Jaap Kampinga

Implantation of cultured thymic fragments in congenitally athymic (nude) rats

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.SummaryCultured 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.

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.

Kinetics of Rat Thymic Dendritic Cells in Bone Marrow-Reconstituted Radiation Chimeras

The thymus meets unique microenvironmental conditions essential for pre-T cell maturation and generation of the complete T cell repertoire. During intrathymic development thymocytes acquire qualities essential for adequate immune functioning of T cells, such as tolerance for self, antigen receptors, and restriction to class I or II MHC antigens. With respects to restriction of T helper cells to class II (Ia) antigens of the MHC, it is more or less generally accepted that class II positive cells in the thymus control this aspect in T cell ‘education’. Potential candidates for regulating restriction of T helper cells to class II molecules are thymic reticular epithelial cells and bone marrow-derived thymic dendritic cells (DC), both known to be Ia+. Attending the question which cell type indeed is controlling class II restriction in the thymus, one can study T helper cell restriction to Ia antigens in allogeneic or semi-allogeneic bone marrow-reconstituted radiation chimeras (BMRRC) or thymus-grafted animals. Results obtained from these studies are conflicting, suggesting on one hand, that class II restriction of T cells is dictated by thymic epithelial cells (1), or on the other hand, that DC (antigen presenting cells) in the thymus are involved in this (2,3). Of crucial importance in such restriction studies is to know at what experimental stage the thymus can be considered class II-chimeric with respects to epithelial cell versus DC, viz. how long does it take in BMRRC for the recipient thymic DC to be replaced by allogeneic or semi-allogeneic donor type DC?

Vascular Thymustransplantation in Rats: A New Method to Study Thymocyte Kinetics

Although many studies have been performed to unravel the cellular kinetics of thymic immigration, intrathymic differentation and thymic emigration, there is still not a generally accepted view on this issue (for review see Scollay (1,2)). An important reason for the contradictory results is the lack of an experimental model mimicking the normal physiological situation of the thymus.

Kinetics of Thymocyte Regeneration in Adult Adriamycin Treated Rats: Evidence for a Bone Marrow Derived Prothymocyte Precursor

The thymus is the central organ for T lymphocyte maturation in which prothymocytes undergo rearrangement of T cell receptor genes, acquire function associated antigens and become “educated ” to respond to foreign antigens in a self MHC class I or class II restricted manner.