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Previous studies have shown that mouse CD8+ T lymphocyte clones (TLC) produce T cell-specific serine proteinase-1 (MTSP-1) as well as a family of six homologous molecules, termed granzymes B - G, which are structurally related to serine proteinases. Of these proteins, only MTSP-1 has been studied in detail. It has been shown to occur in the majority of CD8+ and a fraction of CD4+ T effector cells in vivo and in vitro and has demonstrable enzyme activity in these cells. The presence of the other serine proteinase-like molecules in T cells is less well defined. We have now analyzed the expression of mRNA species specific for granzymes B - G in activated T cell populations using the sensitive polymerase chain reaction which allows the detection of mRNA species from as little as 2 pg of total cytoplasmic RNA. We demonstrate that MTSP-1 and all six serine proteinase-like transcripts are expressed in a panel of four CD8+ and six CD4+ long-term-cultured TLC, though at greatly differing concentrations. In contrast, in vivo primed T cells of both phenotypes, CD4+ and CD8+, and in vitro activated T cells derived from short-term cultures only express mRNA species specific for MTSP-1 and CCP1 and little of those for CCP2, but no transcripts for granzymes D - G. These findings argue against the participation of granzymes D - G in T cell-mediated functions in vivo.

SummaryThe role of antigen presentation as a possible mechanism underlying major histocompatibility complex (MHC) association of autoimmune disease has been studied in non-obese diabetic (NOD) mice. By screening for inhibition of antigen presentation to NOD T cell hybridoma, we have selected a synthetic peptide, yTYTVHAAHAYTYt (small letters denote D amino acids), that efficiently blocks antigen presentation by the NOD class II MHC molecule AoegTA/3g 7 (As 7) in vitro. The inhibition is MHC selective, in that it does not affect antigen presentation by the E d and E k molecules, and has only a marginal effect on presentation by the A d molecule. This peptide also inhibits the priming for As7-restricted T cell responses in vivo, and prevents the spontaneous development of diabetes in female NOD mice, when administered chronically from 3 wk of age on. Chronic treatment with a control peptide, KMKMVHAAHAKMKM, that fails to bind to Ag 7 has no effect on the disease. These data indicate that antigen presentation by the As 7 molecule plays a pivotal role in the induction of autoimmune diabetes. Furthermore, the results demonstrate that interference with antigen presentation by a class II molecule can prevent the onset of spontaneous autoimmune disease associated with the same molecule. Susceptibility to autoimmune diseases is associated with certain class I or II alleles of the MHC (1). Similarly, animal models of autoimmune diseases often exhibit MHC linkage (2-6). Recently, two sets of findings have shed light on the, thus far enigmatic, mechanisms underlying MHC--disease association. First, MHC molecules have been shown to possess a binding site capable of interacting with antigenic peptides in an allele-dependent, selective manner (7-10). Second, the intramolecular mapping of MHC--disease linkages has revealed that in certain instances susceptibility or resistance depends on particular amino acid residues localized within the peptide-binding site (11)(12)(13)(14). Thus, the most likely explanation for MHC-disease association would be that the disease-inducing autoantigen is selectively presented by the particular allelic form of MHC molecule that confers disease susceptibility (15). Since, however, the antigens involved in most autoimmune diseases are unknown, this hypothesis is not directly testable. Here, we have taken the approach of MHC blockade (16--18) to test the above hypothesis experimentally. Using the non-obese diabetic (NOD) mouse as an autoimmune disease model (19), we demonstrate that a peptide capable of blocking antigen presentation by the NOD class II MHC molecule As 7, in vitro and in vivo, can also prevent the spontaneous development of type I diabetes linked to this class II molecule (6). Materials and MethodsMice. NOD mice, originally derived from Dr. E. Leiter's colony (The Jackson Laboratory, Bar Harbor, ME), were bred under specific pathogen-free conditions in the Sandoz animal facilities. Diabetes occurs in 65-72% of female and 15-24% of male mice in this colony.Treatment of NOD Mice with P...

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T cell clones isolated from class II MHC-disparate MLR combinations, and specific for I-Ak and I-Ek molecules, respectively, are shown to induce acute lethal graft-vs-host disease in unirradiated recipients. Cytolytic and noncytolytic clones are equally efficient in this respect. The lethal disease is dependent on recognition of the stimulatory class II molecules in the host. The clones home to lungs and liver, and become activated in these organs as demonstrated by an in vivo thymidine incorporation assay. After activation, a severe vascular leak syndrome develops causing death of the recipients within 5 d after the injection of 5 x 10(6) to 10(7) cloned cells. The disease develops without the participation of secondary host-derived inflammatory mechanisms, such as mast cell degranulation, complement activation, and the release of prostaglandins, oxygen radicals, or proteolytic enzymes. The results raise the possibility that Th cells can directly influence vascular permeability, and control, thereby, the acute inflammatory reaction of blood vessels.

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Autologous sperm are immunogenic and can elicit cell-mediated immunity and antibody responses (1-4). Immune responses against spermatozoa do not occur under normal circumstances, due to the fact that germ cells are efficiently sequestered from the immune system by the blood-testis barrier (5). This barrier, which is established before the immune system becomes competent, creates an immunologically privileged site for sperm and the developing spermatozoa. Therefore, immunological tolerance is not generated to autologous male germ cells. Injuries of the blood-testis barrier (e.g., after vasectomy, infection, or biopsy) can result in the production of autoantibodies and T cell reactivity (1-4).Because the immune system is not normally exposed to germ cells, it is possible that sperm and other germ cells exert regulatory infuences on immune potential. In the present study, the influence of inoculation of germ cells on cellular immune reactions was investigated. The results indicate that male C5 7BL/6 mice injected with syngeneic germ cells exhibited: (a) reduced natural killer (NK) 1 cell activity; (b) reduced mixed lymphocyte reactivity (MLR); (c) enhanced auto-proliferation; and (d) decreased potential to generate cytotoxic T lymphocyte (CTL) responses to modified self and allogeneic antigens. The reduction in CTL potential against trinitrophenyl-modified syngeneic spleen cells (TNP-self) was mediated by radiosensitive suppressor T cells. These findings are discussed with respect to possible effects of exposure to male germ cells on the immune system. Materials and MethodsAnimals. Young adult mice (8-12-wk) were used for all in vitro sensitization studies. The inbred strains C57BI6/J (B6), AKR/J, B10.BR (BR), DBA2/J, and (B6 X DBA/2)F1 (BDFI) were purchased from The Jackson Laboratory, Bar Harbor, ME.Cell Preparations. Testicular cell suspensions were prepared as described elsewhere (6). Briefly, testicular (Te) cells were obtained from the seminiferous tubules by protease treatment (7). After removal of the capsule, the testes were incubated at 37°C for 12 min in 0.1% collagenase (Worthington Biochemical Corp., Freehold, NJ), and dissolved in phosphatebuffered saline (PBS). These elements were then broken up by a 15-min incubation at 37°C in 0.025% trypsin (Gibco Laboratories, Grand Island Biological Co., Grand Island, NY), dissolved * Supported by the Deutsche Forschungsgemeninschaft. ' Abbreviations used in this paper: C,