Will previous palliative surgery for congenital heart disease be detrimental to subsequent pig heart xenotransplantation?

Abstract

Introduction:

Pig heart xenotransplantation might act as a bridge in infants with complex congenital heart disease (CHD) until a deceased human donor heart becomes available. Infants develop antibodies to wild-type (WT, i.e., genetically-unmodified) pig cells, but rarely to cells in which expression of the 3 known carbohydrate xenoantigens has been deleted by genetic engineering (triple-knockout [TKO] pigs). Our objective was to test sera from children who had undergone palliative surgery for complex CHD (and who potentially might need a pig heart transplant) to determine whether they had serum cytotoxic antibodies against TKO pig cells.

Methods:

Sera were obtained from children with CHD undergoing Glenn or Fontan operation (n = 14) and healthy adults (n = 8, as controls). All of the children had complex CHD and had undergone some form of cardiac surgery. Seven had received human blood transfusions and 3 bovine pericardial patch grafts. IgM and IgG binding to WT and TKO pig red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry, and killing of PBMCs by a complement-dependent cytotoxicity assay.

Results:

Almost all children and adults demonstrated relatively high IgM/IgG binding to WT RBCs, but minimal binding to TKO RBCs (p < 0.0001 vs WT), although IgG binding was greater in children than adults (p < 0.01). All sera showed IgM/IgG binding to WT PBMCs, but this was much lower to TKO PBMCs (p < 0.0001 vs WT) and was greater in children than in adults (p < 0.05). Binding to both WT and TKO PBMCs was greater than to RBCs. Mean serum cytotoxicity to WT PBMCs was 90% in both children and adults, whereas to TKO PBMCs it was only 20% and < 5%, respectively. The sera from 6/14 (43%) children were cytotoxic to TKO PBMCs, but no adult sera were cytotoxic.

Conclusions:

Although no children had high levels of antibodies to TKO RBCs, 13/14 demonstrated antibodies to TKO PBMCs, in 6 of these showed mild cytotoxicity. As no adults had cytotoxic antibodies to TKO PBMCs, the higher incidence in children may possibly be associated with their exposure to previous cardiac surgery and biological products. However, the numbers were too small to determine the influence of such past exposures. Before considering pig heart xenotransplantation for children with CHD, testing for antibody binding may be warranted

1. Introduction

Complex congenital heart disease (CHD) is associated with significant morbidity and mortality [1,2]. In many cases, cardiac allotransplantation would be the optimal therapy but, in view of the difficulty in obtaining a suitable human deceased donor heart, these patients are often subjected to a series of palliative operations [3,4]. Although the results of these palliative procedures have improved significantly in recent years, there are significant limitations (e.g., anatomy unsuitable for Fontan procedure, post-operative morbidity and mortality), and the mortality of infants awaiting heart transplantation remains high.

Bridging neonates and infants to cardiac allotransplantation by mechanical circulatory support has been associated with disappointing results [1,5–10]. In this age group, ventricular assist devices are associated with limited long-term survival, and are contraindicated in patients of extreme prematurity, low birth weight, active infection, and when a hypertrophied myocardium can impede function [6]. Taken together, the ineffectiveness and scarcity of current treatment options for children with end-stage heart failure indicates the need for additional novel approaches towards supporting infants with complex CHD. Although as yet untested clinically, cardiac xenotransplantation is one possible alternative.

Using pig heart xenotransplantation as a bridge therapy until a deceased human donor heart becomes available might provide a more successful approach. Protection of a pig heart from the human immune response may be enhanced by the weak and flexible immune system in neonates and infants [11]. Previous studies by our group have demonstrated that infant humans develop high levels of antibody against wild-type (WT, i.e., genetically unmodified) pig xenoantigens during the first few weeks of life [12–14]. However, when expression of the 3 known pig carbohydrate xenoantigens (Gal, Neu5Gc, and Sda) is deleted from the pig cells (Table 1), the great majority of infants do not develop any antibodies against triple-knockout (TKO) pig cells [14].

Table 1.

Known carbohydrate xenoantigens expressed on pig cells.

Carbohydrate (Abbreviation)	Responsible enzyme	Gene- knockout pig
1. Galactose-α1,3-galactose (Gal)	α1,3-galactosyltransferase	GTKO
2. N-glycolylneuraminic acid (Neu5Gc)	CMAH	CMAH-KO
3. Sda	β−1,4 N-acetylgalactosaminyltransferase	β4GalNT2-KO

CMAH = Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH).

