Single Cell Network Profiling (SCNP) Reveals Race-Associated Differences in B Cell Receptor Signaling Pathway Activation

• Published in: Blood 2011-11, Vol.118 (21), p.1125-1125
• Main Authors: Longo, Diane, Louie, Brent, Mathi, Kavita, Pos, Zoltan, Wang, Ena, Hawtin, Rachael E., Marincola, Francesco, Cesano, Alessandra
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V118.21.1125.1125

Abstract 1125 Race-related differences have been documented in the incidence of autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis, in the clinical response to immunotherapies [such as IFNα (in HCV infections) and belimumab (in systemic lupus erythematosus)] and to hematopoietic stem cell transplantation. However, the basis for such race-associated differences remains poorly understood. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry based approach that simultaneously measures intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of peripheral blood mononuclear cells (PBMCs) from 60 healthy donors identified a race-associated difference in αIgD induced levels of pS6 and pAkt in B cells. The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR) in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. 35 BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured by SCNP in PBMCs from 10 healthy donors [5 African Americans (36–51 yrs), 5 European Americans (36–56 yrs), all males]. Cryopreserved PBMCs were thawed, modulated at 37°C in 96-well plates, fixed and permeabilized. Permeabilized cells were stained with fluorochrome-conjugated antibodies that recognize extracellular surface markers and intracellular signaling molecules. The levels of 7 phospho-proteins [pLck (Y505), pSyk (Y352), pAkt (S473), pS6 (S235/S236), p-p38 (T180/Y182), pErk (T202/Y204), and pNFκB (S529)] were measured in CD20+ B cells at 0, 5, 15, 30, and 60 mins post αIgD exposure. CD20 and IgD surface markers were used to determine the frequency of IgD+ B cells. Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower αIgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and Lck as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NFκB pathway (NFκB) (see example for αIgD induced pS6 levels in Fig. 1A). Overall, 4 (pSyk, pS6, pAkt, and pErk) of the 7 BCR pathway components tested (averaged over all timepoints for each donor) showed statistically significant differences in αIgD induced activati