Antibody production and tolerance to the α-gal epitope as models for understanding and preventing the immune response to incompatible ABO carbohydrate antigens and for α-gal therapies

Abstract

This review describes the significance of the α-gal epitope (Galα-3Galβ1-4GlcNAc-R) as the core of human blood-group A and B antigens (A and B antigens), determines in mouse models the principles underlying the immune response to these antigens, and suggests future strategies for the induction of immune tolerance to incompatible A and B antigens in human allografts. Carbohydrate antigens, such as ABO antigens and the α-gal epitope, differ from protein antigens in that they do not interact with T cells, but B cells interacting with them require T-cell help for their activation. The α-gal epitope is the core of both A and B antigens and is the ligand of the natural anti-Gal antibody, which is abundant in all humans. In A and O individuals, anti-Gal clones (called anti-Gal/B) comprise >85% of the so-called anti-B activity and bind to the B antigen in facets that do not include fucose-linked α1-2 to the core α-gal. As many as 1% of B cells are anti-Gal B cells. Activation of quiescent anti-Gal B cells upon exposure to α-gal epitopes on xenografts and some protozoa can increase the titer of anti-Gal by 100-fold. α1,3-Galactosyltransferase knockout (GT-KO) mice lack α-gal epitopes and can produce anti-Gal. These mice simulate human recipients of ABO-incompatible human allografts. Exposure for 2-4 weeks of naïve and memory mouse anti-Gal B cells to α-gal epitopes in the heterotopically grafted wild-type (WT) mouse heart results in the elimination of these cells and immune tolerance to this epitope. Shorter exposures of 7 days of anti-Gal B cells to α-gal epitopes in the WT heart result in the production of accommodating anti-Gal antibodies that bind to α-gal epitopes but do not lyse cells or reject the graft. Tolerance to α-gal epitopes due to the elimination of naïve and memory anti-Gal B cells can be further induced by 2 weeks in vivo exposure to WT lymphocytes or autologous lymphocytes engineered to present α-gal epitopes by transduction of the α1,3-galactosyltransferase gene. These mouse studies suggest that autologous human lymphocytes similarly engineered to present the A or B antigen may induce corresponding tolerance in recipients of ABO-incompatible allografts. The review further summarizes experimental works demonstrating the efficacy of α-gal therapies in amplifying anti-viral and anti-tumor immune-protection and regeneration of injured tissues.

Keywords: ABO-incompatible antigens; alpha-gal epitope; alpha-gal nanoparticles; alpha-gal therapies; anti-Gal; immune accommodation; immune tolerance.

FIGURE 1

Schematic representation of anti-Gal antibody specificities in blood-type O, A, and B individuals. “Pure” anti-Gal (red antibody) is produced in all humans and binds to α-gal epitopes. These epitopes are absent in humans (except as the core of blood-group A and B antigens) but are synthesized in non-primate mammals, lemurs, and New World monkeys. Anti-Gal/B (green antibody) (comprises >85% of the total anti-B activity) is produced in blood-type O and A individuals and binds to the α-gal epitope and the α-gal core in blood-group B. Anti-Gal/AB (black antibody) is produced in blood-type O individuals and binds to the α-gal epitope and the α-gal core in blood-groups A and B. This antibody is present in small amounts in healthy individuals but may increase in O recipients of incompatible blood-group A allograft. “Pure” anti-B antibody (orange antibody) is produced in O and A individuals (comprises <15% of the total anti-B activity) and binds only to blood-group B red cells. “Pure” anti-A antibody (brown antibody) is produced in O and B individuals (comprises >97% of the total anti-A activity) and binds only to blood-group A red cells.

