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Human cytolytic T lymphocyte recognition of miniature swine xenoantigens
Human Cytolytic T Lymphocyte Recognition of Miniature Swine Xenoantigens X.-C. Xu, B. Naziruddin, H. Sasaki, D.M. Smith, S. Shenoy, J. Lowell, T. Howard, and T. Mohanakumar
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RANSPLANTATION rejection across discordant species involves both humoral and cell-mediated immune responses.1,2 The human antiswine xenoresponse is of particular interest because of the potential use of swine organs in clinical transplantation. Both direct and indirect recognition pathways have been shown to be involved in cellmediated recognition of swine xenoantigens.3,4 We studied the mechanism of direct recognition of swine xenoantigens expressed on porcine aortic endothelial cells (PAEC) by human CD81 T cells. MATERIALS AND METHODS The PAEC used in this study were derived from z/z and y/y haplotypes of Yucatan partial inbred miniature swine. Human T-cell lines against PAEC were generated by repeated weekly stimulation in the presence of low concentration of rIL-2 (10 U/mL) as previous described.3 CD81 T cells were purified by magnetic bead separation (Multenyi Biotec, Germany). The direct recognition of porcine xenoantigen by CD81 T cells was monitored by standard CTL assays using 51Cr-labeled PAEC as target cells.3 The human CD81 T cell-xenoantigen interaction was further characterized by antibody-blocking assays3 and by transfection of different swine leukocyte antigen (SLA) class I genes5 into PAEC lines. To determine the role of SLA bound peptides in xenorecognition, the PAEC were briefly treated with citric acid (pH 3.0; 2 min at 4°C) to strip SLA bound peptides.6 In some experiments, acid-treated PAEC depleted of its SLA bound peptides were incubated with naturally processed peptides isolated from affinity purified SLA class I molecules.7
RESULTS AND DISCUSSION
The established human CD81 T cell lines generated against partial inbred miniature swine by repeated weekly stimulation showed SLA haplotype restricted lysis of PAEC targets. T-cell lines generated against z/z PAEC lysed z/z (51% at E:T of 20:1) but not y/y PAEC (4.6%). This SLA haplotype restricted lysis was blocked by anti-SLA class I (41.7% inhibition) and anti-human CD8 (41.6% inhibition) monoclonal antibodies, but not by anti-SLA class II (1.8%), anti-human CD4 (4.6%) or mouse Ig control (6.1%) antibodies. This suggested that the specificity of the human antiswine xenoresponse was directed toward the SLA class I molecules, and human CD8 was involved in this xenorecognition. To directly prove that human CD81 T cells recognized SLA class I allele polymorphism between dif0041-1345/99/$–see front matter PII S0041-1345(98)01834-X
ferent haplotypes of miniature swine, we transfected SLA1z cDNA encoding a SLA class I molecule derived from z/z haplotype into y/y PAEC. Although the y/y PAEC was not lysed (2.6% at E::T of 20:1) by the z/z restricted human CD81 T cells, the SLA1z transfected y/y PAEC was lysed efficiently (39%). y/y PAEC transfected with irrelevant SLA class I allele cDNA, SLA1x was not lysed (0.6%), demonstrating that human TCR recognition of SLA class I is direct and SLA allele specific. It is widely accepted that the major histocompatability (MHC) molecules are associated with bound peptides, and T cell recognizes MHC-peptide complexes. Therefore, we examined the role of SLA-bound peptides in this CD81 restricted xenoresponse. We treated PAEC briefly with citric acid (pH 3.0) to strip SLA of its bound peptides. Using z/z restricted CD81 T cells as effector cells, intact z/z PAEC were lysed 25% at an effector to target ratio of 8:1; z/z PAEC stripped of peptides were lysed 7% (72% inhibition of the lysis). Adding back the purified z/z SLA class I bound peptides, but not y/y SLA bound peptides, restored