Humoral Reactivity of Renal Transplant-Waitlisted Patients to Cells From GGTA1/CMAH/B4GalNT2, and SLA Class I Knockout Pigs

Abstract

Background: Antipig antibodies are a barrier to clinical xenotransplantation. We evaluated antibody binding of waitlisted renal transplant patients to 3 glycan knockout (KO) pig cells and class I swine leukocyte antigens (SLA).

Methods: Peripheral blood mononuclear cells from SLA identical wild type (WT), α1, 3-galactosyltransferase (GGTA1) KO, GGTA1/ cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) KO, and GGTA1/ CMAH /b1,4 N-acetylgalactosaminyl transferase (B4GalNT2) KO pigs were screened for human antibody binding using flow cytometric crossmatch (FCXM). Sera from 820 patients were screened on GGTA1/CMAH/B4GalNT2 KO cells and a subset with elevated binding was evaluated further. FCXM was performed on SLA intact cells and GGTA1/SLA class I KO cells after depletion with WT pig RBCs to remove cell surface reactive antibodies, but leave SLA antibodies. Lastly, human and pig reactive antibodies were eluted and tested for cross-species binding and reactivity to single-antigen HLA beads.

Results: Sequential glycan KO modifications significantly reduce antibody binding of waitlisted patients. Sera exhibiting elevated binding without reduction after depletion with WT RBCs demonstrate reduced binding to SLA class I KO cells. Human IgG, eluted from human and pig peripheral blood mononuclear cells, interacted across species and bound single-antigen HLA beads in common epitope-restricted patterns.

Conclusions: Many waitlisted patients have minimal xenoreactive antibody binding to the triple KO pig, but some HLA antibodies in sensitized patients cross-react with class I SLA. SLA class I is a target for genome editing in xenotransplantation.

FIGURE 1.

Analysis of human immunoglobulin binding to PBMC from genetically modified swine. PBMC were collected, incubated with human serum and analyzed for IgM (y axes) and IgG (x axes) binding by flow cytometry. Dotted lines represent an MFI of 2000. A, A single representative patient is screened on WT, GGTA1 KO, GGTA1/CMAH KO, GGTA1/CMAH/B4GalNT2 KO swine PBMC. B, Sera from 44 randomly selected patients with unknown sensitization were incubated with PBMC from all 4 glycan backgrounds. C, Sera from 820 waitlisted renal patient samples were incubated with GGTA1/CMAH/B4GalNT2 knockout PBMC. Solid dots represent 119 samples that were further analyzed in Figure 3.

FIGURE 2.

Absorption of sera with WT swine RBC minimizes antibody binding to knockout swine PBMC. Porcine-reactive IgG (A) and IgM (B) were depleted from 10 human sera by absorption with WT swine RBC. Immunoglobulin binding to cells deficient in GGTA1, GGTA1/CMAH, or GGTA1/CMAH/B4GalNT2 was analyzed for each serum before (intact) and after (absorbed) absorption. Flow cytometry determined MFIs of antibody binding. Horizontal dotted line appears at MFI 2000. Analysis of depleted sera showed that on average more IgG bound to GGTA1-deficient PBMC than to cells lacking GGTA1/CMAH/B4GalNT2. Wilcoxon matched-pairs rank sum test compared antibody binding preabsorption and postabsorption to each cell type (IgG all comparisons, P = 0.001; IgM GGTA1 KO, P = 0.001; GGTA1/CMAH p = 0.0210, GGTA1/CMAH/B4GalNT2, P = 0.339). Dunnett test was performed to compare IgG binding of depleted sera to the different PBMC (GGTA1 vs GGTA1/CMAH, P = 0.923; GGTA1 vs GGTA1/CMAH/B4GalNT2, P = 0.0239; GGTA1/CMAH vs GGTA1/CMAH/B4GalNT2, P = 0.307).

FIGURE 3.

Some sera contain antibodies which bind class I SLAs. Flow cytometry evaluated IgG binding from 119 sera to PBMC collected from swine lacking GGTA1/CMAH/B4GalNT2 genes before (intact y axis) and after (absorbed x axis) depletion of antibodies recognizing pig-specific carbohydrate xenoantigens. The preabsorption and postabsorption MFI of 73 sera, shown in panel A, exhibited less than 25% variation. Forty-six sera demonstrated more that 25% variation in MFI values and are not shown to simplify analysis. To determine if residual antibodies to TKO swine PBMC-targeted class I SLA molecules, absorbed sera from panel A were incubated with either GGTA1/CMAH/B4GalNT2 knockout (x axis, class I SLA⁺) or GGTA1/SLA1 knockout (y axis, class I SLA⁻) swine PBMC. MFI of IgG binding was determined by flow cytometry. Sera exhibiting greater (B) or less (C) than 25% variation of binding in the presence or absence of class I SLA are shown. Nine sera with high levels of binding to GGTA1/CMAH/B4GalNT2 knockout caused by class I SLA are highlighted (red dots, panels A and B).

FIGURE 4.

Determining the frequency of highly allosensitized human sera containing antibodies Specific for class I SLA molecules. Human sera with panel reactive antibodies greater than 80 were absorbed with WT pig RBC and incubated with PBMC collected from GGTA1 knockout or GGTA1/SLA1 knockout swine. Flow cytometry determined MFIs of binding to these cells for IgG (A) and IgM (B). Thirteen of 22 samples contained class I SLA-specific IgG, and 4 of 17 samples, contained IgM that reacted with class I SLA proteins. Horizontal dotted line appears at MFI = 2000.

FIGURE 5.

Pig-specific human IgG cross-reacts with class I human leukocyte antigens. A, Schema depicting the process of analyzing immunoglobulin cross-reactivity. Flow cytometry determined the MFIs of human IgG binding to beads containing a panel of 98 class I HLA proteins. A single representative patient sera is displayed in panels B to G. B, Shows FCXM of intact serum against porcine PBMC and PBMC from a single human. C, IgG from the intact serum broadly reacts with class I HLA proteins as shown by Incubation of the intact serum with 97 class I HLA alleles, each one linked to a specific bead. IgG binding to each bead was probed by flow cytometry and reported as MFI. D, Antibodies were stripped from the human target cells in panel B. Eluted antibodies were tested for reactivity to porcine PBMC and to human cells. Thick blue histograms represent the MFI of eluted antibodies on each target. Solid gray histograms are the secondary only negative controls. E, The human eluted IgG reactivity to HLA beads was tested. F, IgG eluted from Porcine PBMC were tested for reactivity with pig and human cells. Thick orange histograms represent binding of pig eluted antibodies. Solid gray histograms are the secondary only negative controls. G, Antibodies eluted from swine cells recognize a subset of HLA alleles that were recognized by the human-eluted antibodies (compare binding patterns in panels E and G). HLA single-antigen beads are displayed in the same order from left to right in panels C, E, and G.