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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2002 as doi:10.1096/fj.02-0630fje. |
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1,2-fucosyltransferase 1
Departments of Molecular and Cellular Sciences, Alexion Pharmaceuticals Inc., Cheshire, Connecticut, USA
2Correspondence: Department of Molecular Sciences, Alexion Pharmaceuticals Inc,, 352 Knotter Dr., Cheshire, CT, 06410 USA. E-mail: costac{at}alxn.com
SPECIFIC AIMS
In our laboratory we aim to develop porcine cartilage resistant to delayed tissue rejection for xenotransplant applications. First it was necessary to understand the process of xenogeneic cartilage rejection, particularly the contribution of the Gal
1,3-Gal antigen present in the donor tissue. To this end, in vitro engineered porcine cartilage expressing 1,2-fucosyltransferase (HT) was generated and tested in vivo in wild-type and 1,3-galactosyltransferase knockout (Gal KO) mice.
PRINCIPAL FINDINGS
1. Transgenic expression of HT reduces dramatically the amount of Gal1,3-Gal antigen in porcine cartilage and isolated chondrocytes
We characterized porcine articular chondrocytes (PAC) isolated from control and two lines of HT transgenic pigs (HTAT20 and HTAT21) by flow cytometry. HTAT20-expressing cells showed the most reduction (89%) in Gal1,3-Gal epitope cell surface expression as well as diminished human and Gal KO-mouse natural antibody reactivity. The pattern and level of Gal1,3-Gal expression were investigated in control and HTAT20 transgenic cartilage by immunofluorescence. A generalized decrease in Gal1,3-Gal antigen was detected in both native and in vitro engineered HT transgenic cartilage.
2. CD4+ T cells play a major role in xenogeneic porcine cartilage rejection by inducing an anti-pig antibody response and a monocytic cellular infiltrate
To characterize the process of xenogeneic cartilage rejection in a small animal model, we first studied the contribution of CD4+ T cells to the rejection of HTAT21 porcine cartilage implanted subcutaneously (s.c.) in wild-type mice. The humoral response was evaluated by assessing the anti-PAC antibody reactivity in serum at 3 and 5 wk after transplantation. Treatment with a depleting anti-CD4 antibody completely blocked the elicited antibody response, which was predominantly anti-pig and showed no specific reactivity to the H epitope. The effect of blocking CD4+ T cells on the rejection process was also analyzed by histology. Implants from the control-treatment group showed a predominantly mononuclear cellular infiltrate surrounding the xenograft at 3 and 5 wk. Treatment with anti-CD4-depleting antibody markedly reduced the amount of cellular immune infiltrate at both time points, but a minor lymphocytic infiltrate remained in the implant periphery.
3. HT porcine cartilage is highly resistant to delayed tissue rejection in Gal KO mice
To test the effect of reducing Gal1,3-Gal antigen in the donor tissue, we s.c. transplanted control or HTAT20 engineered cartilage into Gal KO mice. Histological evaluation showed that the nontransgenic porcine cartilage was rejected as early as 2.5 wk, with extensive tissue destruction and a pronounced mononuclear cellular infiltrate penetrating the tissue (Fig. 1
A). The cellular infiltrate progressed and peaked at 5 wk (Fig. 1C
). This rejection process was faster and more aggressive than that observed in grafted wild-type mice, but similarly contained cells from monocyte/macrophage and lymphocyte lineages. In contrast, the HTAT20 transgenic grafts were intact at 2.5 wk (Fig. 1B
) and the cartilage structure was preserved during the course of the study (5 and 10 wk) (Fig. 1D, F
). Rejection was limited to the xenograft periphery, where a minor cellular infiltrate developed.
