In 2014, we pursued 4 separate areas of research: 1) HIV-specific B-cell responses among B-cell subsets isolated from the peripheral blood of HIV-infected individuals;2) plasma cells in the bone marrow of HIV-infected individuals;3) assessment of antibodies generated from single-cell sorting;and 4) B-cell features and functions in non-HIV immunodeficiencies. First, in a study published in the Journal of Clinical Investigation, we used HIV envelope probes and flow cytometry to investigate HIV-specific B cells in the peripheral blood of HIV-infected individuals at different stages of disease. We found that HIV-specific B-cell responses arose early after infection and are blunted by antiretroviral therapy and chronic HIV viremia. Furthermore, we used combinations of envelope probes with mutations in the CD4 and coreceptor (CoR) binding sites (bs) to show that the B cells directed against the CoRbs predominated over those against the CD4bs;yet, it is the latter specificity that has more neutralizing potential. In these multiparameter flow cytometric-based assays, we also included markers that can identify B-cell subsets circulating in the peripheral blood and we performed parallel analyses to investigate B-cell responses to other pathogens, including tetanus and influenza. We found that HIV-specific responses were enriched within abnormal B-cell subsets, namely activated and tissue-like memory B cells that are largely absent in healthy donors. The subset distribution of B-cell responses to HIV was significantly different (i.e., more enriched within abnormal subsets) when compared to responses against non-HIV antigens. These findings demonstrate the unique immunopathogenesis of HIV as it relates to B cells. However, we also found that the HIV-specific B-cell response in individuals who maintained low levels of HIV plasma viremia and immune activation was enriched within the resting memory B cells, the predominant subset in uninfected individuals. Second, in a study that has been completed with a manuscript in preparation, we investigated B cells in the bone marrow of HIV-infected individuals during the chronic, clinically asymptomatic phase of disease. We found that while there were no obvious perturbations in hematopoiesis, alterations in the frequencies of various B-cell subsets were noted when compared to bone marrow cells of HIV-negative individuals, including an increased frequency of plasma cells, the terminally differentiated B-cell subset responsible for making antibodies. This abnormality is consistent with other hallmarks of HIV infection, namely hypergammaglobulinemia and increased frequencies in the periphery of precursors of plasma cells, i.e. plasmablasts. Furthermore, we found that there was a strong correlation between the frequency of HIV-specific plasma cells in the bone marrow and HIV-specific antibodies in the serum of HIV-infected individuals. These findings suggest that despite HIV-induced systemic B-cell perturbations in infected individuals, their bone marrow remains the repository for long-lived plasma cells responsible for antibodies found in circulation. Third, in studies that involve collaborations with the NIAID Vaccine Research Center (VRC) and Yale University, we have used single-cell cloning strategies to express IgG antibodies derived from memory B cell subsets that circulate in the peripheral blood of chronically HIV-infected viremic individuals and in secondary lymphoid tissues. In the peripheral blood, we found that despite a higher number of cell divisions that should correlate with affinity maturation of the antibody response, IgG antibodies derived from the tissue-like memory B-cell compartment displayed similar or reduced levels of somatic hypermutation and HIV neutralization potential compared to their resting memory B-cell counterparts. These findings suggest that HIV-induced B cell exhaustion is associated with a relatively inefficient memory response, which in turn, may help explain why humoral immunity in HIV-infected individuals does little to control disease progression. In preliminary analyses of lymph node B cells, we found that the highest frequency of HIV-specific B cells was within germinal centers;we are currently comparing the antibodies derived from this subset to those derived from other memory B cell subsets in the tissue and in the peripheral blood of the same individuals. Fourth, we have pursued several collaborative studies at NIAID and with other NIH institutes. One study published in Cell involved a large collaborative effort aimed at delineating the various facets of a healthy immune response, using influenza vaccination as a model and systems biology as an approach. In a second large NIAID collaborative effort published in the New England Journal of Medicine, we performed immunologic and virologic assays on B cells and T cells of two siblings with a loss of function mutation in the enzyme manosyl-oligosaccharide glucosidase, involved in N-linked protein glycosylation. We used several well-characterized viruses such as influenza and HIV, to demonstrate that glycosylation-dependent viruses do not replicate efficiently in their cells, providing a likely explanation to their reduced susceptibility to infection despite hypogammaglobulinemia. In another NIAID collaborative effort, published in the Journal of Allergy and Clinical Immunology, we characterized the plasma cells that reside in the gut (sigmoid colon) of patients with a history of colitis (chronic granulomatous disease (CGD) and Crohns disease (CD)), as well as chronically HIV-infected viremic and HIV-uninfected individuals. Gut mucosal inflammation has been described in HIV infection in the absence of a definitive cause and relatively little is known regarding the effects of such inflammatory processes on B cells. We found that CGD/CD-related colitis was associated with the presence of IgG+ plasma cells that expressed CXCR4 whereas the gut of healthy donors was primarily populated with plasma cells that expressed IgA and CCR10, as expected from the literature. In chronically HIV-infected viremic individuals, we found an intermediate profile;while there was an increased frequency of IgG+ plasma cells in the gut when compared to healthy donors, the intensity of CXCR4 remained normal. Thus, our findings demonstrate that changes in the types of plasma cells that reside in the gut reflect a spectrum of inflammatory perturbations. In two other collaborative studies, both published in the New England Journal of Medicine, our group helped delineate B-cells alterations in two disease settings, one that causes early-onset strokes due to mutations in the nucleotide deaminase ADA2 and the other, in an autoinflammatory syndrome linked to a gain in function mutation in the type I interferon gene inducing protein, STING.
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