Identification of new carbohydrate and membrane protein antigens in cardiac xenotransplantation

Abstract

Background: α1,3-Galactosyltransferase gene knockout (GTKO) pigs reduced the significance of antibody to galactose alpha 1,3-galactose (Gal) antigens but did not eliminate delayed xenograft rejection (DXR). We hypothesize that DXR of GTKO organs results from an antibody response to a limited number of non-Gal endothelial cell (EC) membrane antigens. In this study, we screened a retrovirus expression library to identify EC membrane antigens detected after cardiac xenotransplantation.

Methods: Expression libraries were made from GT:CD46 and GTKO porcine aortic ECs. Viral stocks were used to infect human embryonic kidney cells (HEK) that were selected by flow cytometry for IgG binding from sensitized cardiac heterotopic xenograft recipients. After three to seven rounds of selection, individual clones were assessed for non-Gal IgG binding. The porcine complementary DNA was recovered by polymerase chain reaction amplification, sequenced, and identified by homology comparisons.

Results: A total of 199 and 317 clones were analyzed from GT:CD46 and GTKO porcine aortic EC complementary DNA libraries, respectively. Sequence analysis identified porcine CD9, CD46, CD59, and the EC protein C receptor. We also identified porcine annexin A2 and a glycosyltransferase with homology to the human β1,4 N-acetylgalactosaminyl transferase 2 gene.

Conclusion: The identified proteins include key EC functions and suggest that non-Gal antibody responses may compromise EC functions and thereby contribute to DXR. Recovery of the porcine β1,4 N-acetylgalactosaminyl transferase 2 suggests that an antibody response to a SD-like carbohydrate may represent a new carbohydrate moiety involved in xenotransplantation. The identification of these porcine gene products may lead to further donor modification to enhance resistance to DXR and further reduce the level of xenograft antigenicity.

FIGURE 1.

Expression library screening and analysis. (A) The non-Gal antibody response after α1,3-galactosyltransferase gene knockout (GTKO) and GT⁺:CD46 heterotopic cardiac xenotransplantation were determined using flow cytometry by measuring IgG binding to GTKO porcine aortic endothelial cells (PAECs). A comparison of pretransplant (white bars) and necropsy (black bars) IgG binding at 1:20-fold, 1:80-fold, and 1:320-fold dilution is shown. All recipients show an increase in anti-pig non-Gal IgG after transplant. The donor organ genotype (GTKO or GT⁺:CD46) is shown below each data set. (B) Flow cytometry of IgG binding to individual pRETRO-infected HEK clones. Each line represents a different pRETRO-infected clone. The lines are color coded to denote clones with similar levels of antibody binding. The filled histogram is an infected control HEK cell that does not express a non-Gal antigen. (C) Results of polymerase chain reaction (PCR) amplification of the pRETRO-encoded porcine complementary DNA (cDNA) using genomic DNA of individual pRETRO-infected HEK clones. The cDNA product was amplified using PCR primers, which flank the multiple cloning site of the pRETRO vector. These products were cloned and sequenced to identify the non-Gal antigen.

FIGURE 2.

The identification, confirmation, and expression of non-Gal porcine aortic endothelial cell (PAEC) antigens. (A) Flow cytometry of IgG binding to pRETRO-infected HEK cells for individual non-Gal antigens. (B) Flow cytometry showing IgG binding to G418-resistant pcDNA3.1/V5-His-TOPO transfected cell lines expressing individual non-Gal antigens. (C) Reverse-transcriptase polymerase chain reaction analysis of non-Gal gene expression. RNA samples: lane 1, pig heart; lane 2, GTKO PAECs; lanes 3 to 5, independent G418-resistant HEK transformants; lane 6, untransformed HEK cells; lane 7, a flow cytometry negative HEK cell line; and lanes 8 to 10, the same as lanes 3–5 but without reverse transcriptase. Non-Gal antigen expression: (a, g, and m) CD9; (b, h, and n) PROCR; (c, i, and o) CD46; (d, j, and p) CD59; (e, k, and q) ANXA2; and (f, l, and r) B4GALNT2.

FIGURE 3.

A characterization of the specific non-Gal antibody responses detected after pig-to-primate cardiac xenotransplantation. Pretransplant and sensitized necropsy serum from five cardiac xenograft recipients, not treated with T-cell immunosuppression, were individually screened for IgG reactivity to HEK cells expressing porcine non-Gal antigens. The immune response for a single transplant recipient is shown (A and B). The non-Gal antigen names are indicated above each column. (A) Pretransplant serum 1:40 dilution. (B) Necropsy serum 1:40 dilution. Filled histograms represent IgG binding to negative control HEK cells not expressing a non-Gal antigen, and lines indicate specific IgG binding to the indicated antigen. Specific antibody reactivity was calculated as indicated in the Materials and Methods. (C) The average antibody response for all five recipients to each antigen at 1:40, 1:160, and 1:640 serum dilutions (black, white, and gray filled boxes, respectively). Pretransplant serum (PreTx) and necropsy serum (Nec.). For each antigen, the number of recipients out of five showing a positive induction of antibody is indicated. A positive response was considered to be a 2-fold or greater increase in antibody binding in necropsy serum detected in two or more serum dilution.