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4. The elicited antibody response is markedly reduced in Gal KO mice transplanted with HT cartilage grafts
To investigate the mechanism of Gal1,3-Gal-mediated rejection, we assessed the humoral response in the two transplanted cohorts (Fig. 2
). Basal levels of anti-PAC IgM antibody reactivity were detected in nontransplanted Gal KO mice by flow cytometry. The anti-PAC IgM antibody titers rose at 5 wk post-transplantation in the group receiving control cartilage grafts and remained high up 10 wk (Fig. 2A
). In contrast, Gal KO mice receiving HTAT20 cartilage grafts showed no elevation in anti-PAC IgM antibodies above background (Fig. 2A
). Consistent with a predominantly anti-Gal1,3-Gal IgM response in the control graft recipients, reactivity of the IgM subtype to HT transgenic PAC was comparable between experimental groups and was overall lower than to control PAC (Fig. 2C
). With regard to anti-PAC IgG antibodies, no reactivity was detected in serum from nontransplanted Gal KO mice (Fig. 2B, D
). Mice receiving control cartilage mounted an anti-PAC IgG response first detected 2.5 wk after transplantation, peaking at 5 wk (Fig. 2B
). HTAT20 graft recipients showed no detectable anti-PAC IgG at 2.5 wk, lower anti-control-PAC humoral response at 5 wk (2-fold reduction vs. control transplant group), and near-background IgG reactivity at 10 wk (Fig. 2B
). Results with HTAT20 transgenic PAC suggested that the increase in IgG antibody titers in response to the HTAT20 cartilage grafts was predominantly non-anti-Gal1,3-Gal (Fig. 2D
). We later confirmed by ELISA that the elicited anti-Gal1,3-Gal antibody response was absent in Gal KO mice grafted with HT cartilage. Moreover, we assayed splenic B cells for reactivity to the Gal1,3-Gal epitope by flow cytometry. Whereas mice receiving control cartilage showed an increase in the percentage of B cells binding to the Gal1,3-Gal epitope compared with naive mice, this subpopulation of B cells was unchanged in HTAT20-grafted mice.
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CONCLUSIONS AND SIGNIFICANCE
Many human diseases and injuries result in tissue loss or dysfunction. The field of tissue engineering has progressed substantially in recent years to provide a therapeutic solution. Tissue-engineered cartilage is actively being pursued due to the limitations of available treatments to repair cartilage defects. The use of xenogeneic porcine cells or tissues for tissue engineering may lead to further advances and broaden its clinical application, but immunological hurdles need to be identified and addressed appropriately for each type of tissue. To gain insight into the mechanism of xenogeneic cartilage rejection, we first studied the contribution of CD4+ T cells in wild-type mice grafted with porcine cartilage. CD4+-T cell depletion demonstrated that the elicited anti-pig antibody response is T cell dependent in wild-type mice. The grafts showed a dramatic reduction in the amount of cellular immune infiltrate, particularly monocytes/macrophages. These observations agree with previous reports identifying a critical role of CD4+ T cells in rejection of other xenogeneic tissues. Rejection of xenografts is known to be predominantly a Th2 response, and our results indicate that a T cell-mediated humoral response plays a role in rejecting porcine cartilage. We believe that several mechanisms that lead to delayed xenograft rejection in solid organs are common to the rejection process of xenogeneic cells and tissues. The Gal1,3-Gal antigen is the major xenoantigen in porcine tissues recognized by human natural antibodies. The contribution of the Gal1,3-Gal antigen to delayed xenograft rejection and delayed tissue rejection is not well established. To address this question, we transplanted control or HT transgenic engineered cartilage into Gal KO mice. We demonstrated that control cartilage expresses Gal1,3-Gal antigen whereas its expression is dramatically diminished in HT transgenic cartilage. Control porcine cartilage was rejected in several weeks by a prominent cellular immune infiltrate and elevated anti-pig and anti-Gal1,3-Gal antibody titers. In contrast, Gal KO mice receiving the HT cartilage showed a markedly reduced immune response, consistent with a major role of Gal1,3-Gal antigen in mediating delayed rejection of tissues.
