Function Measurements of HLA-II Transgenic Pigs for Xenotransplantation

Hao-Chih Tai1, Ching-Fu Tu2,*, Tien-Shuh Yang2, Jang-Ming Lee1, San-Yuan Huang2,3 and Bao-Tyan Wang4 1Department of Surgery, National Taiwan University Hospital and College of Medicine 2Divisions of Biotechnology and Applied Biology, Animal Technology Institute Taiwan 3Department of Animal Science, National Chung Hsing University, Taichung 4Department of Genomic Medicine, Changhua Christian Hospital, Changhua Taiwan


Introduction
Using xenograft from transgenic (Tg) pigs is a promising approach to lessen the organ shortage for transplantation.Transgenesis such as CD55 or CD46, and CD59 as well as alpha 1, 3-galactosyl transferase gene knockouts shall avoid rejections.Indeed, grafts obtained either from hDAF (CD55) Tg pigs or from alpha 1, 3-galactosyl transferase gene knock-out pigs, all can overcome hyperacute rejection in xenotransplantation since porcine hearts could survive heterotopically in non-human primates more than 6 months (Kuwaki et al., 2005;Tai et al., 2007;).Obstacles are still remaining however, as early inflammation, acute humoral and acute cellular xeno-rejections and thrombotic microangiopathy are the following problems yet to tackle (Tai et al., 2007).
The early inflammation involves up-regulation of pro-inflammatory mediators in the graft and occurs before the T cell responses after engraftment.It is an innate response of NK cells to tissue injury and independent of the adaptive immune system.The major histocompatibility complex (MHC) molecules in human, including human leukocyte antigen (HLA) E, G, and class I molecules, in theory, can inhibit human NK cells xeno-rejection (Sasaki et al., 1999).This is proven by generating the HLA-E/human beta 2-microglobulin Tg pigs that can express transgenes consistently in peripheral blood mononuclear cells and on endothelial cells of organs, including heart and kidney, and these transgenes can provide partial protection against human NK rejections ( Weiss et al., 2009).
In acute cellular rejection, T-cells cytotoxicity is responsible for the major cell-mediated rejections.In human allotransplantation, the donor-recipient match of HLA-II improves graft survivals, especially in kidney transplantation (Sheldon et al., 1999;McKenna et al., 2000).Yet, the roles of HLA-II to attenuate acute cell-mediated xeno-rejections remain uncertain.We have successfully generated HLA-DP, DQ and DR Tg pigs and showed that human-to-pig xenogenic cellular responses could be significantly depressed by expressing

The expression of DP or DQ exogenes in HLA-DP or DQ transgenic pigs reduced human-to-pig cellular responses
In allotransplantation, HLA-II matching could improve graft survivals, especially in kidney transplantation (Sheldon et al., 1999;McKenna et al., 2000), possibly due to better donor lymphocyte survival in recipients, so-called microchimerism (Starzl et al., 1992).However, the roles of HLA-II in porcine xenograft remain to be elucidated.In acute cell-mediated xeno-rejections, both direct and indirect pathways in human T-cell rejections are involving.The direct pathway, presumably the dominant one, engages in the early alloimmune response initiated by direct contact of host T-cells with allo-HLA molecules.Initially, human T cells recognize intact xeno-swine leukocyte antigens (SLA) on the surface of the antigen presenting cells (APC) or endothelial cells (EC) of transplanted pig organs.Then, human T cells identify xeno-SLA molecules bound xeno-peptide as being equivalent in shape to self-HLA bound foreign peptide and, hence, treat the xeno-tissue as foreign.In the proposed indirect pathway, human T cells recognize processed xeno-antigens presented as peptides by human APCs.
The roles of the HLA-II antigen in iso-, allo-, and xenotransplantation have also been studied in HLA-DQ and HLA-DP transgenic mice (Tsuji et al., 1994).The HLA-DP and DQ transgenic pigs were further produced through the technique of microinjection.Genomic DNA clones, including HLA-DP (including both A1 and B1 sequences) (Tu et al., 1999), and HLA-DQA1 and HLA-DQB1 (Tu et al., 2000) were transferred into pronucleus of porcine fresh fertilized eggs by microinjection.The successful integration of both HLA-DP and HLA-DQ transgenes were proven by polymerase chain reaction (PCR), Southern blot (Tu et al., 1999 andTu et al., 2000, respectively), and FISH (Wang et al., 2004).The expression was also revealed by reverse transcriptase-PCR (RT-PCR) and by flow cytometry in HLA-DP (Tu et al., 1999 and1998;Lee et al., 2000 and2002) and HLA-DQ (Tu et al. 2003) transgenesis.To elucidate the function of HLA-DP and DQ antigens, the proliferation of human peripheral blood mononuclear cells (PBMC) to porcine xeno-antigen could be attenuated by primed or direct xenogenic mixed lymphocyte culture (MLC) tests.

