An optimized protocol to quantify signaling in human transitional B cells by phospho flow cytometry.

BACKGROUND AND PURPOSE: Phospho flow cytometry is a powerful technique to analyze signaling in rare cell populations. This technique, however, requires harsh conditions for cell fixation and permeabilization, which can denature surface antigens or antibody-conjugated fluorochromes. These are among several technical limitations which have been a barrier to quantify signaling in unique B cell subsets. One such immature subset, transitional B cells (TrBs), may play a role in suppressing solid organ transplant rejection, graft-versus-host disease, autoimmunity, and even the immune response to malignancy. Here we sought to optimize a protocol for quantification of signaling in human TrBs compared with mature B cell subsets.

RESULTS: TrBs were defined by surface marker expression as CD19+ CD24hi CD38hi . Key parameters optimized included antibody clone selection, sequence of surface epitope labeling in relation to paraformaldehyde-based fixation and methanol-based permeabilization, photomultiplier tube (PMT) voltages, and compensation. Special attention was paid to labeling of CD38 with regard to these parameters, and an optimized protocol enabled reliable identification of TrBs, naïve (CD24+ CD38+ ), early memory (CD24hi CD38- ), and late memory (CD24- CD38- ) B cells. Phospho flow cytometry enabled simultaneous quantification of phosphorylation among at least three different signaling molecules within the same sample. Among normal donors, transitional B cells exhibited diminished mitogen activated protein kinase/extracellular signal-regulated kinase and Akt phospho signaling upon nonspecific stimulation with phorbol 12-myristate 13-acetateand ionomycin stimulation.

CONCLUSIONS: We optimized an effective protocol to quantify B cell subset signaling upon stimulation. Such a protocol may ultimately serve as the basis for assessing dysfunctional B cell signaling in disease, predict clinical outcomes, and monitor response to B cell-directed therapies.