Understanding antigen-specific B cells

Originating in the bone marrow, antigen-specific B cells are critical in infection prevention and control through antibody production, professional antigen presentation and cytokine secretion.

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 as the clinical indication of recovery from HBV.

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. developed this OMIP panel (Optimised Multicolour Immunophenotyping Panel) to understand and characterise global and antigen-specific B cells during HBV infection.

Learn more about antigen-specific b cells and flow cytometry immunophenotyping.

OMIP panel design

In this 24-colour flow cytometry immunophenotyping panel, Cascino et al. seek to characterise 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 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 characterised the B cells based on the expression of markers having 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 labelling strategy to identify HBV-specific B cells. Added at equal concentrations, two Dylight fluorophores served as probes to detect B cells expressing BCRs specific to HBsAg. Only dual labelled 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, Cascino et al. 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.

Learn more about OMIP panels and flow cytometry immunophenotyping.

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 which 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. As well, the added ability to incorporate dual-labelling 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 full study to learn more about this 24-colour OMIP panel.