In 2010, we pursued studies on B cells in HIV-infected and healthy, HIV-uninfected individuals by focusing on 1) B-cell responses to 2009 pandemic H1N1 influenza vaccine;2) changes in phenotypic and functional B-cell properties that occur following initiation of antiretroviral therapy (ART) in HIV-infected individuals;and 3) mechanisms of HIV-associated B-cell exhaustion in HIV-viremic 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 have been submitted to the journal AIDS. In addition to this study, our group has been a major collaborator on a 200-participant clinical protocol undertaken by the Center for Human Immunology that is investigating the innate and adaptive immune responses in healthy individuals following vaccination against both the 2009 pandemic H1N1 and seasonal influenza strains. Our group was responsible for measuring changes in B cells at both the functional and phenotypic levels following vaccination. We found robust early responses to both vaccines followed by the development of memory B-cell responses that were stronger against the seasonal influenza viruses compared to the 2009 pandemic H1N1 influenza strain. Our data are currently being compared with other study parameters including neutralizing antibody titers, modulation of gene expression measured by DNA microarray, modulation of cytokines measured by multiplex and intracellular flow cytometry, and phenotypic changes of other lymphocytic and monocytic cell populations assessed by flow cytometry. In the second study, recently accepted for publication in Blood, we compared phenotypic and functional changes in B cells that occur in HIV-infected individuals who initiate antiretroviral therapy (ART) during the acute or chronic stages of infection. Both groups of HIV-infected individuals had significantly fewer B cells circulating in the blood compared to healthy, HIV-uninfected individuals, and this deficit was reversed and normalized by ART. Furthermore, untreated HIV-infected individuals in the early stage of infection had lower frequencies of immature-transitional and exhausted B cells when compared with untreated HIV-infected individuals in the chronic stage of infection, consistent with these two B-cell subpopulations being associated with advancing disease and chronic viremia respectively. In contrast, early HIV infection was associated with a higher frequency of plasmablasts compared to chronic HIV infection, likely a reflection of the high level of activation and terminal differentiation associated with the rapid dynamics of early infection. After one year of ART, almost all of these B-cell abnormalities were reversed or normalized, except for the resting memory B cells, which were only normalized in HIV-infected individuals treated early in the course of infection. In addition, this loss of memory B cells in infected individuals treated during the chronic stage of infection was associated with lower memory B-cell responses to influenza than those found in infected individuals treated early in the course of infection. There were also indications that early initiation of ART helped preserve B-cell responses against HIV. In the third study, we evaluated the role of multiple inhibitory receptors in HIV-associated B-cell exhaustion that we 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. Downregulation of FCRL4 and Siglec-6 also led to increased secretion of chemokines and cytokines associated with B-cell function. The absence of known ligands for FCRL4 and Siglec-6 suggests these receptors may regulate BCR signaling through their own constitutive signaling. Enhanced BCR-mediated proliferation of B cells following downregulation of FCRL4 was stronger when the costimulus was a Toll-like-receptor-9 (TLR9) compared to CD40 agonist, suggesting that FCRL4 may also regulate TLR9 signaling alone or through its effect on 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.
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