The expression of DR exogenes in HLA-DR transgenic pigs enhanced cyclosporine effect
The integration of HLA-DR15 + transgenes in transgenic pigs (Tu et al., 2001) was directly revealed by FISH which has localized both DRA1 and DRB1 transgenes on pig chromosome 13 near the centromere (unpublished data).The expression of transgenes has been confirmed by flow cytometry and the immunohistochemical stain.Results (unpublished data) have also shown that about 39.2% of porcine peripheral blood mononuclear cells and the endothelial cells on the blood vessel of transgenic pig successfully expressed HLA-DR antigens.
Proteomic approach (Huang et al., 2006) revealed that the HLA-DR15 + transgenic pigs could express more proteins including triosephosphate isomerase, cyclophilin B (CyPB), proteaseme and RhoA than their non-transgenic littermates.It is of great interest to elucidate the association of HLA-DR with CyPB on transgenic pigs especially in xenotransplantation.Triosephosphate isomerase can stimulate lymphocyte proliferation (Richter et al., 1993) and its minor structural change corresponds to substantially enhanced stimulation of a CD4 + tumor-infiltrating lymphocyte line (Sundberg et al., 2002).The proteasome is a multicatalytic complex of proteases involved in T-lymphocyte proliferation and activation and plays an important role in cell-cell interaction during T-lymphocyte activation (Kanaan et al., 2001).The GTP-binding protein RhoA is a member of the Ras GTPase superfamily and has been reported to be actively involved in the regulation of Tlymphocyte morphology and motility (Woodside et al., 2003).The Rho GTPases are molecular switches and are pivotal regulators of antigen-specific T-cell activation by antigen-presenting cells and immunological synapse formation (Deckert et al., 2005).The findings by Mzali et al. (Mzali et al., 2005) also suggested that Rho GTPases play crucial roles in T-lymphocyte functions and proliferation.These proteins are usually involved in Tcell activation or proliferation, except CyPB which belongs to a class of highly conserved proteins that accelerate the folding of proteins and being a cyclosporine A (CsA) binding protein (Schreiber, 1991).This protein is abundant in thymus cytoplasm and appears to be involved in the regulation of T-lymphocyte activation and proliferation (Harding et al., 1986), and inhibits the early T-cell activation (Liu et al., 1991) and prevents graft rejection (Schreiber and Crabtree, 1992).Furthermore, Denys et al. (1998) reported that plasma CyPB may enhance the immunosuppressive activity of CsA through a cell-mediated incorporation of CyPB-complexed CsA with in peripheral blood lymphocytes, and thus contributes to the acceptance and the good maintenance of organ transplantation.It is very interesting to elucidate the association of HLA-DR with CyPB on transgenic pigs especially in xenotransplantation.
By mixed lymphocyte culture, Tg and NTg pigs' lymphocytes (pLC, stimulator) were compared to stimulate activation of human lymphocytes (hLC; effectors) so that the survival of HLA-DR Tg pig endothelial cells (pETC) in contact with xenografting lymphocytosis could be further evaluated.The hLC from HLA-DR15 + or HLA-DR15 -healthy adults with Oor B-type man or women, and from HLA-DR15 + Tg and NTg pLC were harvested from peripheral blood samples by Ficoll-Paque™ Plus and separated from hematopoietic cells by culturing in 35 mm dish for overnight.Porcine LC was inactivated by 10.0 g/mL of mitomycin C for 30 min before mixed with hLC.These xeno-mixed LC reactions were cultivated in RPMI1640 medium adding 0, 0.1 and 1 g/mL of CsA and for 48 hours.The proliferation of hLC was evaluated by MTS assay.Results (Figure 3) thus obtained were: without CsA, the pLC stimulated 1.2 ±2.0 ~ 47.0 ±4.0% of hLC proliferation; while after addition of 0.1 and 1 g/mL CsA, the Tg vs. NTg pLC enhanced inhibition effects on hLC proliferation was 23.8 ±2.5 ~ 23.8 ±2.9% vs. 14.7 ±2.5 ~ 14.9 ±2.5%, and at 47.9 ±2.4 ~ 48.1 ±2.8% vs. 24.9±2.0 ~28.5 ±2.4%, respectively.Result showed that inactivation of hLC by CsA could be enhanced by HLA-DR transgenesis, suggesting that an ameliorated effect has occurred in acute xenograft rejection.The exp r e s s i o n o f C y P B i n H L A -D R t r a n s g e n i c lymphocytes (intracellular) (Huang et al., 2006) could depress hLC activation.However, the improvement of survival rate of pETC (responder) varied highly in Tg pETC as compared to NTg pETC (data not shown).Although CyPB was expressed on endothelial cells (Carpentieret et al., 1999) and capable of enhancing T-cell adhesion to ETC extracellular matrix, then it could significantly attenuate by CsA (Allain et al., 2002).It is worth noting that HLA-DR transgenesis and the expression and secretion of CyPB on pETC will be firstly attached by activated hLC.

