This panel was developed to characterize the phenotypic diversity of human memory B cells, with an emphasis on discriminating cell subsets within both the conventional memory population (CD27+) and the more recently described isotype switched (IgD−) population lacking expression of CD27 (1). It has been tested on fresh and cryopreserved peripheral blood mononuclear cells (PBMC), as well as bone marrow aspirates and tonsillar cells (Table 1). The multicolor panel described herein has been used extensively to analyze large numbers of PBMC samples obtained from healthy controls in steady state and in response to infection (HIV, influenza, respiratory syncytical virus) and vaccination (influenza, tetanus) as well as in hundreds of patients with autoimmune diseases (systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjogren's syndrome, psoriatic arthritis and Type 1 diabetes) and conditions characterized by allogeneic immune responses (renal transplantion and chronic graft versus host disease). This panel is also being applied in a longitudinal study in which 150 SLE patients are to be followed quarterly for a period of 2 years.
Table 1. Summary table for OMIP-003
Phenotyping memory B cells
Fresh or cryopreserved PBMC, bone marrow, and tonsil mononuclear cells
Differentiation, receptor for CD70 on activated T cells
Activation; pro-apoptotic in the presence of activated CD95L T cells
VH4-34- encoded idiotype
Autoreactive B cells
Major human B cell subsets are currently defined by pauci-color flow cytometry protocols that typically include IgD, CD27, CD38, and CD24 to classify the major accepted peripheral blood populations (transitional, naïve, memory, and plasmablast subsets). By and large, the expression of other informative markers (including IgM, CD23, CD10, CD21, and CD95, as well as chemokine receptor expression) is assessed by parallel staining of several sample aliquots with different combinations of the aforementioned markers in four to five color schemes. These approaches suffer from multiple shortcomings including:1) the limited ability of the “defining” markers such as IgD, CD27, and CD38 to properly discriminate major populations;2) the inability to ascertain the actual co-expression of multiple markers in a single population possibly leading to faulty assumptions of extended phenotypes and, by extension, preventing the discovery of new subpopulations; and3) the need for larger number of cells to perform multiple stainings, a major practical limitation when dealing with rare samples. Combined, it seems obvious that limited use of available markers not only fails to differentiate multiple populations within the conventional core subsets, but could potentially lead to erroneous attribution of functional properties. To address these limitations, we have developed several multicolor panels to fully characterize human B cells. These multicolor panels share seven so-called anchor markers. Antibodies against CD19 and CD3, along with the Fixable Aqua Dead Cell Stain, allow the unambiguous identification of live CD19+CD3− B cells. The inclusion of four developmental markers (IgD, CD24, CD27, and CD38) in the same panel makes it feasible to compare and integrate these different classification schemes and provide precise identifications of the core human B cell subpopulations (2).
In addition to these anchor markers, each panel is extended with specific markers for further discrimination of memory, transitional/naïve B cells and plasma cell subsets, respectively. Thus, the incorporation of CD21, CD95, CD45/B220, and CXCR3 in the memory panel (Table 2), as described in this OMIP, provides information regarding the activation status and homing potential of both the CD27+ switched memory and CD27− switched memory B cells (Figure 1). The addition of the rat anti-human Ig idiotype 9G4 antibody completes a 12-color human memory B cell panel, and provides a useful measure of autoreactivity through the identification of B cells expressing autoantibodies encoded by the VH4-34 variable region gene (3).