the capacity of the acid treated z/z PAEC to serve as target cells for the z/z restricted CD81 T cells. These results not only indicate that peptides are involved in xenorecognition, but also that the peptides being recognized are haplotype specific since only relevant peptides restored the lysis of acid striped z/z PAEC. In summary, the results presented in this study indicate that human CD81 T cell lines generated by long-term in vitro culture against PAEC demonstrate direct recognition of SLA class I molecules expressed on swine target cells. We show here that human CD8 directly interacts with SLA class I molecules. We also demonstrate that human CD81 T cell recognition of PAEC is direct and SLA class I allele specific. In addition, the SLA class I bound peptides play an
From the Department of Surgery and Pathology, Washington University School of Medicine (X.-C.X., B.N., H.S., S.S., J.L., T.H., T.M.), St Louis, Missouri and the Department of Pathology, University of Oklahoma Health Science Center (D.M.S.), Oklahoma City, Oklahoma. Supported by NIH grant HL57796. Address reprint requests to Dr T. Mohanakumar, Washington University School of Medicine, Department of Surgery, Box 8109, 3328 CSRB, 600 S. Euclid Ave, St Louis, MO 63110. © 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
916
Transplantation Proceedings, 31, 916–917 (1999)
RECOGNITION OF MINIATURE SWINE XENOANTIGENS
important role in xenorecognition. These data indicate that human CD81 T-cell mediated direct recognition of PAEC is similar to human allo-response, although there may be subtle differences between the two mechanisms. REFERENCES 1. Auchincloss H Jr: Transplantation 46:1, 1988 2. Kaufman CL, Gaines BA, Ildstad ST: Annu Rev Immunol 13:339, 1995
917 3. Shishido S, Naziruddin B, Howard T, et al: Transplantation 64:340, 1997 4. Shishido S, Naziruddin B, Xu XC, et al: Transplantation 65:706, 1998 5. Smith DM, Asbury J, Swearingen W, et al: ASTP Meeting Chicago, IL, 1996:142 (Abstracts) 6. Smith PA, Brunmark A, Jackson MR, et al: J Exp Med 185:1023, 1997 7. Falk K, Rotzschke O, Rammensee HG: Nature 348:248, 1990
T
RANSPLANTATION rejection across discordant species involves both humoral and cell-mediated immune responses.1,2 The human antiswine xenoresponse is of particular interest because of the potential use of swine organs in clinical transplantation. Both direct and indirect recognition pathways have been shown to be involved in cellmediated recognition of swine xenoantigens.3,4 We studied the mechanism of direct recognition of swine xenoantigens expressed on porcine aortic endothelial cells (PAEC) by human CD81 T cells. MATERIALS AND METHODS The PAEC used in this study were derived from z/z and y/y haplotypes of Yucatan partial inbred miniature swine. Human T-cell lines against PAEC were generated by repeated weekly stimulation in the presence of low concentration of rIL-2 (10 U/mL) as previous described.3 CD81 T cells were purified by magnetic bead separation (Multenyi Biotec, Germany). The direct recognition of porcine xenoantigen by CD81 T cells was monitored by standard CTL assays using 51Cr-labeled PAEC as target cells.3 The human CD81 T cell-xenoantigen interaction was further characterized by antibody-blocking assays3 and by transfection of different swine leukocyte antigen (SLA) class I genes5 into PAEC lines. To determine the role of SLA bound peptides in xenorecognition, the PAEC were briefly treated with citric acid (pH 3.0; 2 min at 4°C) to strip SLA bound peptides.6 In some experiments, acid-treated PAEC depleted of its SLA bound peptides were incubated with naturally processed peptides isolated from affinity purified SLA class I molecules.7
RESULTS AND DISCUSSION
