Clarisa M. Buckner

B cells in early and chronic HIV infection: evidence for preservation of immune function associated with early initiation of antiretroviral therapy

Characterization of lymphocytes including B cells during early versus chronic HIV infection is important for understanding the impact of chronic viremia on immune cell function. In this setting, we investigated B cells before and after reduction of HIV plasma viremia by antiretroviral therapy (ART). At baseline, peripheral blood B-cell counts were significantly lower in both early and chronic HIV-infected individuals compared with uninfected controls. Similar to CD4(+) but not CD8(+) T cells, B-cell numbers in both groups increased significantly after ART. At baseline, B cells of early HIV-infected individuals were composed of a higher percentage of plasmablasts and resting memory B cells compared with chronic HIV-infected individuals whose B cells were composed of a higher percentage of immature/transitional and exhausted B cells compared with their early infection counterparts. At 1 year after ART, the percentage of resting memory B cells remained higher in early compared with chronic HIV-infected individuals. This difference translated into a better functional profile in that memory B-cell responses to HIV and non-HIV antigens were superior in early- compared with chronic-treated HIV infected individuals. These findings provide new insights on B cells in HIV infection and how early initiation of ART may prevent irreversible immune system damage.

Attenuation of HIV-associated human B cell exhaustion by siRNA downregulation of inhibitory receptors

Chronic immune activation in HIV-infected individuals leads to accumulation of exhausted tissue-like memory B cells. Exhausted lymphocytes display increased expression of multiple inhibitory receptors, which may contribute to the inefficiency of HIV-specific antibody responses. Here, we show that downregulation of B cell inhibitory receptors in primary human B cells led to increased tissue-like memory B cell proliferation and responsiveness against HIV. In human B cells, siRNA knockdown of 9 known and putative B cell inhibitory receptors led to enhanced B cell receptor-mediated (BCR-mediated) proliferation of tissue-like memory but not other B cell subpopulations. The strongest effects were observed with the putative inhibitory receptors Fc receptor-like-4 (FCRL4) and sialic acid-binding Ig-like lectin 6 (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. Furthermore, the extent of FCLR4 knockdown effects on BCR-mediated proliferation varied depending on the costimulatory ligand, suggesting that inhibitory receptors may engage specific pathways in inhibiting B cell proliferation. These findings on HIV-associated B cell exhaustion define potential targets for reversing the deleterious effect of inhibitory receptors on immune responses against persistent viral infections.

Increased Mucosal Transmission but Not Enhanced Pathogenicity of the CCR5-Tropic, Simian AIDS-Inducing Simian/Human Immunodeficiency Virus SHIVSF162P3 Maps to Envelope gp120

ABSTRACT Through rapid serial transfer in vivo, the chimeric CCR5-tropic simian/human immunodeficiency virus SHIVSF162 evolved from a virus that is nonpathogenic and poorly transmissible across the vaginal mucosa to a variant that still maintains CCR5 usage but which is now pathogenic and establishes intravaginal infection efficiently. To determine whether envelope glycoprotein gp120 is responsible for increased pathogenesis and transmissibility of the variant SHIVSF162P3, we cloned and sequenced the dominant envelope gene (encoding P3 gp120) and characterized its functions in vitro. Chimeric SHIVSF162 virus expressing P3 gp120 of the pathogenic variant, designated SHIVSF162PC, was also constructed and assessed for its pathogenicity and mucosal transmissibility in vivo. We found that, compared to wild-type SHIVSF162 gp120, P3 gp120 conferred in vitro neutralization resistance and increased entry efficiency of the virus but was compromised in its fusion-inducing capacity. In vivo, SHIVSF162PC infected two of two and two of three rhesus macaques by the intravenous and intravaginal routes, respectively. Nevertheless, although peak viremia reached 106 to 107 RNA copies per ml of plasma in some infected animals and was associated with depletion of gut-associated CD4+ lymphocytes, none of the animals maintained a viral set point that would be predictive of progression to disease. Together, the data from this study suggest a lack of correlation between entry efficiency and cytopathic properties of envelope glycoproteins with viral pathogenicity. Furthermore, whereas env gp120 contains the determinant for enhanced mucosal transmissibility of SHIVSF162P3, the determinant(s) of its increased virulence may require additional sequence changes in env gp41 and/or maps to other viral genes.