Our motivation for conducting the current in vitro study was to determine whether previous palliative cardiac surgery would prove detrimental to the outcome of a subsequent pig heart transplant due to sensitization. The objective of this study was (i) to compare antibody binding to red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs in infants and young children, (ii) to investigate the degree of CDC to TKO pig PBMCs (if there is any), and (iii) to determine whether children with complex CHD with previous exposures to biological materials and surgeries develop antibodies that might prove detrimental to a subsequent pig cardiac xenograft.

2. Materials and methods

2.1. Sources of pig RBCs and PBMCs

RBCs and PBMCs were isolated from WT and TKO pigs (all pigs were of blood type O [nonA]) generously provided by Revivicor, Blacksburg, VA. Isolation of the cells has been reported previously [14].

2.2. Sources of human sera

After ethical institutional approval from Children’s (Hospital) of Alabama and the University of Alabama at Birmingham (IRB-300003176), sera were obtained from 14 children with complex CHD who were undergoing either a bi-directional Glenn (n = 6) or Fontan operation (n = 8) at our institution (3–71 months old, mean 35 months; mean weight 13.8 kg) and healthy adults (n = 8). All children had undergone some form of previous cardiac surgery (with or without cardiopulmonary bypass that involved blood transfusion and/or bovine patch grafts). Most common underlying diagnosis for the infants included hypoplastic left heart syndrome, heterotaxy, complex CHD with pulmonary or tricuspid atresia. Seven had received human blood transfusions during cardiopulmonary bypass, and 3 bovine patch grafts. The healthy adults, none of whom had undergone previous surgery or blood transfusion, acted as controls for the children.

2.3. Detection of swine leukocyte antigens (SLA) class I and II expression on pig cells by flow cytometry

Surface expression of SLA class I and class II on pig PBMCs (including CD3⁺, CD21⁺, and CD3⁻ CD21⁻ gated cells) was determined by LSR II flow cytometry (Becton Dickinson, San Jose, CA), and analyzed by FlowJo software (Treestar. Ashland, OR), as previously described [15].

2.4. IgM and IgG antibody binding to WT and TKO pig cells

We examined antibody binding to pig red blood cells (RBCs, that express only carbohydrate xenoantigens) and to pig PBMCs (that express both carbohydrate and protein [e.g., swine leukocyte antigens] xenoantigens). Serum IgM and IgG antibody binding to pig PBMCs (including gated CD3⁺T, CD21⁺B, and CD3⁻ CD21⁻ cells) was performed, as previously described [15]. Briefly, following the antibody assays, PBMCs were stained with PerCP Cy5.5-conjugated anti-pig CD3 (Clone BB23–8E6–8C8, BD) and-human CD21 (Clone B-ly4, BD) for CD3⁺T, CD21⁺B, and CD3⁻ CD21⁻ gated cells.

To run the antibody assays and quantify antibody binding, the same protocol was followed as described previously [14]. Flow cytometry was used to measure IgM and IgG binding to WT and TKO pig RBCs and PBMCs, determined as relative geometric mean (rGM) [14]. For binding to RBCs, rGMs of IgM of <1.2 and of IgG of <1.1 were considered negative, and for binding to PBMCs (rGMs of IgM of <2.0 and of IgG of <1.2), CD3⁺T cells (rGMs of IgM of <1.8 and of IgG of <1.2), CD21⁺B cells (rGMs of IgM of <4.5 and of IgG of <1.6), and CD3⁻ CD21⁻ cells (rGMs of IgM of <2.5 and of IgG of <1.4) were considered negative.

2.5. Serum complement-dependent cytotoxicity (CDC) assay

Serum CDC (at 50% serum concentration) against WT and TKO pig PBMCs was measured. By using human blood group O (negative control) and WT (positive control) PBMCs, as previously described [16], cytotoxicity of <20% was considered negative. IgM and IgG antibody binding to, and CDC of, human blood type O RBCs acted as negative controls.

3. Results

3.1. IgM and IgG binding to WT and TKO pig RBCs (Fig. 1)

Fig. 1.

(A, B) Serum IgM (A) and IgG (B) binding in children and adults to WT RBCs. There was no statistical difference in binding between pediatric and adult sera.

(C, D) Serum IgM (C) and IgG (D) binding in children and adults to TKO RBCs. IgG binding was greater in children than adults (IgG p < 0.05).

IgM (E) and IgG (F) binding were significantly greater to WT than to TKO RBCs in combined pediatric and adult sera (p < 0.0001).

The solid line indicates the median. Levels below the red dotted line are considered negative. (ns = no statistical difference; **p < 0.01, ****p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article

Consistent with our previous studies, almost all children and adults had moderately high levels of IgM and IgG binding to WT RBCs (Fig. 1A, B). Thirteen (13/14, 93%) children and 8/8 (100%) adults exhibited IgM and IgG binding to WT RBCs (mean rGM: IgM: children 25.7, adults 26.6, NS; IgG: children 5.4, adults 9.1, NS) (Fig. 1A,B, respectively).

