Cellular requirements for renal allograft rejection in the athymic nude rat.

This study has examined the ability of adoptively transferred CD4+ and CD8+ T cells to mediate rejection of a fully allogeneic DA renal graft in the PVG nude rat. Transfer, at the time of transplantation, of naive CD4+ T cells caused rapid graft rejection and primed CD4+ cells were several times more potent. In contrast, naive or specifically sensitized CD8+ cells were entirely ineffective at mediating renal allograft rejection. Whereas nonrejecting grafts showed only a mild cellular infiltrate, rejecting grafts in CD4+ reconstituted animals showed a substantial infiltrate and many of the infiltrating cells had a phenotype (MRC OX8+, MRC OX19-), consistent with NK cells. Experiments using a mAb (HIS 41) against an allotypic determinant of the leukocyte common antigen confirmed that the majority (greater than 80%) of the cellular infiltrate in rejecting grafts derived from the host rather than from the CD4+ inoculum. Infiltrating mononuclear cells, obtained from rejecting allografts 7 d after transplantation in CD4+-injected PVG nude hosts, showed high levels of in vitro cytotoxicity against not only kidney donor strain Con A blasts but also third-party allogeneic Con A blasts, as well as against both NK and LAK susceptible targets. When splenocytes from nontransplanted nude PVG rats were tested in vitro they also demonstrated high levels of lytic activity against both NK and LAK susceptible targets as well as allogeneic Con A blasts, which were not susceptible to lysis by spleen cells from euthymic rats. These findings suggest that injected CD4+ cells may cause renal allograft rejection by the recruitment of extrathymically derived, widely alloreactive cells into the kidney in this model of graft rejection.

In vitro and in vivo effects of monoclonal antibodies against T cell subsets on allogeneic and xenogeneic responses in the rat.

The Syrian hamster-to-rat represents an example of a concordant species difference, and therefore organ transplants using the hamster as the donor and the rat as the recipient are not rejected hyperacutely, as in discordant species combinations. Cellular mechanisms of xenogeneic rejection of hamster hearts by rats were studied both in vitro and in vivo, using monoclonal antibodies to rat T cell antigens. The results of this study reveal that CD4-positive cells of rats proliferated in vitro to both allogeneic stimulators and xenogeneic stimulators from a concordant strain, but required accessory cells of the responder phenotype to proliferate to discordant human stimulators. Monoclonal antibody therapy was used to prevent graft rejection in allogeneic and xenogeneic species combinations, using the rat as the recipient. Treatment with anti-CD4 antibodies was effective in prolonging allograft survival across a full MHC mismatch. No rejection occurred during antibody therapy, and long-term graft survival was achieved in 1/3 of transplanted grafts. The same monoclonal antibody therapy led to increased survival of grafts from hamster donors, but all of these grafts were rejected during therapy, and no long-term graft survival was achieved. Anti-CD8 antibody therapy, combined with anti-CD4 did not improve survival of hamster hearts in rats. Addition of cyclosporine to the anti-CD4 regimen also did not improve graft survival. Injection of an anti-T cell receptor antibody was no better than the anti-CD4 antibody in prolonging the survival times of heart grafts from the concordant xenogeneic species. These data suggest that the rejection of concordant xenogeneic tissue is not wholly a T cell-dependent phenomenon.

Simultaneous transplantation and intrathymic tolerance induction. A method with clinical potential.

It has been shown that donor-specific tolerance to cardiac allografts can be induced by pretreating the prospective recipient with injections of donor splenocytes (intrathymically) and antilymphocyte serum (intraperitoneally) weeks or days before the actual transplantation. This procedure, however, lacks clinical relevance in the case of cadaver donors due to the obligatory interval between the start of the tolerance induction protocol and transplantation. We have tried to devise a protocol in which this interval is eliminated, thus allowing allotransplantation simultaneously with tolerance induction. Our results show that simultaneous cardiac allotransplantation and intrathymic tolerance induction by intrathymic injection of donor splenocytes and treatment with antilymphocyte serum is indeed possible in the PVG to AO high-responder rat strain combination, provided that low doses of cyclosporine are given intramuscularly on day 1, 2, and 3 after transplantation. As we now are able to combine the start of tolerance induction with the actual allotransplantation, this procedure may indeed have clinical potential.

Vascular thymus transplantation in rats. Technique, morphology, and function.

A new method of thymus transplantation is introduced, in which the graft is directly connected with the recipient's vascular system. This procedure was used both in euthymic rats and congenitally athymic nude rats. At all tested intervals after transplantation thymus grafts hardly differed from the recipient's own thymus in immunohistology and lymphocyte yield. In athymic nude rats, T cell-dependent immunity, tested by mitogen- and alloantigen-induced T cell responses, as well as by antibody production and delayed-type hypersensitivity after ovalbumin administration, showed that vascular thymus grafts could generate T cell functions to euthymic control levels. We conclude that the technique of vascular thymus transplantation represents a valuable tool, either in fundamental research on thymus function, or for the purpose of immune (re)constitution.

A novel lymphocyte surface antigen recognized by the monoclonal antibody HIS45.