Long-term survival of HLA-DR pig skin in SCID mice after reconstitution with human PBMC and under short-term immunosuppression
To test the role of donor-recipient HLA-II-match in xenotransplantation, the HLA-DR15 + porcine skins were transplanted to SCID mice which were thereafter reconstituted with HLA-DR 15 + or DR15 -hPBMC.The studies were conducted under no immunosuppression (no CsA was given to hPBMC-SCID mice), or under immunosuppression (CsA was given intra-peritoneally to hPBMC-SCID mice for 12 days) to reveal the effectiveness of the graft.
In studies of HLA-DR15 + p o r c i n e s k i n g r a f t s t o h P B M C -S C I D m i c e u n d e r n o immunosuppression (Tu et al., 2008), human CD4 + and CD8 + cells were detected from days 7 to 29 after hPBMC reconstitution in hPBMC-SCID mice.Both CD4 + and CD8 + cells of HLA-DR15 -hPBMC-SCID mice were significantly higher at day 29 post-grafting, compared with that of HLA-DR15 + hPBMC-SCID mice.In HLA-DR15 + hPBMC-SCID mice, the HLA-DR15 + Tg pig skin grafts survived and integrated into mice, and illustrated histopathologically less cellular rejections which showed intact dermis with little lymphocytic infiltration.However, in HLA-DR15 -hPBMC-SCID mice, the HLA-DR15 + transgenic pig skin grafts illustrated more cellular rejections which showed disrupted collagen, as well as mild to moderate lymphocytic infiltration.The results suggested that HLA-DR matching attenuated xenogenic cellular rejection.(Tai et al., 2008), human CD4 + and CD8 + cells were found in hPBMC-SCID mice after reconstitution.Tests of MLC showed more responses of HLA-DR15 -hPBMC against HLA-DR15 + porcine PBMC.HLA-DR15 + porcine skin grafts survived more than 100 days in hPBMC-SCID mice which were reconstituted twice with HLA-DR15 + or HLA-DR15 -hPBMC.In the negative control group, HLA-DR15 + porcine skins were rejected in all non-SCID (Balb/c) mice (data not shown), and the gross pictures showed disappeared porcine skin and growth of murine hair in non-SCID (Balb/c) mice.Histological pictures of transplanted HLA-DR15 + porcine skin grafts showed survived porcine epithelium in remodeling murine dermis (with organized collagen), and little lymphocytes infiltration in murine dermis.Although the results do not suggest that HLA-DR matching attenuated xenogenic cellular rejection, it showed that HLA-DR15 + pig skin grafts could survive over a prolonged period in hPBMC-SCID under a short period of immunosuppression with CsA.The long-term survival of HLA-DR15 + pig skin grafts in either HLA-DR15 + or HLA-DR15 -hPBMC-SCID mice might be due to poor engraftment or function of reconstituted T cells, under immunosuppression with CsA.Because of the gradually decreased number of reconstituted T cells and suppression effect of CsA, HLA-DR15 + pig skin grafts were not rejected and therefore survived more than 100 days.In studies of T cell proliferation responses to porcine aortic endothelial cells (PAEC) either in the presence or absence of CsA, both allogeneic and xenogeneic T cell responses could be inhibited by in vitro (Fig. 3) or by therapeutic levels of CsA in vivo (Batten et al., 1999).

