Flow cytometric assays to investigate b and T cell subsets in primary immunodeficiency

Abstract

The focus of our laboratory has been trying to decipher the development, differentiation and function of B and T cells in humans. We do this by studying these lymphocyte populations in primary immunodeficiencies (PIDs) due to monogenic mutations in critical genes. During the course of our study of PIDs, it has become evident that different cohorts of patients display distinct B and/or T cell phenotypes that are often diagnostic even prior to the discovery of the genetic lesion. For example X-linked lymphoproliferative disease (XLP) is a rare primary immunodeficiency due to mutations in SH2D1A encoding the intracellular adaptor protein SAP. Clinically, XLP patients present with hypogammaglobulinaemia, fulminant infectious mononucleosis, and/or lymphoma. We have previously found SAP is required for the development of NKT cells, which in humans are defined as CD3 + Va24 + Vb11 + cells, and are absent in XLP patients. Furthermore, consistent with hypogammaglobulinaemia, the B cell compartment from XLP patients was found to have a severe reduction in CD27 + memory B cells and Ig class switched cells. More recently we have developed an intracellular staining method for detecting SAP expression. In normal healthy donors SAP can be detected in T cells, but not B cells, as these cells do not express SAP. In XLP patients, SAP is absent in both the B and T cell compartments. Furthermore, XLP carriers can be readily identified, as due to random X-inactivation, they have T cells that are both SAP + as well as SAP – . Similarly, patients with loss of function mutations in STAT3 and DOCK8 present with a distinct lymphocyte phenotype, which can be readily revealed through flow cytometry-based assays; these will be further discussed.