Antigen-Specific B Cells and Chronic Infection: The OMIP Panel
 

Understanding antigen-specific B cells

Antigen-specific B cells originating in the bone marrow prevent and control various infections through antibody production, professional antigen presentation and cytokine secretion.¹

For example, the antibodies produced by B cells against the hepatitis B virus (HBV) envelope protein or the hepatitis B surface antigen (HBsAg) play a key role in the infection response to the virus. The induction of anti-HBsAg is considered to be the clinical indication of recovery from HBV. ¹  While B cells are an important element of infection response, it is unclear why the response fails in some cases of acute HBV infection. Therefore, Cascino et al. (2020) ¹ developed an optimized multicolour immunophenotyping (OMIP) panel to understand and characterize global and antigen-specific B cells during HBV infection.

Learn more about antigen-specific B cells and flow cytometry immunophenotyping>

Explore our predesigned panels for B cells in our interactive cell map>

OMIP panel design

In this 24-color flow cytometry immunophenotyping panel, Cascino et al. seek to characterize antigen-specific B cells for the precise delineation of B cell subsets in chronic infection. Of interest, the authors focused on the antigen specificity of memory B cells in HBV infection. The team extracted samples from cryopreserved human peripheral blood mononuclear cells (PBMCs). They used a dump channel to enable the exclusion of CD3+ T cells, CD14+ monocytes and dead cells to improve the resolution of CD19-CD20- and CD19+CD20+ cells.

The authors characterized B cells based on the expression of markers with a specific purpose. These are:

• HBV-specific B cell tag: HBsAg
• Lineage B cell markers: CD19, CD20
• Differentiation B cell markers: CD10, CD27, CD21, IgM, IgD, CD24, CD38, CD5, CD43, CD86
• Trafficking markers: CXCR3, CXCR5
• Inhibitory/exhaustion markers: CD11c, CD39, PD-1, FcRL5, BTLA, CD22, CD32
• Markers in the dump channel: live/dead CD3, CD14

Cascino et al. used a dual-labeling strategy to identify HBV-specific B cells. Two DyLight™ Fluorophores, added at equal concentrations, served as probes to detect B cells expressing BCRs specific to HBsAg. Only dual-labeled cells could be considered HBV-specific, which increased the overall specificity of the detection method.

Though most markers in this OMIP panel were aimed at conventional B cell subsets, the authors included markers associated with nonclassical B1 B cells (CD43 and CD5) to help identify the best gating scheme to define this population. The interest in B1 B cells stems from their unexpected role in immunosuppression as a source of anti-inflammatory cytokine IL-10.¹

Performance of the OMIP panel in phenotyping antigen-specific B cells

The authors propose that this OMIP panel enables antigen-specific B cell analysis at an unprecedented depth and can potentially uncover new subtypes and phenotypes that could play a role in the progression of infections.

The use of antigen-specific probes in this OMIP panel was also important in assessing HBV-specific B cells. In addition, the added ability to incorporate dual-labeling antigen-specific probes increased the power to make relevant antigen-specific conclusions regarding B cell functions in chronic infections. The team concluded that a similar method can be used to investigate other research questions beyond HBV.

Read the paper to learn more about this 24-color OMIP panel.