The established human CD81 T cell lines generated against partial inbred miniature swine by repeated weekly stimulation showed SLA haplotype restricted lysis of PAEC targets. T-cell lines generated against z/z PAEC lysed z/z (51% at E:T of 20:1) but not y/y PAEC (4.6%). This SLA haplotype restricted lysis was blocked by anti-SLA class I (41.7% inhibition) and anti-human CD8 (41.6% inhibition) monoclonal antibodies, but not by anti-SLA class II (1.8%), anti-human CD4 (4.6%) or mouse Ig control (6.1%) antibodies. This suggested that the specificity of the human antiswine xenoresponse was directed toward the SLA class I molecules, and human CD8 was involved in this xenorecognition. To directly prove that human CD81 T cells recognized SLA class I allele polymorphism between dif0041-1345/99/$–see front matter PII S0041-1345(98)01834-X
ferent haplotypes of miniature swine, we transfected SLA1z cDNA encoding a SLA class I molecule derived from z/z haplotype into y/y PAEC. Although the y/y PAEC was not lysed (2.6% at E::T of 20:1) by the z/z restricted human CD81 T cells, the SLA1z transfected y/y PAEC was lysed efficiently (39%). y/y PAEC transfected with irrelevant SLA class I allele cDNA, SLA1x was not lysed (0.6%), demonstrating that human TCR recognition of SLA class I is direct and SLA allele specific. It is widely accepted that the major histocompatability (MHC) molecules are associated with bound peptides, and T cell recognizes MHC-peptide complexes. Therefore, we examined the role of SLA-bound peptides in this CD81 restricted xenoresponse. We treated PAEC briefly with citric acid (pH 3.0) to strip SLA of its bound peptides. Using z/z restricted CD81 T cells as effector cells, intact z/z PAEC were lysed 25% at an effector to target ratio of 8:1; z/z PAEC stripped of peptides were lysed 7% (72% inhibition of the lysis). Adding back the purified z/z SLA class I bound peptides, but not y/y SLA bound peptides, restored the capacity of the acid treated z/z PAEC to serve as target cells for the z/z restricted CD81 T cells. These results not only indicate that peptides are involved in xenorecognition, but also that the peptides being recognized are haplotype specific since only relevant peptides restored the lysis of acid striped z/z PAEC. In summary, the results presented in this study indicate that human CD81 T cell lines generated by long-term in vitro culture against PAEC demonstrate direct recognition of SLA class I molecules expressed on swine target cells. We show here that human CD8 directly interacts with SLA class I molecules. We also demonstrate that human CD81 T cell recognition of PAEC is direct and SLA class I allele specific. In addition, the SLA class I bound peptides play an
From the Department of Surgery and Pathology, Washington University School of Medicine (X.-C.X., B.N., H.S., S.S., J.L., T.H., T.M.), St Louis, Missouri and the Department of Pathology, University of Oklahoma Health Science Center (D.M.S.), Oklahoma City, Oklahoma. Supported by NIH grant HL57796. Address reprint requests to Dr T. Mohanakumar, Washington University School of Medicine, Department of Surgery, Box 8109, 3328 CSRB, 600 S. Euclid Ave, St Louis, MO 63110. © 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
916
Transplantation Proceedings, 31, 916–917 (1999)
RECOGNITION OF MINIATURE SWINE XENOANTIGENS
important role in xenorecognition. These data indicate that human CD81 T-cell mediated direct recognition of PAEC is similar to human allo-response, although there may be subtle differences between the two mechanisms. REFERENCES 1. Auchincloss H Jr: Transplantation 46:1, 1988 2. Kaufman CL, Gaines BA, Ildstad ST: Annu Rev Immunol 13:339, 1995
917 3. Shishido S, Naziruddin B, Howard T, et al: Transplantation 64:340, 1997 4. Shishido S, Naziruddin B, Xu XC, et al: Transplantation 65:706, 1998 5. Smith DM, Asbury J, Swearingen W, et al: ASTP Meeting Chicago, IL, 1996:142 (Abstracts) 6. Smith PA, Brunmark A, Jackson MR, et al: J Exp Med 185:1023, 1997 7. Falk K, Rotzschke O, Rammensee HG: Nature 348:248, 1990