IgM/IgG binding to TKO RBCs was again consistent with previous studies, as only three children and one adult had minimal binding (Fig. 1C,D). However, IgG binding was greater in pediatric than adult sera (p < 0.01).

The levels of binding were very low when compared to those to WT RBCs (rGM: IgM mean: WT 26.4 vs TKO 1.1, p < 0.0001 [Fig. 1E]; IgG mean: WT 6.8 vs TKO 1.1 p < 0.0001 [Fig. 1F]).

3.2. Expression of SLA class I and II on pig PBMCs

CD3⁺T cells, CD21⁺B cells, and CD3⁻ CD21⁻ cells were gated by staining PBMCs with anti-CD3 and anti-CD21 antibodies (Supplementary Fig. 1A,B). SLA class I was expressed on all populations (Supplementary Fig. 1A). CD3⁺T cells expressed SLA class II although the expression level (i.e., fluorescence intensity) and the frequencies of SLA class II-positive CD3⁺T cells were lower than in CD21⁺B cells (Supplementary Fig. 1B). These data indicated that human antibody binding to pig CD3⁺T cells (by gating) is not only to SLA class I, but also to SLA class II (Supplementary Fig. 1C,D,E,F).

3.3. IgM and IgG binding to WT and TKO pig PBMCs (Fig. 2)

Fig. 2.

Serum IgM (A) and IgG (B) binding in children and adults to WT (A,B) and TKO (C,D) PBMCs. There was no significant difference in IgM and IgG binding to WT PBMCs between pediatric and adult sera, but IgM and IgG binding of pediatric sera to TKO PBMCs was greater than of adult sera (p < 0.05).

Both IgM (E) and IgG (F) binding of combined pediatric and adult sera were significantly greater to WT PBMCs than to TKO PBMCs (p < 0.0001).

The solid line indicates the median. Levels below the red dotted line are considered negative. (ns = no statistical difference; *p < 0.05, ****p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Sera from all children and adults showed relatively high IgM and IgG binding to WT PBMCs (Fig. 2A,B), with no significant difference between children and adults. Thirteen (13/14, 93%) children and 5/8 (63%) adults exhibited some IgM binding to TKO PBMCs although binding was low (mean rGM: children 3.3, adults 2.4) (Fig. 2C). Ten (10/14, 71%) children and 2/8 (25%) adults exhibited minimal IgG binding to TKO PBMCs (mean rGM: children 1.3, adults 1.2) (Fig. 2D). There was significantly less binding of both IgM (p < 0.05) and IgG (p < 0.05) in adult sera than in pediatric sera (Fig. 2C,D). A similar trend of human antibody binding to PBMCs was found in regard to CD3⁺T, CD21⁺B, and CD3⁻ CD21⁻ cells of WT (Supplementary Fig. 2) and TKO (Supplementary Fig. 3) pigs. There was significantly less IgM and IgG binding to TKO PBMCs in both groups compared to binding to WT PBMCs (p < 0.0001) (Fig. 2E,F).

3.4. Comparison of IgM and IgG binding to pig RBCs and PBMCs (Fig. 3)

Fig. 3.

Comparison of IgM and IgG binding to RBCs and PBMCs. IgM (A,C) and IgG (B,D) binding to WT (A,B) and TKO (C,D) of combined pediatric and adult sera were significantly greater to PBMCs than to RBCs, except in regard to IgG binding to WT RBCs and PBMCs (B).

Data show mean ± SD. (ns = no statistical difference; ****p < 0.0001).

Sera from all children and adults showed significantly higher binding to PBMCs than to RBCs (p < 0.0001) (Fig. 3A,C,D), except IgG binding to WT cells (Fig. 3B).

3.5. Serum CDC to WT and TKO pig PBMCs (Fig. 4)

Fig. 4.

Serum (50% concentration) CDC to WT (A) and TKO (B) PBMCs in children and adults. CDC (% killing) of WT and TKO PBMCs were significantly greater in pediatric than in adult sera (WT p < 0.05; TKO p < 0.0001). (C) CDC was significantly greater in combined pediatric and adult sera to WT than to TKO PBMCs (p < 0.0001).

Data show mean ± SD. The solid line indicates the median. Levels below the red dotted line are considered negative. (ns = no statistical difference; *p < 0.05, ****p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Serum CDC of WT PBMCs was high in both children and adults, both with a mean of approximately 90% killing, but was lower in children (p < 0.05) (Fig. 4A). Serum CDC of TKO PBMCs was greatly reduced (p < 0.0001 vs WT) (Fig. 4B,C), but was significantly greater in children (mean 20% killing) than in adults (mean 3% killing; p < 0.0001) (Fig. 4B). The sera from 6/14 (43%) children were cytotoxic to TKO PBMCs, but no adult sera were cytotoxic (p < 0.0001) (Fig. 4B).

