Rat Peripheral T-Cell Receptor V Repertoire in F344-to-B10 (Rat-to-Mouse) Mixed Xenogeneic Chimeras Y. Huang, M. Neipp, S.T. Ildstad, and H. Shirwan W E HAVE previously reported excellent survival of mixed xenogeneic chimerism after the transplanta- tion of 40  10 6 untreated F344 rat bone marrow cells into sublethally irradiated (600 to 700 cGy) B10 mice that received 50 mg/kg cyclophosphamide intraperitoneally (IP) 2 days after transplantation. These chimeras exhibited stable chimerism and evidence for stable donor-specific immune nonresponsiveness. Rat T cells developing in chi- meras exhibit the characteristic phenotypically immature pattern in the thymus (CD4 + CD8 + -TCR + ) and mature pattern (CD4 + CD8 - -TCR + or CD4 - CD8 + -TCR + ) in the periphery. In this report, we characterized the rat T-cell receptor (TCR) -chain repertoire that develops in mixed xenoge- neic chimeras to test whether mouse xenogeneic antigens contribute to the development of the rat peripheral T-cell repertoire. We demonstrate that rat-to-mouse mixed xeno- geneic chimeras exhibit immune nonresponsiveness toward both the recipient and donor antigens as evidenced by the acceptance of donor skin grafts and the lack of graft-vs-host disease (GVHD). The observed immune nonresponsive- ness was associated with the altered pattern of the rat TCR repertoire shaped by deletion and expansion of T cells expressing selected V genes. MATERIALS AND METHODS Animals C57BL/10SNJ mice (H-2 b ) were purchased from the Jackson Laboratory (Bar Harbor, Me). Fischer 344 (RT1 1 ) rats were purchased from Harlan Sprague Dawley (Indianapolis, Ind). Ani- mals were housed in a barrier animal facility. Chimeras B10 recipient mice were irradiated with a single dose of 600 to 700 cGy total body irradiation (TBI) from a cesium source (Nordion, Ontario, Canada) at a dose rate of 117.18 cGy/min. Bone marrow was obtained from F344 rats as previously described. 1 Characterization of Chimeras by Flow Cytometry Thirty days after reconstitution, recipients were characterized for engraftment using two-color flow cytometry to determine the percentage of peripheral blood lymphocytes of host (H-2K b ) and donor (RT1A 1 ) origin as described previously. 1 Rat V Gene Analysis Total RNA was isolated from sorted, rat CD3 + T cells isolated from chimeras and used for analysis with a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) method. 4 Two micrograms of total RNA was converted into the first-strand cDNA using a C-specific oligonucleotide as primer for reverse transcrip- tase (Boehringer Mannheim, Ind). One tenth of the first-strand cDNA was then used as a template for Taq DNA polymerase in PCR amplification using a second anti-sense upstream C primer labeled with 6-carboxyfluorescence and sense primers specific for individual rat V genes as described previously. 2 The PCR prod- ucts were then analyzed using the ABI PRISM 377 DNA Se- quencer and GeneScan™ software (Perkin-Elmer Cetus, Norwalk, Conn). RESULTS The peripheral mature TCR repertoire is shaped by both positive and negative selection events that occur in the thymus during T-cell ontogeny. 3,4 To determine the influ- ence of mouse xenogeneic environment on the develop- ment of the rat TCR repertoire, we analyzed peripheral expression of rat V genes in chimeras. The V repertoire in chimeras was shaped by contraction of V5 (3.4-fold), V7 (1.76-fold), V12 (8.7-fold), V14 (2.0-fold), V16 (7.8-fold), V17 (9.5-fold), and V20 (1.8-fold) and expan- sion of V6 (2.2-fold), V8.2 (3.2-fold), and V9 (1.6-fold) as compared with the levels of expression of these genes in naı ¨ve rats. These observations with RT-PCR were further confirmed by flow cytometry using three available mono- clonal antibodies to rat V8.2, V10, V16 molecules. We next analyzed the pattern of CDR3 expression in chimeras to test if alterations in the V expression also apply to this domain of the TCR. CDR3 expression was analyzed by spectratyping that allows for the assessment of the size heterogeneity of the CDR3 domain. There were no detectable changes in the pattern of the CDR3 domain for  chains containing Vs with altered expression. From the Institute for Cellular Therapeutics, Allegheny Univer- sity of the Health Sciences, Philadelphia, Pennsylvania, USA. Supported in part by grants from the NIH (AI 33587 to H.S.) and the FC (R01-DK 52294-06A2 to S.T.I.). Address reprint requests to Haval Shirwan, PhD, Institute for Cellular Therapeutics, Allegheny University of the Health Sci- ences, 500 Ridgeway Ave, Glenolden, PA 19036. 0041-1345/99/$–see front matter © 1999 by Elsevier Science Inc. PII S0041-1345(98)01865-X 655 Avenue of the Americas, New York, NY 10010 978 Transplantation Proceedings, 31, 978–979 (1999)