Xiao-Chun Xu

Indirect recognition of porcine xenoantigens by human CD4+T cell clones

BACKGROUND Human T-cell response against xenogeneic antigens may occur either by direct recognition of antigens on xenogeneic antigen-presenting cells (APCs) or by an indirect pathway mediated by autologous APCs. METHODS The proliferative response of human CD4+ T cells to porcine aortic endothelial cells (PAECs) was measured. From these T-cell lines, eight CD4+ T-cell clones were obtained by limiting dilution. RESULTS CD4+ T cells, in the absence of monocytes, proliferated in response to PAECs only after swine leukocyte antigen (SLA) class II molecules were induced on PAECs. The proliferation was significantly better when autologous human monocytes were added back as APCs. All of the eight CD4+ T-cell clones demonstrated specific proliferative response when stimulator PAECs, but not PAECs of other porcine origins, were preincubated with autologous human APCs before addition of these clones. These results indicated that the clones are recognizing porcine xenoantigens presented by self-APCs. The proliferative response of CD4+ T-cell clones was blocked by antibodies directed against human leukocyte antigen class II and human CD4, but not by anti-SLA class II monoclonal antibodies. A marked inhibition in proliferation was also noted when human APCs were incubated with chloroquine before addition to the cultures, indicating that xenoantigens had to be processed in order to be recognized by the clones. CONCLUSIONS Human CD4+ T cells can recognize xenoantigens by either a direct or indirect pathway. The CD4+ T-cell clones developed against SLA class II-negative PAECs recognized strain-specific porcine xenoantigens indirectly.

Allele-specific and peptide-dependent recognition of swine leukocyte antigen class I by human cytotoxic T-cell clones.

BACKGROUND The T-cell mediated immune responses play a major role in xenograft rejection. However, the mechanisms behind human T-cell recognition of porcine xenoantigens remain to be elucidated. METHODS Human CD8+ T-cell lines were generated against porcine aortic endothelial cells (PAECs) from y/y and z/z haplotypes of Yucatan inbred swine. T-cell clones were obtained by limiting dilution. The human T-cell receptor (TCR)-swine leukocyte antigen (SLA) class I interaction was characterized. RESULTS The human CD8+ T-cell mediated direct recognition of PAECs was SLA haplotype-specific. The haplotype specificity was restricted by the SLA class I allelic polymorphism. To characterize the role of SLA-bound peptides in the human TCR-SLA class I interaction, we stripped peptides from SLA molecules by a brief acid treatment. Using z/z-specific CD8+ T cells as effectors, we demonstrated that the acid-treatment, which stripped SLA molecules of bound peptides, decreased the lysis of PAECs by 72%. Addition of peptides eluted from affinity purified z/z SLA class I molecules, but not from the irrelevant y/y SLA class I, restored the lysis of acid-treated z/z PAECs. In addition, the lysis of a human HLA class I negative cell line, 721.221, transfected with a relevant SLA class I allele derived from the z/z haplotype, was significantly increased with the addition of relevant z/z peptides. These experiments indicated that both SLA class I and bound peptides were required for recognition by human CD8+ T cells. Cloning studies identified two groups of xenoreactive T-cell clones. Group I clones recognized distinct porcine peptides in the context of SLA class I molecules, whereas group II clones recognized human endogenous cross-reactive peptides presented by SLA class I. CONCLUSIONS Our results demonstrated that, despite the differences in MHC molecules between species, human T-cell recognition of porcine MHC is similar to direct allo-recognition, that is, human TCR recognizes xenogeneic SLA-peptide complexes.

Xenoreactive anti-Galα(1,3)Gal antibodies prevent porcine endogenous retrovirus infection of human in vivo

The discovery of porcine endogenous retroviruses (PERV) has raised concerns regarding the safety of pig to human xenotransplantation. In this study, we examined PERV infection of human cells in vivo. Furthermore, we examined the effect of human xenoreactive natural antibody on in vivo PERV infection. Human peripheral blood leukocyte reconstituted severe combined immunodeficiency mice were transplanted with porcine aortic endothelial cells (PAEC). PERV gene expression was readily detected in human leukocytes after transplantation. In contrast, human leukocytes harvested from mice treated with human serum or anti-Galalpha(1,3)Gal antibody prevented PERV infection in 6 of 8 mice. These results provide the first evidence that PERV can infect human cells in vivo and that natural xenoreactive antibody can prevent this infection.

Activation of Natural Killer Cells and Macrophages by Porcine Endothelial Cells Augments Specific T-Cell Xenoresponse

The rejection of xenografts is characterized by infiltration of monocytes and natural killer (NK) cells into the graft, suggesting an important role for the innate immune system in xenorecognition. In this study, purified human NK or T cells were cocultured with porcine endothelial cells, and cytokines were analyzed by ELISA and intracellular FACS. We demonstrated a vigorous human anti‐porcine xenoresponse that was associated with a strong T‐cell proliferation against porcine endothelial cells. Limiting dilution cloning and T‐cell receptor (TCR) Vβ gene usage revealed a low number of xenoreactive T‐cell precursors. We demonstrated that xenogeneic porcine but not allogeneic human endothelial cells induced the early production of interferon (IFN)‐γ by human NK cells but not by CD3+ T cells. Porcine xenoantigen‐induced IFN‐γ production was only partially dependent on IL‐12. Blocking IL‐12 with neutralizing antibodies or by depletion of human macrophages partially decreased IFN‐γ production by CD56+ NK cells. Three‐color flow cytometry revealed that IL‐12 was produced through a species‐specific activation of human macrophages by porcine endothelial cells. Our results indicate that the direct activation of NK cells and macrophages by porcine endothelial cells provides a unique pathway of xenorecognition that augments downstream specific T‐cell immunity and represents a powerful effector mechanism in xenograft rejection.

