M. Jeyakanthan

Chemical Basis for Qualitative and Quantitative Differences Between ABO Blood Groups and Subgroups: Implications for Organ Transplantation.

Blood group ABH(O) carbohydrate antigens are carried by precursor structures denoted type I–IV chains, creating unique antigen epitopes that may differ in expression between circulating erythrocytes and vascular endothelial cells. Characterization of such differences is invaluable in many clinical settings including transplantation. Monoclonal antibodies were generated and epitope specificities were characterized against chemically synthesized type I–IV ABH and related glycans. Antigen expression was detected on endomyocardial biopsies (n = 50) and spleen (n = 11) by immunohistochemical staining and on erythrocytes by flow cytometry. On vascular endothelial cells of heart and spleen, only type II–based ABH antigens were expressed; type III/IV structures were not detected. Type II–based ABH were expressed on erythrocytes of all blood groups. Group A1 and A2 erythrocytes additionally expressed type III/IV precursors, whereas group B and O erythrocytes did not. Intensity of A/B antigen expression differed among group A1, A2, A1B, A2B and B erythrocytes. On group A2 erythrocytes, type III H structures were largely un‐glycosylated with the terminal “A” sugar α‐GalNAc. Together, these studies define qualitative and quantitative differences in ABH antigen expression between erythrocytes and vascular tissues. These expression profiles have important implications that must be considered in clinical settings of ABO‐incompatible transplantation when interpreting anti‐ABO antibodies measured by hemagglutination assays with reagent erythrocytes.

Chemical Basis for Qualitative and Quantitative Differences Between ABO Blood Groups and Subgroups

Blood group ABH(O) carbohydrate antigens are carried by precursor structures denoted type I–IV chains, creating unique antigen epitopes that may differ in expression between circulating erythrocytes and vascular endothelial cells. Characterization of such differences is invaluable in many clinical settings including transplantation. Monoclonal antibodies were generated and epitope specificities were characterized against chemically synthesized type I–IV ABH and related glycans. Antigen expression was detected on endomyocardial biopsies (n = 50) and spleen (n = 11) by immunohistochemical staining and on erythrocytes by flow cytometry. On vascular endothelial cells of heart and spleen, only type II–based ABH antigens were expressed; type III/IV structures were not detected. Type II–based ABH were expressed on erythrocytes of all blood groups. Group A1 and A2 erythrocytes additionally expressed type III/IV precursors, whereas group B and O erythrocytes did not. Intensity of A/B antigen expression differed among group A1, A2, A1B, A2B and B erythrocytes. On group A2 erythrocytes, type III H structures were largely un‐glycosylated with the terminal “A” sugar α‐GalNAc. Together, these studies define qualitative and quantitative differences in ABH antigen expression between erythrocytes and vascular tissues. These expression profiles have important implications that must be considered in clinical settings of ABO‐incompatible transplantation when interpreting anti‐ABO antibodies measured by hemagglutination assays with reagent erythrocytes.

ABH‐glycan microarray characterizes ABO subtype antibodies: fine specificity of immune tolerance after ABO‐incompatible transplantation

Organ transplantation from ABO blood group–incompatible (ABOi) donors requires accurate detection, effective removal and subsequent surveillance of antidonor antibodies. Because ABH antigen subtypes are expressed differently in various cells and organs, measurement of antibodies specific for the antigen subtypes in the graft is essential. Erythrocyte agglutination, the century‐old assay used clinically, does not discriminate subtype‐specific ABO antibodies and provides limited information on antibody isotypes. We designed and created an ABO‐glycan microarray and demonstrated the precise assessment of both the presence and, importantly, the absence of donor‐specific antibodies in an international study of pediatric heart transplant patients. Specific IgM, IgG, and IgA isotype antibodies to nonself ABH subtypes were detected in control participants and recipients of ABO‐compatible transplants. Conversely, in children who received ABOi transplants, antibodies specific for A subtype II and/or B subtype II antigens—the only ABH antigen subtypes expressed in heart tissue—were absent, demonstrating the fine specificity of B cell tolerance to donor/graft blood group antigens. In contrast to the hemagglutination assay, the ABO‐glycan microarray allows detailed characterization of donor‐specific antibodies necessary for effective transplant management, representing a major step forward in precise ABO antibody detection.