3.6. Effect of prior blood transfusion or bovine tissue transplantation

Mean rGM of serum IgM and IgG binding to TKO PBMCs in those children with a previous blood transfusion (during cardiopulmonary bypass) (n = 7) was IgM: 3.6, IgG: 1.4 compared to IgM: 3.0, IgG: 1.3 in those with no previous blood transfusion (n = 7, IgM and IgG: ns) (Fig. 5A,B). In those with a bovine patch (n = 3), serum IgM and IgG binding to TKO PBMCs was IgM: 4.4, IgG: 1.5 compared to IgM: 3.0, IgG: 1.3 in those without a patch (n = 11, IgM: p < 0.05, IgG: ns) (Fig. 5C,D). Mean serum CDC of TKO PBMCs in those children with a previous blood transfusion (n = 7) was 23% compared to 16% in those with no previous blood transfusion (n = 7, ns) (Fig. 5E). In those with a bovine patch (n = 3), serum CDC was 28% compared to 18% in those without a patch (n = 11, ns) (Fig. 5F).

Fig. 5.

Comparison of serum anti-TKO IgM and IgG antibody binding and CDC in those children with/without a previous blood transfusion (A,B,E) or those with/without a bovine patch (C,D,F).

The solid line indicates the median. Levels below the red dotted line are considered negative. (ns = no statistical difference; *p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

Several conclusions can be drawn from this small study. First, the results relating to IgM and IgG binding to WT and TKO pig RBCs were consistent with our previous study [14]. These data suggest that TKO pigs (+/− added human protective transgenes) should provide optimal sources of hearts for transplantation in immunologically-naïve infants.

Second, pediatric patients who had undergone prior cardiac surgical procedures (with or without blood transfusion and/or bovine patch implantation) had higher levels of IgM and IgG binding to, and serum cytotoxicity of, TKO PBMCs than did the control adults. This suggests that all such children should be tested for the presence of cytotoxic antibodies before considering pig heart xenotransplantation as a bridge to allotransplantation. If serum antibody binding to, and/or serum cytotoxicity of, TKO PBMCs are demonstrated to be absent, then the previous surgery may not be detrimental to the outcome of TKO pig heart xenotransplantation. However, if there is significant antibody binding to, or killing of, TKO pig PBMCs in vitro, this indicates a greater possibility that a TKO pig graft may be rejected.

The xenogeneic targets for the increased cytotoxic antibody binding to TKO pig PBMCs remain unknown, but are possibly SLA expressed on the PBMCs. SLA have been demonstrated to be xenoantigens [17,18]. However, the observation that a small number of children had serum antibodies to TKO pig RBCs (that do not express SLA) suggests that possibly some antibodies were directed to (unknown) carbohydrate antigens other than Gal, Neu5Gc, and Sda.

One limitation of our study is that we did not have a control group of sera from age-matched children who had not undergone previous cardiac surgical procedures. Furthermore, there was some variability in the time that had elapsed between the previous cardiac surgical procedure and the performance of the assays. However, we suggest that the data obtained from testing sera from healthy adults reflect the data that would have been obtained from sera from immunologically naïve children.

Greater numbers of sera from children who received blood transfusions or bovine patch grafts need to be tested to determine the role of blood transfusion and/or bovine patch implantation in the increased antibody binding and cytotoxicity observed in the children in the present study.

It has been reported that Gal and Neu5Gc expressed in both pig and bovine cardiac tissue [19–22]. In contrast, except in the pig [22–24], the expression of Sda has not been detected in the heart tissues of other mammals including bovines [19,24]. Therefore, it is difficult to determine whether the children who received bovine patches were exposed to Sda antigens or not. The presence of a bovine patch (that expresses Gal) would seem more likely to be associated with an increase in cytotoxic antibodies in the child than a previous human ABO-compatible blood transfusion.

In conclusion, the data suggest that all pediatric patients who have undergone prior cardiac surgical procedures, particularly if this includes the implantation of a bovine patch graft, should be tested for the presence of cytotoxic antibodies before considering pig heart xenotransplantation as a bridge therapy.

Abbreviations:

CDC

complement-dependent cytotoxicity

CHD

congenital heart disease

PBMCs

peripheral blood mononuclear calls

RBCs

red blood cells

SLA

swine leukocyte antigens

TKO

triple-knockout, i.e., with deletion of expression of the 3 known pig carbohydrate xenoantigens against which humans have anti-pig antibodies

WT

wild-type, i.e., genetically unmodified