FIGURE 2

Anti-Gal immune response to α-gal epitopes on mammalian cells. Anti-Gal titers are presented as reciprocals of a serum dilution yielding half the maximum binding in ELISA using synthetic α-gal epitopes linked to BSA as solid-phase antigens. (A) Anti-Gal titers following three intraperitoneal infusions of 6 × 10⁹ 3T3-derived packaging mouse fibroblasts containing a replication-defective virus as part of a gene therapy experiment [modified from Galili et al. (2001)]. Note the >100-fold increase in anti-Gal titer within 14 days after infusion, the 10-fold decrease in Week 7, and the increase after the second and third infusions performed 7 weeks apart. (B) Increase in anti-Gal titer in a representative patient with a ruptured anterior cruciate ligament (ACL) who was implanted with a porcine patellar tendon enzymatically treated to remove α-gal epitopes from the tendon and then partially crosslinked by glutaraldehyde. Cell membranes presenting α-gal epitopes continuously leached out of the remodeled bone plugs attached to the tendon. The cells presenting α-gal epitopes within the bone cavities retained α-gal epitopes because the processing enzyme did not reach the bone cavities for eliminating these epitopes [modified from Stone et al. (2007)]. Note that anti-Gal activity remained elevated for several months and subsequently decreased as a result of the remodeling of the porcine bone into autologous human bone. From “Galili U. book “The natural anti-Gal antibody as foe turned friend in medicine.” Academic Press/Elsevier, London, 2018, with permission. pp. 13–16.

FIGURE 3

Analysis of anti-Gal/B antibodies produced in healthy individuals with blood types A and O. The activity (titer representing reciprocal serum dilution) of anti-Gal/B antibodies was derived from the decrease in agglutination of blood-group B red cells following adsorption of sera on an equal volume of packed rabbit red cells presenting natural α-gal epitopes (open columns) or on synthetic α-gal epitopes on silica beads (gray columns). Original anti-B activity in sera is presented as closed columns. The changes in B red cell agglutination after adsorption indicate that anti-Gal/B antibodies comprise ∼85%–95% of the so-called anti-blood-group B antibodies. Reproduced from Galili U. book “The natural anti-Gal antibody as foe turned friend in medicine,” Academic Press/Elsevier, London, 2018, with permission, pp. 50–52, and based on Galili et al. (1987b).

FIGURE 4

Induction of tolerance to the α-gal epitope on syngeneic WT mouse lymphocytes, as indicated by no rejection of heterotopically grafted mouse WT heart. (A,C) Hyperacute rejection within 30–60 min of WT heart grafted in GT-KO mice, which received 4 weekly PKM immunizations prior to the heart grafting. (A) The hearts were rejected as indicated by the occlusion of blood vessels and edema in peri-vascular regions. (C) The immunostained tissue displayed anti-Gal IgM binding to the endothelial cells of the grafted WT heart. Similar results were obtained with anti-IgG staining. (B,D) Hearts transplanted into mice tolerized by WT lymphocytes presenting α-gal epitopes that were administered 4 weeks prior to transplantation. The hearts were harvested 2 months after transplantation and were functioning despite three additional weekly PKM immunizations starting 1 week after grafting. (B) Normal myocardial structure. (D) No binding of IgM indicated by immunostaining with an anti-mouse IgM antibody. Similarly, no IgG binding was observed. (A,B) Hematoxylin–eosin staining (H&E); (C,D) immunostained with peroxidase coupled anti-mouse IgM antibodies (×200). From Ogawa et al. (2003), with permission.

