Immunoglobulin profile and B-cell frequencies are altered with changes in the cellular microenvironment independent of the stimulation conditions.

INTRODUCTION: B-cells are essential in the defense against Mycobacterium tuberculosis. Studies on isolated cells may not accurately reflect the responses that occur in vivo due to the presence of other cells. This study elucidated the influence of microenvironment complexity on B-cell polarization and function in the context of tuberculosis disease. METHODS: B-cell function was tested in whole blood, peripheral blood mononuclear cells (PBMCs), and as isolated cells. The different fractions were stimulated and the B-cell phenotype and immunoglobulin profiles analyzed. RESULTS: The immunoglobulin profile and developmental B-cell frequencies varied for each of the investigated sample types, while in an isolated cellular environment, secretion of immunoglobulin isotypes immunoglobulin A (IgA), IgG2, and IgG3 was hampered. The differences in the immunoglobulin profile highlight the importance of cell-cell communication for B-cell activation. Furthermore, a decrease in marginal zone B-cell frequencies and an increase in T1 B-cells was observed following cell isolation, indicating impaired B-cell development in response to in vitro antigenic stimulation in isolation. CONCLUSION: Our results suggest that humoral B-cell function and development was impaired likely due to a lack of costimulatory signals from other cell types. Thus, B-cell function should ideally be studied in a PBMC or whole blood fraction.

Regulatory B lymphocytes: development and modulation of the host immune response during disease.

The role of B lymphocytes (B cells) in immunogenic responses has become increasingly important over the past decade, focusing on a new B-cell subtype: regulatory B-cells (Bregs). These Bregs have been shown to possess potent immunosuppressive activities and have identified as key players in disease control and immune tolerance. In this review, the occurrence of Breg type in various conditions, along with evidence supporting discovered functions and proposed purposes will be explored. An example of such regulatory functions includes the induction or suppression of various T lymphocyte phenotypes in response to a particular stimulus. Should Bregs prove effective in mediating immune responses, and correlate with favorable disease outcome, they may serve as a novel therapeutic to combat disease and prevent infection. However, the induction, function and stability of these cells remain unclear and further investigation is needed to better understand their role and therapeutic efficacy.

Isolation of B-cells using Miltenyi MACS bead isolation kits.

This article describes the procedures used to isolate pure B-cell populations from whole blood using various Miltenyi magnetic-activated cell sorting (MACS) bead Isolation kits. Such populations are vital for studies investigating the functional capacity of B-cells, as the presence of other cell types may have indirect effects on B-cell function through cell-cell interactions or by secretion of several soluble molecules. B-cells can be isolated by two main approaches: 1) Negative selection-in which B-cells remain "untouched" in their native state; this is advantageous as it is likely that B-cells remain functionally unaltered by this process. 2) Positive selection-in which B-cells are labelled and actively removed from the sample. We used three Negative B-cell isolation kits as well as the Positive B-cell isolation kit from Miltenyi and compared the purity of each of the resulting B-cells fractions. Contamination of isolated B-cell fractions with platelets was the conclusive finding for all of the isolation techniques tested. These results illustrate the inefficiency of current available MACS B-cell isolation kits to produce pure B-cell populations, from which concrete findings can be made. As such we suggest cell sorting as the preferred method for isolating pure B-cells to be used for downstream functional assays.