Serum Antibody Binding and Cytotoxicity to Pig Cells in Chinese Subjects: Relevance to Clinical Renal Xenotransplantation

Abstract

Kidney xenotransplantation is expected to contribute to resolving the shortage of kidneys from deceased human donors. Although progress in experimental life-supporting pig renal xenotransplantation has been encouraging, there are still issues to be considered before a clinical trial can be initiated. We attempted to clarify some of these by an in vitro study. Blood was drawn from healthy volunteers (Volunteers, n=20), patients with end-stage renal disease (ESRD, n=20) pre-operation (Pre), and on Day 1 (POD 1) and Day 14 (POD 14) after renal allotransplantation, brain-dead organ donors (DBD, n=20), and renal allotransplant recipients who were currently experiencing T cell-mediated rejection (Allo-TCMR, n=20). Serum IgM/IgG binding to, and complement-dependent cytotoxicity (CDC) of, PBMCs and RBCs from (a) wild-type (WT), (b) α1,3-galactosyltransferase gene-knockout (GTKO), (c) GTKO/beta-1,4-N-acety1 galactosaminyltransferase 2-knockout (GTKO/β4GalNT2KO), (d) GTKO/cytidine monophosphate-N-acetylneuraminic acid hydroxylase-knockout (GTKO/CMAHKO), and (e) GTKO/β4GalNT2KO/CMAHKO/hCD55 (TKO/hCD55) pigs were measured by flow cytometry. We obtained the following results: (i) Serum IgM/IgG binding and CDC in Volunteers were significantly greater to WT, GTKO, and GTKO/β4GalNT2KO PBMCs or RBCs than to GTKO/CMAHKO and TKO/hCD55 cells; (ii) ESRD, DBD, and Allo-TCMR serum antibody binding and CDC to WT pig PBMCs were significantly greater than to GTKO, GTKO/β4GalNT2KO, GTKO/CMAHKO, and TKO/hCD55 cells; (iii) antibody binding to GTKO/CMAHKO pig cells was significantly lower in hemodialysis than peritoneal dialysis patients. (iv) Two of twenty allotransplantation recipients’ serum IgG binding to GTKO pig PBMCs increased on POD14 compared with Pre, but IgG binding to GTKO pig RBCs did not; (v) In all sera, the lowest antibody binding and CDC were to GTKO/CMAHKO and TKO/CD55 pig cells. We conclude (i) CMAHKO in the pig may be critical to the success of clinical pig kidney xenotransplantation, and may be the most important after GTKO, at least in Chinese patients; (ii) subjects with ESRD, or who are immunosuppressed after kidney allotransplantation, and DBD, have lower levels of antibody binding and CDC to genetically-engineered pig cells than do volunteers; (iii) TKO pigs with selected human ‘protective’ transgenes, e.g., CD55, are likely to prove to be the optimal sources of kidneys for clinical xenotransplantation.

What Have We Learned From In Vitro Studies About Pig-to-primate Organ Transplantation?

“…In humans, depletion of Gal was associated with significantly reduced antibody binding to pRBCs (Figure 3A), and this was further reduced after additional deletion of expression of Sda and Neu5Gc (ie, TKO). 21,22,41,53 Serum antibody binding to TKO pRBCs was not significantly different from that to human blood type O RBCs (controls). 41,53 54 ) CMAHKO, cytidine monophospho-N-acetylneuraminic acid hydroxylase gene-knockout; DKO, double-knockout; Gal, galactose-α1,3-galactose; GM, geometric mean; GTKO, α1,3-galactosyltransferase gene-knockout; Ig, immunoglobulin; Neu5Gc, N-glycolylneuraminic acid; pRBC, pig red blood cell; RBC, red blood cell; TKO, triple-knockout; WT, wild type.…”

“…The expression levels of the 3 known pig carbohydrate xenoantigens on peripheral blood mononuclear cells (PBMCs) from pigs and primates are illustrated in Figure 1 and are summarized in Table 2. [20][21][22][23] All 3 carbohydrate xenoantigens (Gal, Neu5Gc, and Sda) are expressed on wild-type (WT, ie, genetically unmodified) pig (p) PBMCs (Figure 1, right).…”

“…44,48 The presence or absence of antibody binding can be confirmed by a CDC assay using pPBMCs as the target cells. 22,23,31,37,38 The CDC assay should involve cells from pigs that lack "protective" human transgenes (eg, human complement-regulatory proteins [hCRPs]), as these additional genetic modifications can protect against cell lysis and therefore complicate interpretation of the result. However, the benefit of the expression of one or more hCRPs can be measured by comparing the CDC of pig cells that do or do not express hCRPs.…”

“…IgM and IgG Binding and CDC to WT, GTKO, and TKO pPBMCs in Selected Primates pPBMCs express not only carbohydrate antigens, but also major histocompatibility complex class I and II, 27,47 and so are also used for measurement of antibody binding and CDC in primates. 22,23,28,31,32 Sera from OWNHPs, and NWNHPs were tested in addition to human sera. The results were similar to those obtained with pRBCs, 21 suggesting that the carbohydrate xenoantigens are of major importance.…”

“…Presence of Abs Anti-Gal>>anti-Neu5Gc>anti-Sda Abs (No/minimum anti-4th antigen Abs) [20][21][22]27,29,31,36,43,53 Anti-SLA class I and II also involved as xenoantigens in some high PRA patients 27,46 Anti-Gal>anti-4th antigen* Abs>anti-Sda Abs (*Unknown, but expressed when CMAH is KO in pigs) 3,20,21,23,28,31,41 Anti-Neu5Gc Abs, anti-Sda Abs (No anti-Gal Abs, minimum anti-4th antigen Abs) 31,56 Complementdependent cytotoxicity WT>>GTKO>TKO 22,31,38,40,41 WT>*TKO>GTKO>GTKO/ β4GalNT2KO (*Anti-TKO Abs are highly cytotoxic) 23,28,31 WT,GTKO>>TKO 31…”