FIGURE 5

Induction of immune tolerance to α-gal epitopes in GT-KO mice by elimination of memory anti-Gal B cells following administration of WT lymphocytes presenting α-gal epitopes. (A) Timeline for the induction of tolerance on memory anti-Gal B cells. (B) Time required for tolerance induction. Irradiated GT-KO mice received 20 × 10⁶ lymphocytes, including memory anti-Gal B-cells, naïve GT-KO bone marrow cells, and WT lymphocytes or no WT lymphocytes (control group). The mice further received two PKM immunizations, the first of which was at 1, 3, 10, and 14 days. The second PKM immunization and ELISA and ELISPOT (both with α-gal BSA as a solid-phase antigen) were performed as in (A). Absorbance values are presented at a serum dilution of 1:100. Each column represents one out of five mice in each group. (C) ELISPOT analysis of anti-Gal secretion in tolerized versus control mouse spleen cells was performed with α-gal BSA as a solid-phase antigen. Mice tolerized by WT lymphocytes (○), or control mice receiving no WT lymphocytes (●). Means ± SE (n = 5). (D) Flow cytometry identification of anti-Gal B cells among B cells by double staining with FITC-α-gal BSA (green) and PE-anti-mouse Ig (red-staining of all B cells). Control and tolerized mice, as in (C). Note that as many as ∼1% of B cells bound α-gal epitopes of α-gal BSA in the control mice (i.e., anti-Gal B cells), whereas almost no such B cells were detected in the tolerized mice. (E) Tolerance induction on memory anti-Gal B cells does not affect B cells producing anti-blood-group A antibody. The study was performed as in (A). However, both experimental and control mice received a mixture of memory anti-Gal B cells and memory anti-blood-group A B cells from mice immunized four times with blood-group A red cells. The first of the two PKM and blood-group A red cell immunizations was delivered on Day 14. Anti-blood-group A antibody production was assayed by ELISA with A red cell membranes as a solid-phase antigen. Anti-Gal antibody production was assayed by ELISA with α-gal BSA as a solid-phase antigen. (□, ○) Experimental mice also receiving WT lymphocytes. (■, ●) Control mice receiving no WT lymphocytes. (□, ■) Anti-blood-group A IgG production. (○, ●) Anti-Gal IgG production. Note that anti-Gal B cells were tolerized by the WT lymphocytes, whereas anti-blood-group A B-cell-produced anti-A antibodies were not affected. Means ± SE (n = 5). From Mohiuddin et al. (2003b), with permission.

FIGURE 6

Intradermal recruitment of macrophages in anti-Gal-producing GT-KO mice by 10 mg α-gal nanoparticles. (A) Macrophage recruitment 24 h after injection of α-gal nanoparticles. The injection site is the empty area in which nanoparticles were eliminated during the fixation process (H&E ×100). (B) Identification of the recruited cells as macrophages by immunostaining on Day 4 after injection with the macrophage-specific peroxidase coupled-anti-F4/80 antibody (×200). (C) Macrophages at the injection site on Day 7. Macrophages are large with ample cytoplasm (H&E ×400). (D) Macrophages recruited into a polyvinyl alcohol sponge disc containing 10 mg α-gal nanoparticles 7 days after subcutaneous implantation into a GT-KO mouse. (Wright staining, ×1,000). Reproduced from Galili U. book “The natural anti-Gal antibody as foe turned friend in medicine,” Academic Press/Elsevier, London, 2018, with permission.

FIGURE 7

Anti-Gal-mediated targeting of α-gal presenting human lymphoma cells to APC. (A) Synthesis of α-gal epitopes on human tumor cells studied. (Left chain) A representative N-linked carbohydrate chain capped by sialic acid (SA). (Center chain). Sialic acid is removed by neuraminidase, thereby exposing the penultimate Galβ1-4GlcNAc-R called N-acetyllactosamine (LacNAc) (Center chain). The recombinant glycosylation enzyme α1,3-galactosyltransferase (rα1,3-GT) links galactose provided by sugar donor uridine diphosphate galactose (UDP-Gal) to the carbohydrate chain, resulting in the synthesis of α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R), which readily bind the anti-Gal antibody (Left chain). A similar glycoengineering for the expression of α-gal epitopes can be performed in enveloped viruses. (B) In vitro demonstration of anti-Gal-mediated uptake of human lymphoma cells by autologous APC. Freshly obtained lymphoma cells were subjected to α-gal epitope synthesis, as described in (A). The lymphoma cells with or without α-gal epitopes were incubated with autologous anti-Gal for 30 min then for 2 h at 37°C with autologous macrophages or dendritic cells. Triangles mark the nuclei of the APC. The macrophage incubated with α-gal presenting lymphoma cells internalized nine cells, and the dendritic cell internalized one α-gal presenting lymphoma cell. No uptake of lymphoma cells lacking α-gal epitopes was observed (×1,000). Adapted with permission from Manches et al. (2005).