A hybridoma producing the monoclonal antibody HIS45 was isolated from a fusion between spleen cells from a Balb/c mouse immunized with rat bone marrow cells and the fusion partner SP2/0. This antibody recognizes a determinant present on the majority of peripheral T and B cells and a small percentage of thymocytes. In a xenogeneic mixed leucocyte reaction HIS45 completely inhibits the proliferation of responding cells. HIS45 does not inhibit natural killer cell-mediated lysis. Comparison with other antibodies which have been reported to effect lymphocyte function fail to reveal any which have similar properties.

Turnover of ED2-expressing macrophages in the thymus cortex of rats.

To investigate the turnover of thymic ED2+ cortical macrophages, vascular thymus transplantation in RT7-congenic rats were performed. Thymus graft cell suspensions were analyzed using ED2 in combination with congenic markers. Immunohistology of thymus graft sections was performed to demonstrate the immigration and persistence of these macrophages at several time points after transplantation. In contrast to other mobile thymus cells like thymocytes and interdigitating cells, most ED2+ cortical macrophages showed a slow turnover rate. At 76 days after transplantation more than 30% of ED2+ macrophages were still of donor origin. The migration properties of these macrophages are discussed in relation to their presumed role in thymocyte maturation and proliferation.

T-cell development in the fetus and the invariant series hypothesis.

Comparison of the timing of appearance of certain T-cell markers in the intrathymic development of T cells during gestation reveals a common sequence of expression in several species. Here Richard Aspinall and colleagues put forward a hypothesis concerning this 'invariant series' of markers that shares the same timing of expression across species barriers. It is proposed that T-cell markers that are members of the invariant series are very important in deciding the fate of a developing thymocyte.

Differences in turnover between thymic medullary dendritic cells and a subset of cortical macrophages.

To investigate the turnover of thymic accessory cells, we performed vascular thymus transplantation in RT7 congenic rats. mAb specific for one of the two allelic variants of the RT7 molecule, as well as mAb specific for either medullary interdigitating cells or a subset of cortical macrophages (M phi), were used on cryostat sections and cell suspensions prepared from grafted thymuses to monitor the turnover of these two cell types. In contrast to the complete turnover of interdigitating cells within 3 wk after transplantation, ED2-labeled cortical M phi showed a very slow turnover. Seventy-six days after transplantation, more than 30% of these M phi were found to be still of donor origin. The different turnover rates of these thymic accessory cells could reflect their function in T cell development.

Prolonged survival of skin grafts following treatment with an antibody to a putative cell triggering molecule, QCA-1.

The QCA-1 molecule (quiescent cell antigen-1) appears to be involved in the differentiation events undergone by T cell following occupancy of the antigen receptor. Here we show that modulation of the QCA-1 antigen from the surface of the cell normally follows activation, and that treatment of animals with the antibodies against the QCA-1 molecule inhibits the normal response to an allograft without appearing to alter the number of peripheral T cells or the expression by these cells of the alpha/beta T cell antigen receptor.

Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats.

In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.

Reversibility of thymic atrophy induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and bis(tri-n-butyltin)oxide (TBTO).

We studied the reversibility of thymic atrophy induced by intubation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 10 days after a single dose of 50 micrograms/kg, or bis(tri-n-butyltin)oxide (TBTO), 4 days after a single dose of 75 mg/kg. This was done by an experimental design in which the atrophic thymus was placed in an in vivo situation in which the toxic chemical was no longer present, e.g. by transplantation of atrophic thymic lobes in untreated normal rats with connection to the vasculature of the recipient. At 20 days after the transplantation, the atrophic thymus showed the morphology and architecture of a normal uninvoluted thymus: lymphocyte counts and phenotypic expression of markers on lymphocytes, epithelium, and macrophages in the transplanted lobe did not differ from those in untreated donor rats or those in the normal uninvoluted thymus. Considering the mechanism of action of the toxic chemical, TBTO has been claimed to affect preferentially (passenger) lymphocytes in the thymus: the recovery after transplantation therefore is explained on the mere influx of newly-recruited precursor cells from the bone marrow. For TCDD a toxic action on the stationary epithelial component of the thymus has been claimed. We conclude that this epithelial damage is reversible within the 3-week period of the present experiment, with respect to both the morphology and immunologic phenotype of epithelium and other cell populations, as well as the recruitment of lymphocytes.

Implantation of cultured thymic fragments in congenitally athymic (nude) rats. Ultrastructural characteristics of the developing microenvironment.

Cultured thymic fragments correspond to the thymic microenvironment depleted of lymphocytes and dendritic cells. When these fragments are implanted under the kidney capsule of congenitally athymic rats, lymphocytes and dendritic cells of host origin enter the graft and induce thymus-dependent immunity in the recipient. This paper describes the ultrastructure of the fragments and the changes that occur during the restoration of normal thymic architecture. At the end of the culture period of 6-9 days and in the early stages after implantation, the grafts consist of keratin-containing epithelial cells of unusual morphology that can be labelled with antibodies raised against the epithelium of the mid/deep cortex and the subcapsule/medulla. Normal thymic architecture develops, including nerves and blood vessels, as lymphocytes populate the environment, and by 4-6 weeks the epithelial cells are the same phenotypically and ultrastructurally as those found in normal rat thymus. However, some areas without lymphocytes still contain the atypical epithelial cells seen before implantation. Large multinucleated giant cells are also present with a few associated epithelial cells of subcapsular/medullary phenotype. In conclusion, the cultured thymic fragments contain a hitherto unknown precursor epithelial cell with an atypical ultrastructure and phenotype that is not seen in normal development.