StemCell Therapy

Proc Natl Acad Sci U S A. 2000 Aug 15; 97(17): 95559560.

Immunology

Departments of *Medicine and Pathology, Stanford University School of Medicine, Stanford, CA 94305

Contributed by Irving L. Weissman

Engraftment of allogeneic bone marrow (BM) has been shown to induce tolerance to organs genotypically matched with the BM donor. Immune reconstitution after BM transplantation therefore involves re-establishment of a T cell pool tolerant to antigens present on both donor and host tissues. However, how hematopoietic grafts exert their influence over the regenerating immune system is not completely understood. Prior studies suggest that education of the newly arising T cell pool involves distinct contributions from donor and host stromal elements. Specifically, negative selection is thought to be mediated primarily by donor BM-derived antigen-presenting cells, whereas positive selection is dictated by radio-resistant host-derived thymic stromal cells. In this report we studied the effect of highly purified allogeneic hematopoietic stem cells (HSCs) on organ transplantation tolerance induction and immune reconstitution. In contrast to engraftment of BM that results in near-complete donor T cell chimerism, HSC engraftment results in mixed T cell chimerism. Nonetheless we observed that HSC grafts induce tolerance to donor-matched neonatal heart grafts, and one way the HSC grafts alter host immune responses is via deletion of newly arising donor as well as radiation-resistant host T cells. Furthermore, using an in vivo assay of graft rejection to study positive selection we made the unexpected observation that T cells in chimeric mice rejected grafts only in the context of the donor MHC type. These latter findings conflict with the conventionally held view that radio-resistant host elements primarily dictate positive selection.

Keywords: bone marrow transplantation, MHC restriction, mice

Transplantation of allogeneic bone marrow (BM) is known to alter immune responses in recipients so that tolerance is established to tissues matched with the genotype of the BM donors (13). Thus, the process of regeneration of the hematopoietic system involves the re-establishment of parameters that identify self- from nonself-antigens. The way in which BM grafts affect these changes is not completely understood. However, because T cells control antigen-specific immune responses the pathways that lead to regeneration of the peripheral T cell pool are central to immune reconstitution. T cell development after BM transplantation (BMT) is thought to recapitulate normal T cell ontogeny, which begins with the migration of BM-derived hematopoietic stem cells (HSCs) or more differentiated progenitors to the thymus (4). Within the thymus, under the influence of a specialized stromal microenvironment, progenitor T cells expand, differentiate, and undergo the rigorous processes of positive and negative selection (58). Positive selection results in survival of T cells with antigen receptors that corecognize self-MHC molecules plus foreign peptides. T cells whose receptors do not detect self-MHC molecules die, presumably by failure to receive critical differentiating signals. Negative selection involves the removal of potentially autoreactive T cells that interact too well with self-MHC molecules plus self-peptides.

Classic BM and thymus grafting studies by Zinkernagel et al. (9) and Bevan and Fink (10, 11) showed that the radio-resistant elements in the host thymus dictate MHC restriction of killer T cells. They proposed, and many experiments followed to support, the notion that these positively selecting elements in the thymus are epithelial cells (5, 6, 8). Subsequent studies refined these observations by tracking T cell development via expression of V type or expression of a single transgenic T cell receptor and showed that both CD8+ and CD4+ T cells are likely to be positively selected on a subpopulation of epithelial cells located in the thymic cortex (5, 6, 8). In contrast, negative selection primarily is mediated by BM-derived antigen-presenting cells (APCs) (7, 12, 13). The absoluteness with which these stromal components dictate the selection processes continues to be challenged by discordant observations (1417). In the setting of an MHC-mismatched allogeneic BMT, this schema of T cell selection predicts that the resultant host will be immunodeficient, insofar as the developing cells will be educated in the thymus to respond to antigens in the context of host MHC type, but will encounter BM-derived APCs in the periphery with the donor MHC type.

In the studies presented here we examined the issues of tolerance induction and immune reconstitution after transplantation of highly purified MHC-disparate HSCs in mice. HSCs are devoid of contaminating differentiated cell populations and thus, unlike most radiation BM chimeras, the effects of the donated immune system that arises from the HSC grafts are solely the result of de novo hematopoiesis. The HSC-transplanted mice also differ from BM chimeras because the former retain a significant proportion of radio-resistant host T cells (18). We found that HSCs induce tolerance to donor-matched organs and that such grafts can mediate negative selection of both developing donor T cells and residual T cells from the host. Furthermore, we made the unexpected observation that analysis of MHC restriction by an in vivo assay suggests that in chimeric mice the donor, not the host-type MHC, predominates in controlling heart graft rejection, a measure of T cell responsiveness. These studies, and the studies by Zinkernagel and Althage (17), reopen the issues of how, where, and on which cell types developing T cells learn MHC restriction and suggest that immunoincompetence in the post-BMT setting, a known clinical problem, is not completely explained by disparity between the MHC type of the donor versus the host.

Three different C57BL/Ka congenic mouse lines were used as donors or recipients. C57BL/Ka were mice H-2b, Thy-1.2, CD45.2; congenic Thy-1.1 mice were H-2b, Thy-1.1, CD45.2 (C57BL/Ka.Thy-1.1) and designated as BA throughout the text; and congenic CD45.1 mice were H-2b, Thy-1.1, CD45.1 (C57BL/Ka.Thy-1.1.CD45.1) and designated BA.CD45.1 throughout the text. HSC or BM recipients were 7- to 10-week-old BALB/c (H-2d, Thy 1.2), BALB/k (H-2k, Thy 1.2), or C57BL/Ka mice. HSC and BM donors were BA or AKR/J mice (H-2k, Thy 1.1). For the neonatal heart transplantation experiments donors were 1- to 24-h-old neonates derived from BA, BA.CD45.1, BALB/c, C3H.SW (H-2b) or DBA.2 (H-2d) strain mice. All mice were bred and maintained at Stanford University.

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Purified hematopoietic stem cell grafts induce tolerance ...

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