In 2012, we investigated 1) B-cell responses to influenza vaccine;2) dysregulation of B cells in gut mucosal tissues;3) terminally differentiating B cells in the blood of HIV-infected individuals at different stages of disease;and 4) HIV-specific B-cell responses and antibodies obtained from single-cell analyses of B cells isolated from HIV-infected individuals. In the first study, we investigated influenza-specific memory B-cell responses in individuals with chronic granulomatous disease (CGD), an inherited immunodeficiency that is associated with low frequencies of CD27+/CD21hi classic memory B cells in the blood, similar to that observed in HIV-infected individuals. However, in contrast to reduced influenza-specific memory B-cell responses observed in HIV-infected individuals, the responses in the CGD group were not significantly different compared to age-matched healthy controls. In HIV disease, we have described two subsets of aberrant memory B cells, namely exhausted (CD27-/CD21lo) and activated (CD27+/CD21lo) memory B cells that are present at very low levels in HIV-uninfected individuals. While the frequencies of these two subsets were not elevated in the blood of CGD patients, we found an increased frequency of CD27-/CD21hi B cells that expressed IgG, the Ig isotype responsible for the majority of influenza-specific B cells. We also found evidence that these unconventional IgG+/CD27-/CD21hi memory B cells contribute to the influenza-specific response and that this may help explain the observations that influenza-specific B-cell and antibody responses are normal in CGD patients despite reduced numbers of conventional CD27+ memory B cells in their blood. These findings further demonstrate that different disease settings may induce alterations in the memory B-cell compartment that may (CGD with unconventional resting memory B cells) or may not (HIV with exhausted and activated memory B cells) be beneficial to the host. A manuscript of this study is being revised for resubmission to Blood. In the second study, completed with a manuscript in preparation for submission to Mucosal Immunology, we investigated B cells in the gut (sigmoid colon) of patients with a history of colitis (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;however, little is known relative to effects of such inflammatory processes on B cells. We found that CGD/CD-related colitis was associated with the presence of IgG+ plasma cells (PC) that expressed CXCR4 whereas the gut of healthy donors was primarily populated with (PC) that expressed IgA and CCR10, the latter being consistent with reports in the literature. In chronically HIV-infected viremic individuals, we found an intermediate profile;while there is a predominance of IgA+/CCR10+ PC in their gut mucosa, we observed a higher frequency of IgG+/CXCR4+ PC when compared to healthy donors yet lower when compared to CGD/CD patients with colitis. Given that pathology reports of the tissue samples also suggested an intermediate profile of inflammation in the HIV-viremic individuals, more inflammation compared to healthy donors but less compared to CGD patients, we concluded that the frequency of IgG+/CXCR4+ PC in the gut mucosa represents a good predictor of inflammation in this tissue compartment. Whether these cells are a driving force or are a consequence of gut inflammation remains to be determined. In the third study, completed with a manuscript in preparation for submission to the Journal of Virology, we investigated terminally differentiating (TD) B cells in the blood of HIV-infected and HIV-uninfected individuals. In a previous study, we described a higher frequency of TD B cells in the blood of early compared to chronic HIV-infected untreated individuals, and frequencies among both these groups were higher compared to HIV-uninfected and ART-treated infected individuals. In this study, we further characterized these cells in an effort to understand their origin and association with HIV pathogenesis. TD B cells represent less than 1% of blood B cells in healthy donors and, with the primary isotype being IgA, these circulating TD B cells are thought to originate from mucosal sites. Following parenteral immunization there is a short-lived burst of TD B cells in the blood, and in this case the primary isotype is IgG. These IgG+ TD B cells are thought to originate from draining lymph nodes. In early HIV infection, the TD B cells present at high frequencies in untreated individuals were primarily IgA, although IgG frequencies were higher than in HIV-uninfected individuals. In contrast to other acute viral infections, the frequency of virus-specific TD B cells in the blood of HIV-infected individuals was very low, less than 1% of total TD B cells. In chronically HIV-infected viremic individuals, a higher than normal frequency of TD B cells was maintained and these were primarily of the IgG isotype, consistent with high serum IgG also observed in this group. The percentage of HIV-specific B cells did not increase during the chronic phase of HIV viremia, suggesting as others have, that TD B cells in the blood of HIV-infected viremic individuals are primarily a consequence of polyclonal activation of B cells induced by ongoing viral replication. In this regard, we also found correlations between IgG levels and other soluble factors associated with HIV-induced immune activation. Thus, while the increasing frequencies of IgG+ TD B cells in the blood of HIV-viremic individuals may reflect gut perturbations (2nd study), they are also likely to be a reflection of systemic immune activation and possibly involvement of other lymphoid tissues, as suggested by the similarities with IgG+ TD B cells in the blood following parenteral immunization. In a fourth study, new approaches involving flow cytometry, single-cell sorting, and HIV-specific probes are being employed to investigate HIV-specific B cells in the blood of HIV-infected individuals at various stages of disease. Markers that define B-cell subsets and flow cytometric analyses are being used to evaluate HIV-specific B-cell responses and determine whether HIV-specific B cells show evidence of incremental maturation involving multiple rounds of proliferation and selection of higher affinity clones. Preliminary findings suggest that HIV-specific B cells arise early after infection, decrease with ART and are better maintained in early compared to chronic-treated individuals. Unexpectedly, we found that B cells that recognize the CD4 binding site (CD4bs) of the HIV envelope also appeared early after infection and we found little evidence at the population level that these cells had undergone several rounds of proliferation and selection. Of note, the CD4bs-specific response was enriched within the tissue-like memory B-cell compartment of HIV-viremic individuals. These cells are largely absent in healthy donors, suggesting that a vaccine strategy directed at this site may prove difficult. Finally, we used single-cell cloning to express IgG antibodies derived from two memory B cell subsets found in the blood of chronically HIV-infected viremic individuals: the abnormal tissue-like memory B cells associated with B-cell exhaustion and their normal resting memory B cell counterparts. Preliminary indications are that IgG antibodies derived from the tissue-like memory B-cell compartment displayed less somatic hypermutation and higher poly/autoreactivity than those derived from the resting memory B-cell subset. These findings suggest that HIV-induced B cell exhaustion is associated with the generation of aberrant antibodies that may help explain the ineffectiveness of the antibody response in controlling HIV viremia.
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