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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cytometry A. Author manuscript; available in PMC Nov 1, 2012.
Published in final edited form as:
PMCID: PMC3199331
NIHMSID: NIHMS315080

OMIP-003: Phenotypic Analysis of Human Memory B Cells

Purpose and Appropriate Sample Types

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. 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, RSV) and vaccination (influenza, tetanus) as well as in hundreds of patients with autoimmune diseases (Systemic Lupus Erythematosus, 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 two years.

Background

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 4–5 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 sub-populations; and 3) 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, 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. 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).

Figure 1
Classification of core human B cell subsets and phenotypic characterization of memory B cells in healthy subjects and patients with Systemic Lupus Erythematosus
Table 1
Summary table for OMIP-000.
Table 2
Reagents used for OMIP-000.

Supplementary Material

Supp Figure S1

Supp Figure S2

Supp Figure S3

Supplementary Data

Acknowledgments

This work was supported by National Institutes of Health Grants R01 AI049660-01A1 and U19 Autoimmunity Center of Excellence AI56390 to I.S.

Literature Cited

1. Wei C, Anolik J, Cappione A, Zheng B, Pugh-Bernard A, Brooks J, Lee E-H, Milner ECB, Sanz I. A New Population of Cells Lacking Expression of CD27 Represents a Notable Component of the B Cell Memory Compartment in Systemic Lupus Erythematosus. J Immunol. 2007;178(10):6624–6633. [PubMed]
2. Sanz I, Wei C, Lee FE-H, Anolik J. Phenotypic and functional heterogeneity of human memory B cells. Seminars in Immunology. 2008;20(1):67–82. [PMC free article] [PubMed]
3. Cappione A, 3rd, Anolik JH, Pugh-Bernard A, Barnard J, Dutcher P, Silverman G, Sanz I. Germinal center exclusion of autoreactive B cells is defective in human systemic lupus erythematosus. J Clin Invest. 2005;115(11):3205–16. [PMC free article] [PubMed]
4. Moir S, Ho J, Malaspina A, Wang W, DiPoto AC, O’Shea MA, Roby G, Kottilil S, Arthos J, Proschan MA, et al. Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals. J Exp Med. 2008;205(8):1797–1805. [PMC free article] [PubMed]
5. Jacobi AM, Reiter K, Mackay M, Aranow C, Hiepe F, Radbruch A, Hansen A, Burmester GR, Diamond B, Lipsky PE, et al. Activated memory B cell subsets correlate with disease activity in systemic lupus erythematosus: delineation by expression of CD27, IgD, and CD95. Arthritis Rheum. 2008;58(6):1762–73. [PubMed]
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