Background: ABO-incompatible (ABOi) heart transplantation (HTx) can be performed safely in infants and results in B cell tolerance to donor ABO antigens by mechanisms that remain unclear. We showed that the inhibitory molecule CD22 is more highly expressed on splenic CD27+IgM+ B cells, which contain the vast majority of ABO-specific antibody secreting cell precursors, compared to other splenic B cell subsets. CD22 expression was highest on infant CD27+IgM+ B cells and decreased with age. Binding of CD22 to sialic acids (CD22L) on the same cell (cis-binding) or different cells (trans-binding) regulates B cell activation and may lead to B cell tolerance. Here, we studied whether CD22-CD22L cis-binding affects activation of CD27+IgM+ and CD27-IgM+ B cells from pediatric and adult spleen.
Methods: CD27+IgM+ and CD27-IgM+ B cells were isolated via fluorescence-activated cell sorting from pediatric (n=3; age range: 0–2.5 yr) and adult (n=3; age range: 21–46 yr) spleen obtained from organ donors. To ‘break’ CD22-CD22L cis-binding, sorted B cells were treated with neuraminidase (NEU) to cleave surface sialic acids. Activation of B cells was defined by analyzing intracellular phosphorylation of PLCγ2 (pY759) after stimulation with anti-IgM antibodies using ‘phospho-flow’ cytometry.
Results: Kinetics of pY759 signaling after stimulation were similar between untreated CD27+IgM+ and CD27-IgM+ B cells as well as pediatric and adult samples. At peak response (1–3 min after stimulation), the median fluorescence intensity (MFI) of pY759 in pediatric CD27+IgM+ B cells was comparable to that in pediatric CD27-IgM+ B cells (350–614 vs. 259–565), whereas in adult cells the MFI was higher in CD27+IgM+ B cells than in CD27-IgM+ B cells (540–912 vs. 248–328). NEU treatment had a strong effect on kinetics and magnitude of pY759 signaling in both pediatric and adult CD27+IgM+ B cells: the MFI of pY759 increased to 506–2023 and 1086–2051, respectively, at peak response (4–8 min after stimulation). In contrast, treatment had minimal effect on pY759 signaling in CD27-IgM+ B cells.
Conclusion: NEU treatment increased the magnitude of pY759 signaling in stimulated CD27+IgM+ B cells, but not CD27-IgM+ B cells, suggesting that CD22-CD22L cis-binding may play a role in control of activation of CD27+IgM+ B cells. This finding may contribute to unraveling mechanisms of B cell tolerance in ABOi HTx in children.
Canadian National Transplant Research Program (CNTRP) investigator.