Donor-Specific Isohemagglutinins: Measuring the Unknown

ABO-incompatible (ABOi) heart transplantation is not routinely performed due to the high risk of hyperacute antibody-mediated rejection elicited by preformed natural antibodies against non-self-blood group antigens. In infants, however, ABOi transplantation can be performed safely due to low or absent natural antibodies. Since our initial report (1) of the first cohort of 10 intentional infant ABOi heart transplants, it is estimated that more than 150 such transplants have been performed in young children worldwide with excellent long-term outcomes. Posttransplant investigation of an early cohort of children revealed a notable paucity of natural antibodies against the donor blood type; further immunological studies demonstrated acquired B-cell tolerance to donor blood type antigens (2).

Failure of neonatal B-cell tolerance induction by ABO-incompatible kidney grafts in piglets.

Background ABO-incompatible (ABOi) infant heart transplantation results in B-cell tolerance to graft A/B antigens, confirming human susceptibility to acquired immunologic or “neonatal” tolerance as described originally in murine models. Starting with this clinical observation, we sought to model neonatal ABOi organ transplantation to allow mechanistic studies of tolerance. Methods Plasma anti-A/B antibodies were measured over time in piglets to establish developmental antibody kinetics. Blood group O piglets received kidney allografts from group A (AO-incompatible) or group O (AO-compatible) donors under cyclosporine immunosuppression. Anti-A antibodies were measured serially after transplantation; A/H antigen expression and allograft rejection were assessed in graft biopsies. Results Anti-A antibodies developed in naïve piglets in a kinetic pattern analogous to human infants; anti-B remained low. After transplantation, anti-A antibodies developed similarly in AO-incompatible and AO-compatible groups and were not suppressed by cyclosporine. A/H antigen expression was persistent in all graft biopsies; however, A/H antigens were not detected in vascular endothelium. Cellular and antibody-mediated rejection was absent or minimal in early and late biopsies in both groups, with one exception. Conclusions Naturally delayed isohemagglutinin production in piglets is analogous to the developmental kinetics in human infants. However, in contrast to deficient anti-A antibody production as seen long-term after “A-into-O” infant heart transplant recipients, normal anti-A antibody production after “A-into-O” piglet kidney transplantation indicates that tolerance did not develop despite graft A antigen persistence. These findings suggest that the impact on the host immune system of exposure to nonself ABH antigens during early life in human heart versus porcine kidney grafts may depend on expression in vascular endothelium.

Conjugation of A and B Blood Group Structures to Silica Microparticles for the Detection of Antigen-Specific B Cells

Silica microparticles were functionalized with A and B blood group carbohydrate antigens (A type I, A type II, B type I, and B type II) to enable the detection and monitoring of ABO antigen-specific B cells. Microparticles were prepared via the Stöber synthesis, labeled with an Alexafluor fluorescent dye, and characterized via TEM and fluorescence microscopy. The silica microparticles were functionalized with (3-aminopropyl)trimethoxysilane (APTMS), followed by the use of an established fluorenylmethyloxycarbonyl (Fmoc)-protected PEG-based linker. The terminal Fmoc moiety of the PEG-based linker was then deprotected, yielding free amino groups, to which the A and B antigens were coupled. The carbohydrate antigens were synthesized with a p-nitrophenol ester to enable conjugation to the functionalized silica microparticles via an amide bond. The number of free amine groups available for coupling for a given mass of PEG-functionalized silica microparticles was quantified via reaction with Fmoc-glycine. The antigen-functionalized microparticles were then evaluated for their specificity in binding to A and B antigen-reactive B-cells via flow cytometry, and for blocking of naturally occurring antibodies in human serum. Selective binding of the functionalized microparticles to blood group-reactive B cells was observed by flow cytometry and fluorescence microscopy. The modular approach outlined here is applicable to the preparation of silica microparticles containing any carbohydrate antigen and alternative fluorophores or labels. This approach therefore comprises a novel, general platform for screening B cell populations for binding to carbohydrate antigens, including, in this case, the human A and B blood group antigens.