B CELLS AND ANTIBODY RESPONSE

S. Urschel¹, L.A. Ryan², M. Jeyakanthan³, I.M. Larsen³, D.B. Ross⁴, L.J. West^1
1Department Of Pediatrics, Cardiac Transplant Research, University of Alberta, Edmonton/CANADA, ²Cardiac Transplant Research, University of Alberta, Edmonton/AB/CANADA, ³Department Of Pediatrics, Cardiac Transplant Research, University of Alberta, Edmonton/AB/CANADA, ⁴Cardiac Surgery, University of Alberta, Edmonton/CANADA

Body: Introduction:
Transplantation of blood group (ABO) incompatible solid organs in adults requires aggressive antibody removal strategies to avoid (hyper) acute and accelerated chronic rejection. ABO-incompatible heart transplantation was never successfully performed in adults, but was found to be safe in early childhood. In contrast to adults the majority of these children show persistent tolerance of the donor blood group antigens defined as absence of antibodies and specific B-cells. Mechanisms underlying this unique immune state are not yet determined. We hypothesized that differences in presence or phenotype of memory B-cells influences the capability to develop this unique form of tolerance towards polysaccharide antigens in young children.

Methods and Materials:
Children undergoing heart transplantation were included in a prospective study approved by the institutional ethical review board. Peripheral blood lymphocytes were analyzed for expression of surface markers with flow-cytometry. B-cells were subtyped for CD27, IgM, and IgD; expression levels of CD21, CD81, CD5 and CD1d were quantified. In vitro proliferation was assessed on using donor-spleen cells of children and adults. Cells were labelled with Carboxyfluorescein succinimidyl (CFSE) and proliferation rate was determined for different lymphocyte subtypes in response to stimulation with mitogens, erythrocytes and synthetic ABO antigens.

Results:
We investigated 55 samples from heart transplant recipients (age at transplant 4.2 months [0.03-20.4], age at sample collection 14.6 mo. [0.04-51.3], 53% ABO-incompatible). Significant correlation was found between age of the patients and prevalence of both, IgM^high memory B-cells (CC: 0.45, p=0.001) and switched IgM^low (CC: 0.31 p=0.027) memory B-cells. CD21^high cells were less frequent in the first 6 mo. but showed constant presence thereafter; CD81 expression was not age-dependent.
In adult spleen CD21^high marginal zone B-cells comprised the main pool of CD27+ memory B-cells, whereas in infants this reservoir was nearly absent. In CFSE-assays memory B-cells proliferated more than CD27- B-cells (p<0.05), especially in isolated B-cell cultures without T-cell support. When stimulated with ABO-antigens, specific proliferation was exclusively found in CD27+IgM+ B-cells.

Conclusions:
CD27+ memory B-cells are crucial for T-independent immune responses to polysaccharides such as ABO-antigens. Their limited presence in early childhood provides the environment to develop tolerance towards the donor blood group. This is further facilitated by lower CD21 expression. Phenotyping and quantification of memory B-cells using our approach provides an additional diagnostic tool for evaluation of eligibility for ABO-incompatible heart transplantation in childhood and for understanding mechanisms of tolerance in this setting.

Disclosure: All authors have declared no conflicts of interest.