 2ϩ and PL 1 (1). Doc2 consists of two isoforms, Doc2␣ and Doc2␤ (1, 2). Doc2␣ is specifically expressed in neuronal cells, whereas Doc2␤ is ubiquitously expressed (1-3). Both isoforms have at least the N-terminal Doc2-specific region and C-terminal two C2 domains. We have moreover shown that overexpression of the N-terminal fragment of Doc2␣ or its C-terminal fragment including the C2 domains in PC12 cells inhibits Ca 2ϩ -dependent exocytosis (4).These results suggest that Doc2␣ is involved in Ca 2ϩ -dependent exocytosis and interacts with another component of Ca 2ϩ -dependent exocytotic machinery. To clarify the mode of action of Doc2␣ in Ca 2ϩ -dependent exocytosis, it is important to isolate its interacting protein(s). We have attempted here to isolate a Doc2␣-interacting protein from a rat brain cDNA library by use of the yeast two-hybrid system and isolated Munc13 as a Doc2␣-interacting protein.Munc13 has been isolated as a mammalian homologue of Caenorhabditis elegans unc-13, which is implicated in Ca 2ϩ -dependent neurotransmitter release (5, 6). Munc13 has three isoforms, Munc13-1, -2, and -3. All the isoforms have two C2 domains and Munc13-1 has another atypical C2 domain. They have moreover one C1 domain that interacts with PE or DAG and PL (5-7). Munc13 is specifically expressed in neuronal cells, and Munc13-1 is localized at the presynaptic plasma membrane (6).We describe here that Doc2␣ directly interacts with Munc13-1 in a DAG-dependent manner and that the Doc2␣-Munc13-1 interactions play an important role in Ca 2ϩ -dependent exocytotic machinery. EXPERIMENTAL PROCEDURESTwo-hybrid Assay-The N-terminal fragment (1-90 aa) of human Doc2␣ cDNA (1) was inserted into the pBTM116 (pLexA-Doc2␣N). The yeast reporter strain L40 was transformed with pLexA-Doc2␣N and a rat brain cDNA library constructed in pGAD10 (CLONTECH). Library plasmids from positive clones were analyzed by transformation tests and DNA sequencing. Overlapping clones containing the full-length coding region of Munc13-1 were isolated by screening the rat brain cDNA library. The cDNA fragments encoding several Munc13-1 deletion mutants were constructed from the overlapping clones and inserted into pGAD424. The cDNA fragments encoding several Doc2␣ deletion mutants were inserted into pBTM116. After co-transformation into yeast strain L40, ␤-galactosidase activity was assayed by liquid and filter assays (8,9).Preparation of Recombinant Proteins-The cDNA fragments encoding the N-terminal fragment (1-90 aa) of human Doc2␣ (1) and Munc13-1-...

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We previously isolated a new protein having two C2-like domains which interacted with Ca 2؉ and phospholipid and named Doc2 (Double C2). Because Doc2 was abundantly expressed in brain where it was highly concentrated on the synaptic vesicle fraction, we have examined here whether Doc2 is involved in Ca