Quantitative analysis of influenza virus-specific B cell memory generated by different routes of inactivated virus vaccination.

We consider both Ab-secreting cell (ASC) and memory B cell (B(Mem)) populations in a quantitative analysis of virus-specific B cell memory generated by intramuscular or intranasal vaccination of mice with inactivated influenza virus. After both forms of vaccination, the memory phase was characterized by localization of ASCs in the bone marrow and dispersion of B(Mem) to organized lymphoid tissues. The stronger IgG response to intramuscular vaccination correlated with larger numbers of IgG ASCs in the bone marrow and IgG B(Mem). IgA production was only prominent in the response to intranasal vaccination and was associated with IgA ASC localization in the lung and IgA B(Mem) formation. Notably, few IgG ASCs or B(Mem) localized in the lung after intramuscular vaccination, in contrast to the situation following influenza pneumonia. Our analysis links the nature of immunization to characteristics of the state of B cell memory that may relate to protective immunity.

The generation of influenza-specific humoral responses is impaired in ST6Gal I-deficient mice.

Posttranslational modification of proteins, such as glycosylation, can impact cell signaling and function. ST6Gal I, a glycosyltransferase expressed by B cells, catalyzes the addition of alpha-2,6 sialic acid to galactose, a modification found on N-linked glycoproteins such as CD22, a negative regulator of B cell activation. We show that SNA lectin, which binds alpha-2,6 sialic acid linked to galactose, shows high binding on plasma blasts and germinal center B cells following viral infection, suggesting ST6Gal I expression remains high on activated B cells in vivo. To understand the relevance of this modification on the antiviral B cell immune response, we infected ST6Gal I(-/-) mice with influenza A/HKx31. We demonstrate that the loss of ST6Gal I expression results in similar influenza infectivity in the lung, but significantly reduced early influenza-specific IgM and IgG levels in the serum, as well as significantly reduced numbers of early viral-specific Ab-secreting cells. At later memory time points, ST6Gal I(-/-) mice show comparable numbers of IgG influenza-specific memory B cells and long-lived plasma cells, with similarly high antiviral IgG titers, with the exception of IgG2c. Finally, we adoptively transfer purified B cells from wild-type or ST6Gal I(-/-) mice into B cell-deficient (microMT(-/-)) mice. Recipient mice that received ST6Gal I(-/-) B cells demonstrated reduced influenza-specific IgM levels, but similar levels of influenza-specific IgG, compared with mice that received wild-type B cells. These data suggest that a B cell intrinsic defect partially contributes to the impaired antiviral humoral response.

Broad dispersion and lung localization of virus-specific memory B cells induced by influenza pneumonia.

Although memory B cells (B(Mem)) contribute significantly to resistance to infection, B(Mem) population characteristics that may relate to protective efficacy have received little attention. Here, we report a comprehensive quantitative analysis of virus-specific IgG and IgA B(Mem) dispersion after transient influenza pneumonia in mice. From early in the response, B(Mem) circulated continuously and dispersed widely to secondary lymphoid tissues. However, a complicated picture emerged with B(Mem) frequency differences between secondary lymphoid tissues indicating an influence of local tissue factors on trafficking. B(Mem) numbers increased and stabilized at tissue-specific frequencies without contraction of the B(Mem) pool during the period of analysis. The lung was notable as a nonsecondary lymphoid tissue where a rapid influx of IgG and IgA B(Mem) established relatively high frequencies that were maintained long term. Our findings provide insights into the pattern of B(Mem) dispersion, and emphasize the lung as a complex repository of immune memory after local infection.

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.

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.

Production of monoclonal antibodies and immunohistochemical studies of brain myo-inositol monophosphate phosphatase.

Five monoclonal antibodies that recognize porcine brain myo-inositol monophosphate phosphatase (IMPase) have been selected and designated as mAb IMPP 9, IMPP 10, IMPP 11, IMPP 15, and IMPP 17. These antibodies recognize different epitopes of the enzyme and one of these inhibited the enzyme activity. When the total proteins of the porcine brain homogenate separated by SDS-PAGE were probed with monoclonal antibodies, a single reactive protein band of 29 kDa, co-migrating with the purified porcine brain IMPase, was detected. Using the anti-IMPase antibodies as probes, the cross reactivities of the brain IMPase from human and other mammalian tissues, as well as from avian sources, were investigated. Among the human and animal tissues tested, the immunoreactive bands on Western blots appeared to have the same molecular mass of 29 kDa. In addition, there was IMPase immunoreactivity in the various neuronal populations in the rat brain. These results indicate that mammalian brains contain only one major type of immunologically similar IMPase, although some properties of the enzymes that were previously reported differ from each another. The first demonstration of the IMPase localization in the brain may also provide useful data for future investigations on the function of this enzyme in relation to various neurological diseases.

Immunohistochemical studies of brain pyridoxine-5'-phosphate oxidase.

A total of six hybridoma cell lines, which produce monoclonal antibodies (mAbs) against the sheep brain pyridoxine-5'-phosphate oxidase (PNP oxidase), were established. Isotype analysis revealed that all antibodies corresponded to the IgG 2B kappa subclass. Immunoblotting with various tissue homogenates indicated that all the mAbs specifically recognize a single protein band of 30 kDa. They also appear to be extensively cross-reactive among different mammalian and avian sources. These results demonstrated that only one type of immunologically similar PNP oxidase is present in all of the mammalian tissues tested. When the purified PNP oxidase was incubated with the mAbs, the enzyme activity was inhibited up to a maximum of 81%. Furthermore, these antibodies were successfully applied in immunohistochemistry in order to detect PNP oxidase in various regions of rat brain tissues. The immunoreactive neurons in PNP oxidase were found in cerebellar cortex, hippocampus, amygdala, paraventricular nucleus, cerebral cortex and ependyma. This result suggests that PNP oxidase may play an important role in the neuronal metabolism.