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Published on: May 27, 2011
A strategy for selective, CD4+ T cell-independent activation of virus-specific memory B cells for limiting dilution
Xiaofeng Li1, Daisy J Vanitha, Hye Mee Joo
1Department of Microbiology, M409 Walters Life Sciences Bldg, University of Tennessee, Knoxville, 37996, USA.
Insights
Accurate quantitation of memory B cells (MBCs) is crucial for understanding immune responses. This study found that beta-propiolactone-inactivated virus particles selectively activate MBCs for antibody secretion, enabling sensitive measurement without T cell influence.
Area of Science:
- Immunology
- Virology
- Cellular Biology
Background:
- Accurate quantification of memory B cell (MBC) pools is essential for characterizing immune responses to infection and vaccination.
- Current methods like limiting dilution analysis (LDA) rely on in vitro stimulation of MBCs, but their sensitivity depends heavily on optimal activation conditions.
Purpose of the Study:
- To develop a sensitive and reproducible limiting dilution assay (LDA) for measuring influenza-specific MBC frequencies.
- To evaluate strategies for optimal in vitro activation of MBCs for quantitative analysis.
Main Methods:
- Evaluated in vitro stimulation strategies for influenza-specific MBCs using an ELISPOT assay to enumerate IgG antibody-secreting cells (ASCs).
- Compared T cell-dependent activation with influenza-infected splenocytes versus T cell-independent activation using beta-propiolactone (BPL)-inactivated influenza virus particles (BPL-flu).
- Extended the methodology to herpes simplex virus (HSV)-specific MBCs using BPL-inactivated HSV.
Main Results:
- Optimal MBC activation for LDA sensitivity was achieved using BPL-inactivated influenza virus particles (BPL-flu) in a T cell-independent manner.
- BPL-flu selectively activated MBCs without stimulating naive B cells or MBCs of other specificities.
- The BPL-flu method provided a sensitive and reproducible basis for measuring influenza-specific MBC frequencies in various anatomical locations.
- Similar selective, T cell-independent activation was observed for HSV-specific MBCs using BPL-inactivated HSV.
Conclusions:
- BPL-inactivated viral particles are effective tools for the selective, T cell-independent activation of virus-specific MBCs in vitro.
- This approach eliminates the confounding influence of T cell-associated factors, improving the accuracy of MBC frequency determination.
- The findings establish a robust method for quantifying MBC responses critical for vaccine and infection studies.
Abstract:
Complete characterization of the B cell response to infection or vaccination is dependent on accurate quantitation of the memory B cell (MBC) pool. An established method for measuring MBC frequencies is limiting dilution analysis based on in vitro stimulation of MBCs to divide and differentiate into antibody-secreting cells (ASCs). The presence of specific antibody then serves to identify cultures positive for precursor MBCs. The sensitivity of this approach is critically dependent on optimal in vitro MBC activation. To develop a limiting dilution assay (LDA) for measuring influenza-specific MBC frequencies, we evaluated strategies for the in vitro stimulation of influenza-specific MBCs. An ELISPOT assay to enumerate influenza-specific IgG ASCs was used as the readout for MBC activation. Culture of influenza-specific MBCs with influenza-infected splenocytes was effective for MBC activation, but T cell-associated factors were required for optimal LDA sensitivity and clonal expansion of activated MBCs. However, optimal influenza-specific MBC activation was T cell-independent when MBCs were simply cultured with beta-propiolactone (BPL)-inactivated influenza virus particles (BPL-flu). BPL-flu did not stimulate naïve B cells to produce influenza-specific IgG, demonstrating that only MBCs were activated. In addition, BPL-flu acted selectively and only activated influenza-specific MBCs, not MBCs of other specificities. Analysis of influenza-specific MBC frequencies in different anatomical locations in influenza-immune mice established that in vitro stimulation with BPL-flu provided the basis for a sensitive and reproducible LDA. Extending our studies to the herpes simplex virus (HSV) system, we demonstrated that HSV-specific MBCs cultured with BPL-inactivated HSV were selectively activated to IgG secretion in the absence of T cells. Our studies identify BPL-inactivated viral particles as a valuable tool for selective, T cell-independent activation of virus-specific MBCs in vitro. This strategy eliminates the influence of poorly defined T cell-associated factors on MBC frequency determinations.
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