To understand the Gal1,3-Gal-mediated tissue rejection process, we need to keep in mind how the anti-Gal1,3-Gal antibody response is generated in Gal KO mice. The anti-Gal1,3-Gal IgM response is independent of T cell help, but Gal1,3-Gal binding B cells that recognize glycoproteins containing this antigen need to bind to T cells for further activation and isotype switching. We have observed an increase in the quantity of Gal1,3-Gal binding B cells in spleen of control grafted mice, but not in Gal KO mice receiving HT grafts. An increase in this population of B cells has been described in Gal KO mice immunized with Gal1,3-Gal-containing antigens. Moreover, these cells are major producers of anti-Gal1,3-Gal IgM antibodies. As there was no anti-Gal1,3-Gal antibody response of either IgM or IgG subtypes in the HT-grafted Gal KO mice, the response was most likely absent due to insufficient Gal1,3-Gal-mediated B cell activation. Furthermore, the anti-pig antibody response was markedly reduced in this cohort. Apart from Gal1,3-Gal, no other carbohydrates seemed to play a major role in the elicited IgM response in this animal model. The lower anti-pig IgG antibody response of the HT-grafted Gal KO mice could be explained by a decreased presentation of xenoantigens to T cells. Our results imply that the Gal1,3-Gal binding B cells play a role as antigen-presenting cells that amplify the T cell response. In summary, different pathways may have contributed to the survival of the HT grafts in the Gal KO mice. First is a reduction in natural antibody reactivity, as we determined a decrease in Gal KO-mouse XNA deposition on HT PAC relative to control cells. Second, the anti-Gal1,3-Gal antibody response was eliminated and the anti-pig antibody response was reduced. A concomitant decrease in complement activation and antibody-dependent cell-mediated cytotoxicity would also be expected. Other mechanisms such as reduced cell interactions to HT grafts by NK cells and macrophages due to specific carbohydrate recognition may have played a role, but those direct cellular reactions remain to be elucidated in Gal KO mice.
Our observations have clear relevance to the pig-to-human clinical setting, although there are probably differences to be considered. In some cases, the human system has been better characterized than the mouse. We have observed that transgenic expression of HT decreases human NK cell-mediated cytolysis of fibroblasts and monocyte cell adhesion to PAC. To evaluate these effects, this strategy should ultimately be tested in a primate model. Nevertheless, our results are consistent with those of Stone et al., where porcine cartilage was treated with -galactosidase to remove the Gal1,3-Gal antigen and transplanted into Cynomolgus monkeys. However, they observed no reduction in the anti-pig antibody titers relative to control-cartilage recipients. Our approach is advantageous because it does not involve treatment of the tissue and prevents the cells from producing more Gal1,3-Gal antigen. It is also preferable to other strategies based on modifying the recipient. We believe that expression of HT by genetic engineering of the animal donor has real potential for contributing to xenograft acceptance in cell- and tissue-based clinical applications. Numerous applications may benefit from combining the progress in the fields of tissue engineering and xenotransplantation. Porcine cartilage either as cartilage plugs, in vitro engineered with/without scaffolds, or isolated chondrocytes injected with hydrogels could be used for orthopedic and reconstructive surgery. Here we show that reduction of the Gal1,3-Gal antigen on cartilage tissue by high expression of HT inhibits the elicited antibody response and delays rejection. Additional genetic modifications of the donor tissues designed to inhibit the cellular immune responses may eventually eliminate the need of immunosuppression.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0630fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0630fje 
This article has been cited by other articles:
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  J. J. Ramsoondar, Z. Machaty, C. Costa, B. L. Williams, W. L. Fodor, and K. R. Bondioli Production of {alpha}1,3-Galactosyltransferase-Knockout Cloned Pigs Expressing Human {alpha}1,2-Fucosylosyltransferase Biol Reprod, August 1, 2003; 69(2): 437 - 445. [Abstract] [Full Text] [PDF]
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