Characterization of ABH-subtype donor-specific antibodies in ABO-A-incompatible kidney transplantation.

ABO-incompatible (ABOi) transplantation requires preemptive antibody reduction; however, the relationship between antibody-mediated rejection (AMR) and ABO-antibodies, quantified by hemagglutination (HA), is inconsistent, possibly reflecting variable graft resistance to AMR or HA assay limitations. Using an ABH-glycan microarray, we quantified ABO-A antigen-subtype (A-subtype)-specific IgM and IgG in 53 ABO-O recipients of ABO-A kidneys, before and after antibody removal (therapeutic plasma exchange [TPE] or ABO-A-trisaccharide immunoadsorption [IA]) and 1-year posttransplant. IgM binding to all A-subtypes correlated highly (R2  ≥ .90) and A-subtype antibody specificities was reduced equally by IA versus TPE. IgG binding to the A-subtypes (II-IV) expressed in kidney correlated poorly (.27 ≤ R2  ≤ .69). Reduction of IgG specific to A-subtype-II was equivalent for IA and TPE, whereas IgG specific to A-subtypes-III/IV was not as greatly reduced by IA (p < .005). One-year posttransplant, IgG specific to A-II remained the most reduced antibody. Immunostaining revealed only A-II on vascular endothelium but A-subtypes II-III/IV on tubular epithelium. These results show that ABO-A-trisaccharide is sufficient for IgM binding to all A-subtypes; this is true for IgG binding to A-II, but not subtypes-III/IV, which exhibits varying degrees of specificity. We identify A-II as the major, but importantly not the sole, antigen relevant to treatment and immune modulation in adult ABO-A-incompatible kidney transplantation.

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.

Sex, T Cells, and the Microbiome in Natural ABO Antibody Production in Mice.

BACKGROUND: "Natural" ABO antibodies (Abs) are produced without known exposure to A/B carbohydrate antigens, posing significant risks for hyperacute rejection during ABO-incompatible transplantation. We investigated anti-A "natural" ABO antibodies versus intentionally induced Abs with regard to the need for T-cell help, the impact of sex, and stimulation by the microbiome. METHODS: Anti-A was measured by hemagglutination assay of sera from untreated C57BL/6 wild-type (WT) or T cell-deficient mice of both sexes. Human ABO-A reagent blood cell membranes were injected intraperitoneally to induce anti-A Abs. The gut microbiome was eliminated by maintenance of mice in germ-free housing. RESULTS: Compared with WT mice, CD4 + T-cell knockout (KO), major histocompability complex-II KO, and αß/γδ T-cell receptor KO mice produced much higher levels of anti-A nAbs; females produced dramatically more anti-A nAbs than males, rising substantially with puberty. Sensitization with human ABO-A reagent blood cell membranes did not induce additional anti-A in KO mice, unlike WT. Sex-matched CD4 + T-cell transfer significantly suppressed anti-A nAbs in KO mice and rendered mice responsive to A-sensitization. Even under germ-free conditions, WT mice of several strains produced anti-A nAbs, with significantly higher anti-A nAbs levels in females than males. CONCLUSIONS: Anti-A nAbs were produced without T-cell help, without microbiome stimulation, in a sex- and age-dependent manner, suggestive of a role for sex hormones in regulating anti-A nAbs. Although CD4 + T cells were not required for anti-A nAbs, our findings indicate that T cells regulate anti-A nAb production. In contrast to anti-A nAbs, induced anti-A production was T-cell dependent without a sex bias.

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.

Inhaled nitric oxide for modulation of ischemia-reperfusion injury in lung transplantation.

BACKGROUND: The prophylactic administration of inhaled nitric oxide (NO) during reperfusion after lung transplantation has been shown to reduce neutrophil-induced injury in animal models. There remain questions regarding efficacy in the clinical setting and concerns regarding increased free radical injury. We sought to assess the efficacy of NO in reducing neutrophil infiltration and associated injury if administered from the very onset of reperfusion in clinical lung transplantation. METHODS: Twenty bilateral sequential lung transplant recipients were randomized to receive 20-ppm inhaled NO (NO group) or a standard anesthetic gas mixture (control group) from the onset of ventilation. Bronchoalveolar lavage was performed immediately prior to implantation and after 30 minutes of reperfusion and analyzed for inflammatory cytokine levels and free radical surrogates. Primary graft dysfunction (PGD) scoring was performed prospectively for 72 hours post-transplant. RESULTS: The prophylactic administration of NO during the first 30 minutes of reperfusion had no statistically significant effect on the development of Grade II to III PGD (5 of 10 in NO group and 7 of 10 in control group, p = 0.36) or gas exchange (area under the curve: 429 +/- 296 vs 336 +/- 306; p = 0.64) in the NO and control groups, respectively. Pulmonary neutrophil sequestration, as measured by the transpulmonary arteriovenous neutrophil difference, was not influenced by the administration of NO. Prophylactic NO did not significantly alter the concentration of interleukin-8, myeloperoxidase or nitrotyrosine during transplantation. CONCLUSIONS: This study could not demonstrate a significant effect of inhaled NO during the first 30 minutes of reperfusion in the prevention of neutrophil injury and primary graft dysfunction after lung transplantation.