Biomedical subjects

Mark Y Sangster

Publications and source records attributed to Mark Y Sangster.

4 recordsLinked to original sources

PubMed2006

Quantitative analysis of herpes simplex virus type 1-specific memory B cells generated by different routes of infection.

We compared the herpes simplex virus type 1 (HSV)-specific memory B cell (MBC) populations generated by footpad and intranasal infection in mice. Both routes of infection generated transient antibody-secreting cell responses in the draining lymph nodes and spleen, and sustained circulating IgG. HSV-specific IgG MBCs, analyzed by limiting dilution assay approximately 8 weeks after infection, were distributed in a range of lymph nodes and in the spleen and Peyer's patches. Overall, the route of infection had little effect on the MBC frequency in each anatomical location. Interestingly, after both routes of infection there was a trend towards preferential MBC accumulation in the mediastinal lymph node. Intravaginal challenge of mice primed by footpad or intranasal infection generated similar secondary IgG responses. Our findings indicate that the widespread dispersion of MBCs to lymphoid tissues throughout the body is largely independent of the route of infection, but may be influenced by tissue-specific factors.

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PubMed2006

A strategy for selective, CD4+ T cell-independent activation of virus-specific memory B cells for limiting dilution analysis.

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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PubMed2005

Pathogenesis of Hong Kong H5N1 influenza virus NS gene reassortants in mice: the role of cytokines and B- and T-cell responses.

The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92-->Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50%) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1alpha, IL1beta, IL6, IFN-gamma and chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.

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PubMed2003

An early CD4+ T cell-dependent immunoglobulin A response to influenza infection in the absence of key cognate T-B interactions.

Contact-mediated interactions between CD4+ T cells and B cells are considered crucial for T cell-dependent B cell responses. To investigate the ability of activated CD4+ T cells to drive in vivo B cell responses in the absence of key cognate T-B interactions, we constructed radiation bone marrow chimeras in which CD4+ T cells would be activated by wild-type (WT) dendritic cells, but would interact with B cells that lacked expression of either major histocompatibility complex class II (MHC II) or CD40. B cell responses were assessed after influenza virus infection of the respiratory tract, which elicits a vigorous, CD4+ T cell-dependent antibody response in WT mice. The influenza-specific antibody response was strongly reduced in MHC II knockout and CD40 knockout mice. MHC II-deficient and CD40-deficient B cells in the chimera environment also produced little virus-specific immunoglobulin (Ig)M and IgG, but generated a strong virus-specific IgA response with virus-neutralizing activity. The IgA response was entirely influenza specific, in contrast to the IgG2a response, which had a substantial nonvirus-specific component. Our study demonstrates a CD4+ T cell-dependent, antiviral IgA response that is generated in the absence of B cell signaling via MHC II or CD40, and is restricted exclusively to virus-specific B cells.

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