In 2011, we pursued studies on B cells in HIV-infected and HIV-uninfected individuals by completing studies on 1) B-cell responses to 2009 pandemic H1N1 influenza vaccine;and 2) mechanisms of HIV-associated B-cell exhaustion in HIV-viremic individuals;as well as pursuing the investigation of 3) hallmarks of B-cell hyperactivity both in the peripheral blood and mucosal tissues of HIV-infected individuals, healthy individuals, and individuals with primary immunodeficiencies involving mucosal inflammation;and 4) the effects of B-cell exhaustion on the antibody response against HIV in infected individuals. In the first study, we investigated the B cell responses in individuals vaccinated with the 2009 pandemic H1N1 influenza vaccine in the absence or presence of an oil-in-water adjuvant (AS03). One group of HIV-infected, aviremic individuals received a low dose of the vaccine in the presence of the AS03 adjuvant, while another group of HIV-infected individuals (matched to the first group in age, HIV viral load and CD4+ T-cell count) as well as a group of HIV-uninfected individuals (matched for age) received a standard dose of non-adjuvanted vaccine. Our findings indicate that the memory B-cell response against the 2009 pandemic H1N1 influenza virus was significantly higher in the HIV-infected group that received the adjuvanted vaccine compared to the two groups that received the same vaccine without adjuvant. Antibody titers against 2009 pandemic H1N1 influenza did not differ significantly among the three groups. The findings of this study were published in the journal AIDS. In the second study, recently published in The Journal of Clinical Investigation, we evaluated the role of multiple inhibitory receptors in HIV-associated B-cell exhaustion that we first described in 2008 in The Journal of Experimental Medicine. Given that B-cell exhaustion is a property of tissue-like memory B cells, an aberrant subpopulation of B cells that arises in the setting of persistent HIV viremia characterized by the overexpression of multiple inhibitory receptors, we focused on this subpopulation. We used gene-specific small interfering RNA (siRNA) molecules to downregulate the expression of nine known or putative B-cell inhibitory receptors and then evaluated the effect of this targeted downregulation on B-cell proliferation and effector function. We found that downregulation of each of the nine inhibitory receptors significantly increased the proliferation of tissue-like memory but not other B-cell subpopulations. The strongest effects were observed after downregulating putative inhibitory receptors Fc-receptor-like-4 (FCRL4) and sialic acid-recognizing Ig-superfamily lectin (Siglec)-6. Inhibitory receptor downregulation also led to increased levels of HIV-specific antibody-secreting cells and B cell-associated chemokines and cytokines. The absence of known ligands for FCRL4 and Siglec-6 suggests these receptors may regulate BCR signaling through their own constitutive or tonic signaling. In support for this notion, we found that inhibiting the BCR signaling pathway by coligation with a strong known antagonist, namely the inhibitory Fc receptor CD32b, reversed the gain in function that occurred following the downregulation of FCRL4. In addition, B-cell costimuli, including Toll-like-receptor-9 (TLR9) and CD40 agonist, had differential effects on BCR-mediated enhancement of proliferation following downregulation of FCRL4, suggesting that FCRL4 may affect distinct pathways associated with BCR signaling. These findings provide insights into HIV-associated B-cell exhaustion and potential targets for reversing the unresponsiveness of B and T cells in the setting of persistent viral infections. In a third ongoing study, we focused on terminally differentiating B cells, the frequency of which is increased in the blood of HIV-viremic individuals and of healthy individuals transiently following vaccination. In our previous study published in 2010 in Blood, we described a higher frequency of terminally differentiating 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 the current study, we extend these observations by showing that in addition to differences in frequencies of terminally differentiating B cells, there are also distinctions in the class of immunoglobulins secreted by these cells. Whereas terminally differentiating B cells in the blood of chronic HIV-viremic individuals primarily express IgG, similar to what is found in the blood shortly and transiently after vaccination, IgA remains the primary isotype of terminally differentiating B cells of early HIV-viremic individuals, similar to what is found in the blood of healthy donors at steady state and in HIV-aviremic individuals. Given that terminally differentiating B cells in the blood of healthy donors at steady state are thought to originate from the intestinal mucosa and that this lymphoid tissue is perturbed in HIV-infected individuals, we investigated whether the gut associated lymphoid tissue (GALT) was the source of IgG-expressing terminally differentiating B cells found in the blood of chronic HV-viremic individuals. The answer was no;IgA remained the major isotype in the GALT of these individuals despite IgG being largely responsible for hypergammaglobulinemia and the predominant isotype of terminally differentiating B cells in their blood. This is in contrast to findings in a separate study where we investigated GALT B cells of patients with severe intestinal inflammation associated with a primary immunodeficiency called chronic granulomatous disease (CGD), and found IgG to be the predominant isotype of terminally differentiated B cells. Given that IgA has anti-inflammatory properties, these latter findings may help explain the pathogenesis of CGD. However, for chronic HIV infection, IgG does not appear to be a major contributor to HIV-induced mucosal inflammation and further, our findings suggest that the gut is not the source of IgG hypergammaglobulinemia and terminally differentiating B cells circulating in the blood of chronic HIV-viremic individuals. In a fourth ongoing study, single-cell cloning has been used to express and compare properties of IgG antibodies derived from two memory B cell subsets found in the blood of chronic HIV-viremic individuals: the abnormal tissue-like memory B cells responsible for HIV-associated 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, which represents the majority of HIV-specific B cells in the peripheral blood, displayed lower levels of somatic hypermutation and higher levels of polyreactivity and 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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