Specific B‐cell tolerance toward donor blood group antigens develops in infants after ABO‐incompatible heart transplantation, whereas their immune response toward protein antigens such as HLA has not been investigated. We assessed de novo HLA‐antibodies in 122 patients after pediatric thoracic transplantation (28 ABO‐incompatible) and 36 controls. Median age at transplantation was 1.7 years (1 day to 17.8 year) and samples were collected at median 3.48 years after transplantation. Antibodies were detected against HLA‐class I in 21 patients (17.2%), class II in 18 (14.8%) and against both classes in 10 (8.2%). Using single‐antigen beads, donor‐specific antibodies (DSAs) were identified in six patients (all class II, one additional class I). Patients with DSAs were significantly older at time of transplantation. In patients who had undergone pretransplant cardiac surgeries, class II antibodies were more frequent, although use of homografts or mechanical heart support had no influence. DSAs were absent in ABO‐incompatible recipients and class II antibodies were significantly less frequent than in children with ABO‐compatible transplants. This difference was present also when comparing only children transplanted below 2 years of age. Therefore, tolerance toward the donor blood group appears to be associated with an altered response to HLA beyond age‐related effects.
Physical activity is central in prevention and treatment of metabolic syndrome. High‐intensity aerobic exercise can induce larger energy expenditure per unit of time compared with moderate‐intensity exercise. Furthermore, it may induce larger energy expenditure at post‐exercise recovery. The aim of this study is to compare the excess post‐exercise oxygen consumption ( EPOC ) in three different aerobic exercise sessions in men with metabolic syndrome. Seven men (age: 56.7 ± 10.8) with metabolic syndrome participated in this crossover study. The sessions consisted of one aerobic interval (1‐ AIT ), four aerobic intervals (4‐ AIT ), and 47‐min continuous moderate exercise ( CME ) on separate days, with at least 48 h between each test day. Resting metabolic rate ( RMR ) was measured pre‐exercise and used as baseline value. EPOC was measured until baseline metabolic rate was re‐established. An increase in O 2 uptake lasting for 70.4 ± 24.8 min (4‐ AIT ), 35.9 ± 17.3 min (1‐ AIT ), and 45.6 ± 17.3 min ( CME ) was observed. EPOC were 2.9 ± 1.7 L O 2 (4‐ AIT ), 1.3 ± 1.1 L O 2 (1‐ AIT ), and 1.4 ± 1.1 L O 2 ( CME ). There were significant differences ( P < 0.001) between 4‐ AIT , CME , and 1‐ AIT . Total EPOC was highest after 4‐ AIT . These data suggest that exercise intensity has a significant positive effect on EPOC in men with metabolic syndrome.
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. 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.
Regulatory T cell (Treg)–based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3+ Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood. Regulatory T cell (Treg)–based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3+ Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood.
Introduction: Infant heart transplant (HTx) recipients have better graft survival than patients transplanted at older ages. Nonetheless, due to lifelong need for therapy, infant HTx recipients can expect to carry an immunosuppressive heavier burden, resulting in substantial morbidities from adverse drug effects. Development of a cellular therapy using regulatory T cells (Tregs) to suppress graft-directed immune responses would greatly benefit these infants. A major challenge for such a therapy, however, is generating a large quantity of stable, highly suppressive Tregs. Infants undergoing HTx usually have thymectomy in order to gain adequate exposure of the retrosternal operative field. We investigated the potential of explanted thymic tissue as a source for isolation and expansion of highly suppressive CD4+CD25+CD127lowFOXP3+ Tregs. Methodology: Thymic tissue (n=3) was obtained from thymectomy during pediatric cardiac surgery and thymocytes were recovered through mechanical dissociation. FOXP3+ cells were isolated by automated magnetic cell separation of CD4+CD127lowCD25+ thymocytes. CD4+CD127low-depleted cells were used as controls. Cells were expanded for two weeks by stimulation with anti-CD3, IL-2, rapamycin and CD32+ L cells. FOXP3 and intracellular cytokine staining was performed to define characteristics of expanded cells. The suppressive capacity after expansion was determined by co-culturing the expanded cells with PKH-labeled anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMC) and analyzing proliferative responses by flow cytometry. Results: CD4+CD25+CD127lowFOXP3+ cell frequency within the total thymocyte population ranged from 2.8 to 7.9%. Isolated CD4+CD25+CD127low cell populations were 65-84% positive for FOXP3. After two weeks of culture, we observed a 4 to 40-fold expansion of CD4+CD25+CD127low cells with >95% viability; 0.4 to 25-fold expansion was observed for control cells with 49-88% viability. The expanded CD4+CD25+CD127low cells were >95% FOXP3+ and produced no IL-2 or IFN-γ, whereas control cells were < 14% FOXP3+ and 58-65% produced IFN-γ. Moreover, in contrast to control cells, expanded CD4+CD25+CD127lowFOXP3+ cells were highly potent suppressors, efficiently suppressing the frequency of proliferating PBMC >80% even at a 1:20 ratio of Tregs:PBMC. Conclusion: In this preliminary study, we showed that highly suppressive FOXP3+ Tregs can be expanded from CD4+CD25+CD127low T cells isolated from pediatric thymic tissue, indicating that explanted thymuses may be a potential source for isolation and expansion of Tregs for cellular therapy. Future work includes defining the stability of expanded Tregs and their suppressive capacity of alloantigen immune responses.