In studies of HLA-DR15 + p o r c i n e s k i n g r a f t s t o h P B M C -S C I D m i c e u n d e r immunosuppression with CsA
In the studies of Hagihara et al. (1996), in vitro MLC and in vivo skin grafting were conducted by using HLA-DP Tg mice (B6-DP mice).Xenogenic iso-(B6-DP to B6 mice) MLC showed positive but much less responses when compared to allo-MLC responses.Nevertheless, B6-DP skin grafts were rejected in a similar time period as allo-skin grafts.Further studies of in vitro cytotoxic lymphocyte responses and delayed-type hypersensitivity reactions indicated that xenogeneic HLA-DP antigens could act as significant transplantation antigens equivalent to alloantigens despite their less stimulative activity in vitro.Results also support the interpretation that DP antigens act like a minor histocompatibility antigen beyond the difference of species (Hagihara et al., 1996).In our studies, xenogenic HLA-DR15 + antigens which act as minor histocompatibility antigens and swine leukocyte antigens (SLA) contributed simultaneously to exert acute cellular rejection of porcine skins in hPBMC-SCID mice.
During allogenic skin rejection, the destruction of critical dermal structures that determine the ultimate viability of the skin graft is highly antigen-specific and is almost certainly accomplished by cytotoxic T cells.Whereas destruction of non-critical epidermal structures of the skin allograft is antigen-nonspecific and can be accomplished by inflammatory cells or their secreted products.The MHC antigens are specific antigens for skin rejection.Matched MHC antigens of donors and recipients may improve survivals of allogenic skin graft.The in vivo skin-grafting in HLA-DR15 + hPBMC-SCID mice, matched HLA-DR15 + transgenic pig skin grafts displayed less cellular rejections.

Summary and conclusion
Our results from studies on the HLA-DPw0401 and HLA-DQw0601 Tg pig support the concept that increasing the similarity of MHC class II determinants between pig and human using an HLA-II Tg pig could reduce the xenogenic cellular response and attenuate human lymphocytic proliferation in xeno-mixed lymphocyte cultures and human-to-pig xenogenic cellular responses.In addition, proteomic approaches revealed that the HLA-DR15 + transgenic pigs could express more proteins including triosephosphate isomerase, cyclophilin B (CyPB), proteaseme and RhoA than their nontransgenic littermates.These proteins are involved in T-cell activation or proliferation, except CyPB which belongs to a class of highly conserved proteins that accelerate the folding of proteins and being a cyclosporine A (CsA) binding protein.This protein is abundant in thymus cytoplasm and appears to engage in the regulation of T-lymphocyte activation and proliferation, and inhibits the early T-cell activation and prevents graft rejection.Furthermore, the plasma CyPB may enhance the immunosuppressive activity of CsA through a cell-mediated incorporation of CyPB-complexed CsA within peripheral blood lymphocytes, and thus contributes to the acceptance and the good maintenance of organ transplanted.By using in vitro mixed xeno-lymphocytes culture, the inactivation of hLC by CsA could be enhanced by HLA-DR transgenesis, suggesting that an ameliorated effect has occurred in acute xenograft rejection.Further studies using in vivo skin-grafting in HLA-DR15 + hPBMC-SCID mice, HLA-DR15 + Tg pig skin grafts displayed less cellular rejections due to additional histocompatibility factor, HLA-DR15 + , especially at the administration of CsA.

Fig. 1 .
Fig. 1.Stimulating indexes (SI) of direct xenogenic mixed lymphocyte culture (MLC) test with responder of HLA-DPw4 + human lymphocytes.In direct xenogenic MLC tests with responders from HLA-DPw0401 + humans, the PBMCs from the HLA-DPw0401 transgenic (Tg) pigs, as compared with those from the non-transgenic (NTg) pigs, induced a lower degree of xenogenic cellular responses to human PBMCs.(n=4).The cellular proliferation of human PBMC under stimulation by porcine PBMC was reduced in the presence of HLA-DQ molecules expressed on the porcine cells, as compared to that in the presence of the NTg littermate control.The human-to-porcine xenogenic Th1 response, as represented by the production of INF-gamma, was also attenuated by stimulation with HLA-DQ transgenic pig cells (Lee et al., 2003).These evidences were demonstrated in human PBMCs with or without the HLADQw0601 allele.The studies on the HLA-DPw0401 and DQw0601 transgenic pigs supported the concept that increasing the similarity of MHC class II determinants between HLA-II Tg pigs and human beings shall reduce the xenogenic cellular responses.

Fig. 2 .
Fig. 2. Stimulating indices (SI) (A1 and A2) and TH1 response (INF  production) (B1 and B2) of direct xenogenic mixed lymphocyte culture (MLC) tests with matched or mismatched responders of HLA DQw0601(+ or -) human PBMCs.A1 and A2: the cellular proliferation of human PBMC under stimulation by porcine PBMC could be reduced in the presence of HLA-DQ molecules expressed on the porcine cells, as compared to that in the presence of the NTg littermate control.B1 and B2: the human-to-porcine xenogenic Th1 response, as represented by the production of INF-gamma, was also attenuated by stimulation with HLA-DQ transgenic pig cells.Tg: HLA-DQ transgenic pig; NTg: non-transgenic littermate; WP: wild pigs; and each group n=3.* p < 0.05 as compared to Tg; # using the cpm of Tg as referent (SI=1).