Antiviral Cytotoxic Activity Across a Species Barrier in Mixed Xenogeneic Chimeras: Functional Restriction to Host MHC 1 Yolonda L. Colson,* Ralph A. Tripp, ² Peter C. Doherty, ‡ Sherry M. Wren, § Michael Neipp, § Ashraf Y. Abou El-Ezz, § and Suzanne T. Ildstad 2§ Reconstitution of lethally irradiated mice with a mixture of mouse and rat bone marrow cells (mouse  rat3mouse) results in mixed xenogeneic chimerism and donor-specific tolerance. The current study demonstrates that mouse and rat T lymphocytes that have developed in xenogeneic chimeras are restricted to Ag presentation by mouse, but not rat, APC. Restriction to host Ags results in functional immunocompetence with generation of antiviral cytotoxic activity in vivo, within and across species barriers. These data demonstrate for the first time that the host thymus is sufficient to support development and positive selection of functional cross-species T lymphocytes. The superior immunocompetence, as compared with fully xenogeneic (rat3mouse) chi- meras, may prove to be of significant benefit in the clinical application of xenotransplantation to solid organ transplantation and immune reconstitution for AIDS. The Journal of Immunology, 1998, 160: 3790 –3796. T lymphocytes recognize conventional Ags presented in the context of self MHC molecules (1– 8). Studies from semi- allogeneic radiation bone marrow chimeras demonstrated that negative selection, or clonal deletion, of T cells is mediated by the APC of the bone marrow donor (9, 10). In contrast, the positive selection of T lymphocytes necessary for the recognition of con- ventional Ags is determined by the MHC stromal phenotype of the host environment in which the T cells have developed (1, 4 –7). Tolerance to MHC determinants, however, was not, in itself, suf- ficient to result in the recognition of those determinants for the purpose of Ag presentation. Singer et al. demonstrated that unfrac- tionated spleen cells from fully allogeneic bone marrow chimeras (A3 B), which were tolerant to both host and donor Ags, did not recognize an antigenic stimulus, such as trinitrophenol (TNP 3 ), when presented in the context of donor MHC molecules (strain A) (1). This immunoincompetence could be overcome with the direct addition of host-type APC (3, 4), or with the preparation of mixed allogeneic chimeras (B + A3 B), in which the genotypically ap- propriate host APC (strain B) are present in the bone marrow in- oculum at the time of transplantation (1). The failure of donor T cells to recognize Ag was therefore due to the absence of host-type APC to which the donor T cells were restricted, and not due to an inherent defect in T cell function. Therefore, T cells specifically recognize those self MHC determinants expressed on radiation- resistant host elements encountered by developing T cells in the thymus at a critical point in development. This phenomenon was also shown in vivo using a model of intranasal infection of mice with Hk31 influenza A virus. Intranasal infection results in se- vere pneumonia and a massive enlargement of mediastinal lymph nodes (MLN) that drain the respiratory tract (11, 12). However, this infection is nonlethal in immunocompetent recipients, as clear- ance of the virus occurs through the generation of viral-specific CTL activity and the recruitment of influenza A-specific CTL pre- cursors (CTLp). Viral-specific CTLp are not detected (10 6 ) within MLN and spleen before infection, but rapidly increase fol- lowing infection in immunocompetent recipients (13). Mixed al- logeneic and syngeneically reconstituted recipients survive due to generation of viral-specific CTLs, while fully allogeneic chimeras succumb to infection (14). Until recently, the lack of a model for T cell development and tolerance induction in a xenogeneic stromal environment has not permitted similar analyses for xenoantigens. Whether positive, as well as negative selection events associated with the development of a functional T cell repertoire can occur across a species barrier has remained an uncertainty. Clearly, the successful clinical ap- plication of xenogeneic bone marrow chimerism requires the pres- ence of both donor-specific tolerance and immunocompetence to respond to infectious agents. We have developed a model to achieve stable multilineage fully xenogeneic chimerism (rat3mouse) through reconstitution of mice conditioned with 950 centigray of total body irradiation with rat bone marrow (15). Recipient mice exhibit phenotypically nor- mal rat T cell development, and are functionally tolerant to donor xenoantigens in vivo since donor-specific skin and islet xenografts were permanently accepted, while MHC-disparate third-party mouse and rat grafts are promptly rejected (15, 16). Mixed xeno- geneic chimeras, in which T cell-depleted syngeneic mouse bone marrow is coadministered with either untreated or T cell-depleted rat bone marrow (mouse + rat3mouse), demonstrate similar func- tional donor-specific tolerance for skin, pancreatic islet, and car- diac xenografts in vivo (17–19). The rat- and mouse-derived T lymphocytes develop in a phenotypically normal fashion, and lym- phocytes from the chimeras exhibit donor-specific functional tol- erance in vitro by proliferative MLR assays (20). We have now applied the model of mixed xenogeneic chimer- ism (mouse + rat3mouse) to examine T cell repertoire selection *Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15261; ² Centers of Disease Control and Prevention, Atlanta, GA 30033; ‡ Department of Immunology, St. Jude’s Research Hospital, Memphis, TN 38101; and § Institute for Cellular Therapeu- tics, Allegheny University of Health Sciences, Philadelphia, PA 19102 Received for publication September 11, 1997. Accepted for publication December 22, 1997. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by National Institutes of Health RO1 AI30615 and DK4390l, Juvenile Diabetes Foundation 19911433, American Heart Association, American Diabetes Association, and American College of Surgeons Resident Scholar. 2 Address correspondence and reprint requests to Dr. Suzanne T. Ildstad, Institute for Cellular Therapeutics, Allegheny University of the Health Sciences, Broad & Vine Streets, Mail Stop, Philadelphia, PA 19102. 3 Abbreviations used in this paper: TNP, trinitrophenol; CML, cell-mediated lym- pholysis; CTLp, cytolytic T lymphocyte precursors; MLN, mediastinal lymph nodes. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00