FIGURE 8

Amplification of viral vaccine immunogenicity by anti-Gal-mediated targeting of the vaccinating virus to APC. Influenza virus glycoengineered to present α-gal epitopes is used as an illustrative example for α-gal inactivated whole virus vaccine. Anti-Gal IgM and IgG bind at the vaccination site to α-gal epitopes on the vaccinating virus. This anti-Gal/α-gal epitope interaction activates the complement system, resulting in the release of complement cleavage chemotactic peptides C5a and C3a that recruit APC, such as dendritic cells and macrophages, to the vaccination site. Anti-Gal IgG coating the virus mediates its extensive uptake by the recruited APC via Fc/Fcγ receptors (FcγR) interaction. C3b/C3b receptor interaction on APC also may contribute to the extensive uptake of the virus vaccine. APC transport the internalized virus vaccine to the regional lymph nodes and process and present the viral immunogenic peptides on class I and class II MHC molecules for the activation of virus-specific CD8⁺ and CD4⁺ T cells, respectively. HA, hemagglutinin; NA, neuraminidase; TCR, T-cell receptor. Reproduced from Galili U. book “The natural anti-Gal antibody as foe turned friend in medicine,” Academic Press/Elsevier, London, 2018, with permission.

FIGURE 9

Mechanism for the regenerative effects of α-gal nanoparticles in injuries. (A) Schematic section in α-gal nanoparticles illustrating the phospholipids forming a lipid bilayer of the nanoparticle wall, in which multiple glycolipids with α-gal epitopes (rectangles) are anchored. Upon administration into various injured tissues, the anti-Gal antibody, which is abundant in the serum, readily binds to the α-gal epitopes on the α-gal nanoparticles. (B) Steps in the activity of α-gal nanoparticles administered to injuries. 1) Binding of natural anti-Gal to α-gal nanoparticles activates the complement system and results in the formation of the chemotactic complement cleavage peptides C5a and C3a. 2). The chemotactic factors C5a and C3a induce extensive recruitment of macrophages to the site of α-gal nanoparticles. 3) The recruited macrophages bind via their Fcγ receptors (FcγR) the Fc portion of anti-Gal coating the α-gal nanoparticles. 4) This interaction induces polarization of the cells into pro-regenerative macrophages that secrete a wide range of cytokines and growth factors, which accelerate the healing of the treated injuries and prevent scar formation. Reproduced from Galili U. book “The natural anti-Gal antibody as foe turned friend in medicine.” Academic Press/Elsevier, London, 2018, with permission.

FIGURE 10

The effects of repeated encounters between anti-Gal B cells and α-gal epitopes on endothelial cells of syngeneic wild-type (WT) heart grafts in GT-KO mice, in the absence of T-cell help, on the production of anti-Gal. When T-cell help is provided within 24 h by immunization with PKM, exposure to α-gal epitopes activates anti-Gal B cells into plasma cells producing cytolytic anti-Gal antibodies. If T-cell help is provided to anti-Gal B cells only after 7–14 days of repeated encounters with α-gal epitopes, these B cells differentiate into plasma cells producing accommodating anti-Gal antibodies. Repeated encounters of naïve or memory anti-Gal B cells for >14 days, in the absence of T-cell help, result in tolerance to the α-gal epitope due to the elimination of anti-Gal B cells either by deletion or by Ig receptor editing. BCR, B-cell receptor. Modified from Galili U. book “The natural anti-Gal antibody as foe turned friend in medicine.” Academic Press/Elsevier, London, 2018” with permission and based on Galili (2004).