The maintenance of transplantation tolerance induced in adult mice after short-term treatment with nonlytic monoclonal antibodies to CD4 and CD8 was investigated. CD4+ T cells from tolerant mice disabled naïve lymphocytes so that they too could not reject the graft. The naïve lymphocytes that had been so disabled also became tolerant and, in turn, developed the capacity to specifically disable other naïve lymphocytes. This process of "infectious" tolerance explains why no further immunosuppression was needed to maintain long-term transplantation tolerance.
CD3 antibodies are proven immunosuppressants capable of reversing transplant rejection episodes. Their general application has been limited both by their immunogenicity and, in particular, by the "first-dose" cytokine-release syndrome experienced by patients after the initial administration of antibody. We have produced a set of variants of the humanized YTH 12.5 CD3 monoclonal antibody (mAb) (Routledge et al., Eur. J. Immunol. 1991. 21: 2717) bearing different human heavy (H) chain constant regions, with the intention of finding a form of the antibody that is not able to activate T cells. Comparison of the variants having gamma 1, gamma 2, gamma 3 and gamma 4 H chains in a competitive binding assay showed that antibody avidity was not affected by IgG subclass. Using a sensitive indicator of FcR binding activity (the capacity of the CD3 mAb to redirect cytotoxic T cells to kill the monocytic cell line U-937) we demonstrated a functional hierarchy of gamma 1 = gamma 4 > alpha 2 =/> gamma 3 mb >> gamma 2. An aglycosyl version of the gamma 1 CD3 mAb, produced by site-directed mutagenesis (Asn297 to Ala), still had considerable activity in this assay (intermediate to the gamma 1 and alpha 2 CD3 mAb), albeit at a level approximately 10-fold lower than that of the parental gamma 1 form. When we tested their ability to stimulate T cell proliferation in vitro in the presence of 5% human serum, all of the wild-type immunoglobulin isotypes were found to be active, although there were T cell donor-dependent variations in the extent of the responses. The aglycosyl gamma 1 mAb was, however, completely non-mitogenic in all of ten donors tested, unless the assay was performed in IgG-free medium. Despite being non-stimulatory, this mAb was also able to inhibit the mixed lymphocyte reaction responses of both naive and primed T cells. Comparison of the gamma 1 and aglycosyl gamma 1 mAb in an experimental mouse model for CD3 mAb-induced cytokine release indicated that removal of the carbohydrate moiety from the gamma 1 constant region reduced the in vivo tumor necrosis factor-alpha response by a factor of at least 16-fold. These data suggest that the aglycosyl gamma 1 CD3 mAb is a promising candidate for immunosuppressive therapy without "first dose" side effects.
Mechanisms that allow potentially autoreactive T cells to escape central tolerance and persist in the peripheral lymphoid organs of healthy individuals are poorly defined. It has been proposed that such cells are specific for epitopes which normally are not well presented to the immune system or, in other words, are cryptic. We have used synthetic peptides to define potential T cell epitopes within the N-terminal portion of myelin basic protein (MBP). These were defined in terms of their relative affinity for the MHC-restriction element I-Au and their ability to activate T cells in mice of the H-2(u) haplotype. Three epitopes were identified, one of which corresponded to the known dominant N-terminal epitope (Ac1-9). The other two epitopes (9-20 and 5-20) bound to their MHC-restriction element with relatively high affinity but were cryptic, as defined by the poor response to these epitopes following immunization with intact MBP. Even the longer of these two epitopes did not induce autoimmune encephalomyelitis in H-2(u) mice. These results demonstrate that antigen processing can control both the induction of and effector function of autoreactive T cells, and is therefore a principal mechanism involved in limiting the autoreactive T cell repertoire.
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