Abstract
Purpose: Immune immaturity allows ABO-incompatible heart transplantation (ABOi HTx) to be performed safely in infants and generally results in B-cell tolerance to donor ABO antigens. ABO antibodies are thought to arise in a T-independent (TI) manner; TI B-cell activation has been shown to be inhibited by the interaction of the inhibitory B-cell co-receptor CD22 with sialic acids on cells and tissues, leading to B-cell tolerance. It is unknown whether CD22 plays a role in regulating B-cell responses to ABO antigens in the transplant setting. Due to the generally reduced immune response to TI-antigens in early childhood, we hypothesized an enhanced role for CD22 during infancy compared to later in life. In this study we examined CD22 expression on various B-cell subsets. In addition, we performed functional assays to determine CD22 expression after B-cell stimulation and to assess the presence of ABO antigen-specific IgM antibody-secreting cells (ASC). Methods: We analyzed human splenocytes isolated from organ donors (n=41; ages 4 days - 74 years). Flow cytometric analysis was performed to quantify the expression levels of CD22, CD27, CD38, IgM and IgG on the surface of CD19+ B cells. CD27+IgM+ B cells and CD27−IgM+ B cells were isolated from human splenocytes (n=5) by automated magnetic activated cell sorting, labelled with Cell Proliferation Dye eFluor450 and stimulated with CpG plus IL-2, IL-10 and IL-15. After one week, CD22 expression was examined by flow cytometry and the frequency of ABO antigen-specific ASC detected by ELISPOT. Results: Significant differences were observed when comparing the median fluorescence intensity (MFI) of CD22 amongst various B-cell subsets (p< 0.0001; Kruskal Wallis test). Post testing using the Dunn's multiple comparison test revealed that CD27+IgM+ B cells had higher expression (p< 0.001) than other B-cell subsets. Furthermore, the MFI of CD22 on the CD27+IgM+ B cells was inversely correlated with age (p=0.001), with infant samples having the highest level of CD22, and expression decreasing with increasing age. After culture, down-regulated CD22 expression was observed in proliferating CD27+IgM+ B cells compared to non-proliferating cells. ELISPOT analysis showed that the vast majority of ABO antigen-specific ASC were derived from the CD27+IgM+ B-cell population. Conclusion: Based on previous studies showing the inhibitory role of CD22 on B-cell activation, our findings suggest that the increased expression of CD22 on the CD27+IgM+ B-cell subset, which includes precursors of ABO antigen-specific ASC, may cause these infant B cells to be more susceptible to down-regulation of B-cell signaling leading to subsequent inactivation. CD22 may therefore play an inhibitory role in immune responses to ABO antigens in the ABOi HTx setting. Studies are underway to determine the role of CD22 in B-cell signalling and regulation during infancy.
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