Infant B-Cell Signaling after ABO-Incompatible Heart Transplantation (ABOi HTx): Defining the Role of the Inhibitory Molecule CD22

Abstract

Purpose Immune immaturity allows ABOi HTx to be performed safely in infants and results in B-cell tolerance to donor ABO antigens (Ags). In experimental settings, T-independent (TI) B-cell activation has been shown to be inhibited by binding of the B-cell co-receptor CD22 to CD22 ligands leading to B-cell tolerance. Since immunity to TI Ags, including ABO Ags, are generally reduced in early childhood, we hypothesize a role for CD22, possibly contributing to ABOi HTx tolerance. Previously, we showed that CD22 expression is higher on CD27+IgM+ B-cells in infants when compared to older individuals. In this study we performed functional assays with CD27+IgM+ B-cells to assess the presence of ABO Ag-specific IgM antibody (Ab)-secreting cells (ASC). In addition, we established a phospho-flow assay (PPF) to assess B-cell signaling. Methods and Materials CD27+IgM+ and CD27-IgM+ B-cells were isolated from human splenocytes (n=3 infant, n=6 adult) by cell sorting and stimulated with CpG plus IL-2, IL-10, and IL-15. After 1 week the frequency of ABO Ag-specific ASC was detected by ELISPOT. The PPF was optimized using a Ramos B-cell line and intracellular protein phosphorylation of CD22(pY822) and PLCγ2(pY759) were examined after stimulation with anti-IgM Abs. Results The total number of ASC was lower in infant samples compared to older individuals and ELISPOT analysis revealed that the majority of ABO Ag-specific ASC were derived from CD27+IgM+ B-cells in both groups. Preliminary results by PPF (n=8) showed pY759 signaling beginning at 0.5min and peaking at 4min, with a 7-fold increase in median fluorescence intensity (MFI) of pY759. pY822 signaling began at 2min with a modest peak at 12min, with a 1.6-fold increase in MFl of pY822. Conclusions The overall increased expression of CD22 on infant CD27+IgM+ B-cells, which include ABO Ag-specific ASC precursors, may increase their susceptibility to decreased signaling, leading to inactivation. CD22 may therefore play an inhibitory role in infant immune responses to ABO Ags after ABOi HTx.