Natural Antibodies Prevent In Vivo Transmission of Porcine Islet-Derived Endogenous Retrovirus to Human Cells

The discovery of porcine endogenous retroviruses (PERV) has raised concerns regarding the safety of porcine xenotransplantation. However, transmission of PERV had not been observed in humans exposed to porcine tissue. We examined whether PERV derived from porcine pancreatic islet cells could infect human cells in vivo and the role of natural antibodies in inhibiting PERV infection. In vivo infective potential of PERV was studied in SCID mice reconstituted with human peripheral blood leucocytes. Porcine islets were transplanted under the kidney capsule. PERV infection was determined by analyzing PERV gene expression in graft infiltrating lymphocytes (GIL) harvested 21 days posttransplantation. Mice were administered normal human serum prior to and 2 days posttransplantation to study their role in protection of human cells against PERV infection. PERV genes were expressed in all porcine tissues examined, including purified porcine islets. PERV expression was detected in GILs from three of five human-SCID mice. Administration of human serum blocked PERV infection in GILs in five of five human-SCID mice. These results indicate that PERV from porcine islets can infect human cells in vivo. Normal human serum blocks transmission of retrovirus in vivo, suggesting that natural xenoreactive antibodies can prevent PERV infection.

HUMAN IMMUNE RESPONSES TO PORCINE ENDOGENOUS RETROVIRUS-DERIVED PEPTIDES PRESENTED NATURALLY IN THE CONTEXT OF PORCINE AND HUMAN MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I MOLECULES: IMPLICATIONS IN XENOTRANSPLANTATION OF PORCINE ORGANS

Background. Porcine endogenous retroviruses (PERV) have been shown to infect human cells, raising concerns regarding safety of xenotransplantation. In patients exposed to porcine tissues, no PERV infection has been observed. This study was designed to develop human CD8+cytotoxic T lymphocytes (CTL) against PERV-derived peptides presented in the context of human leukocyte antigen (HLA) or swine leukocyte antigen (SLA) class I molecules and to define dominant epitopes contributed by PERV. Methods. Human CD8+CTL were generated against porcine aortic endothelial cells (PAEC). Peptides presented on SLA class I molecules were acid eluted and fractionated by reverse-phase high-performance liquid chromatography. Peptide fractions that restored lysis of acid-stripped PAEC were sequenced by tandem mass spectrometry. Human CD8+CTL were generated against PERV envelope-derived peptides and PERV-infected human cells to identify immunodominant PERV-derived epitopes. Results. We identified two peptides derived from retroviral transactivating regulatory protein (AHQD-PLPEQP) and retroviral transcription factor (PQK-PFVT) recognized by human CD8+CTL in the context of SLA class I. Computer-assisted analysis identified nine PERV-envelope–derived 9-mer peptides with high affinity for the HLA-A2 molecule (Env-1–9). PERV-specific CD8+CTL generated in vitro identified the immunodominant Env-5 peptide (303–311, KLFS-LIQGA) and demonstrated HLA-A2–restricted cytotoxicity against PERV-infected human cells. Conclusions. Our results indicate that PERV-derived peptides are presented naturally on porcine and human major histocompatibility complex class I molecules. CD8+CTL responses elicited against dominant SLA and HLA class I-restricted PERV-derived epitopes may play an important role in xenograft rejection and in containment of PERV infection of human cells after xenotransplantation.

Tissue-specific peptides influence human T cell repertoire to porcine xenoantigens.

BACKGROUND Human CD8+ T cells elicit a vigorous response to allo- or xenogeneic MHC class I molecules. However, the influence of a given MHC-bound peptide to the responding allo- or xenoreactive T cell repertoire is not clear. METHODS In this study, we analyzed individual T cell responses to unique tissue epitopes presented on syngeneic porcine endothelial and lymphoblastoid cells by limiting dilution analysis and analyzed the responding T cell repertoire by T cell receptor beta (TCR Vbeta) chain spectrotyping. RESULTS Both porcine endothelial and lymphoblastoid cells were able to elicit swine leukocyte antigen (SLA) class I restricted and peptide-dependent cytotoxic T lymphocyte (CTL) responses. The responding human CD8+ T cells showed a heterogenous but limited TCR Vbeta gene usage. Interestingly, although a large portion of the selected TCR Vbeta gene usage in response to endothelial and lymphoblastoid cells were shared (i.e., Vbeta-1, 2, 6.1, 13), unique Vbeta usage was noted in T cells that respond to either endothelial (Vbeta-5.3) or lymphoblastoid cells (Vbeta-5.1, 11), suggesting that porcine tissue-specific epitopes play a role in modulating the responding T cell repertoire. Limiting dilution cloning analysis revealed that a majority (89%) of the CTL clones stimulated by porcine endothelial cells recognized shared peptides presented by both endothelial cells and syngeneic lymphoblastoid cells. However, a significant portion (11%) of the CTL clones recognized unique peptides presented only in the context of SLA class I molecules on endothelial cells. CONCLUSION These results provide evidence for the first time that tissue-specific peptides can directly influence T cell repertoire in response to the xenogeneic stimulus.