Abnormal B cell memory subsets dominate HIV-specific responses in infected individuals

Recently, several neutralizing anti-HIV antibodies have been isolated from memory B cells of HIV-infected individuals. Despite extensive evidence of B cell dysfunction in HIV disease, little is known about the cells from which these rare HIV-specific antibodies originate. Accordingly, we used HIV envelope gp140 and CD4 or coreceptor (CoR) binding site (bs) mutant probes to evaluate HIV-specific responses in peripheral blood B cells of HIV-infected individuals at various stages of infection. In contrast to non-HIV responses, HIV-specific responses against gp140 were enriched within abnormal B cells, namely activated and exhausted memory subsets, which are largely absent in the blood of uninfected individuals. Responses against the CoRbs, which is a poorly neutralizing epitope, arose early, whereas those against the well-characterized neutralizing epitope CD4bs were delayed and infrequent. Enrichment of the HIV-specific response within resting memory B cells, the predominant subset in uninfected individuals, did occur in certain infected individuals who maintained low levels of plasma viremia and immune activation with or without antiretroviral therapy. The distribution of HIV-specific responses among memory B cell subsets was corroborated by transcriptional analyses. Taken together, our findings provide valuable insight into virus-specific B cell responses in HIV infection and demonstrate that memory B cell abnormalities may contribute to the ineffectiveness of the antibody response in infected individuals.

Characterization of Plasmablasts in the Blood of HIV-Infected Viremic Individuals: Evidence for Nonspecific Immune Activation

ABSTRACT Terminal differentiation of B cells and hypergammaglobulinemia are hallmarks of B-cell hyperactivity in HIV disease. Plasmablasts are terminally differentiating B cells that circulate transiently in the blood following infection or vaccination; however, in HIV infection, they arise early and are maintained at abnormally high levels in viremic individuals. Here we show that only a small fraction of plasmablasts in the blood of viremic individuals is HIV specific. Assessment of plasmablast immunoglobulin isotype distribution revealed increased IgG+ plasmablasts in early and most prominently during chronic HIV viremia, contrasting with a predominantly IgA+ plasmablast profile in HIV-negative individuals or in aviremic HIV-infected individuals on treatment. Of note, IgG is the predominant immunoglobulin isotype of plasmablasts that arise transiently in the blood following parenteral immunization. Serum immunoglobulin levels were also elevated in HIV-infected viremic individuals, especially IgG, and correlated with levels of IgG+ plasmablasts. Several soluble factors associated with immune activation were also increased in the sera of HIV-infected individuals, especially in viremic individuals, and correlated with serum immunoglobulin levels, particularly IgG. Thus, our data suggest that while plasmablasts in the blood may contribute to the HIV-specific immune response, the majority of these cells are not HIV specific and arise early, likely from indirect immune-activating effects of HIV replication, and reflect over time the effects of chronic antigenic stimulation. Such B-cell dysregulation may help explain why the antibody response is inadequate in HIV-infected individuals, even during early infection.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2–responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.

Humans with chronic granulomatous disease maintain humoral immunologic memory despite low frequencies of circulating memory B cells.

CD27(+) memory B cells are reduced in the blood of patients with chronic granulomatous disease (CGD) for reasons and consequences that remain unclear. Here we confirm not only decreased CD27(+) but also IgG(+) B cells in the blood of CGD patients compared with healthy donors (HDs). However, among IgG(+) B cells, the ratio of CD27(-) to CD27(+) was significantly higher in CGD patients compared with HDs. Similar to conventional memory B cells, CD27(-)IgG(+) B cells of CGD patients expressed activation markers and had undergone somatic hypermutation, albeit at levels lower than their CD27(+) counterparts. Functional analyses revealed slight reductions in frequencies of total IgG but not influenza-specific memory B-cell responses, as measured by Elispot in CGD patients compared with HDs. Serum IgG levels and influenza-specific antibodies were also normal in these CGD patients. Finally, we provide evidence that influenza-specific memory B cells can be present within the CD27(-)IgG(+) B-cell compartment. Together, these findings show that, despite reduced circulating CD27(+) memory B cells, CGD patients maintain an intact humoral immunologic memory, with potential contribution from CD27(-) B cells.

HIV-tat alters Connexin43 expression and trafficking in human astrocytes: role in NeuroAIDS

BackgroundHIV-associated neurocognitive disorders (HAND) are a major complication in at least half of the infected population despite effective antiretroviral treatment and immune reconstitution. HIV-associated CNS damage is not correlated with active viral replication but instead is associated with mechanisms that regulate inflammation and neuronal compromise. Our data indicate that one of these mechanisms is mediated by gap junction channels and/or hemichannels. Normally, gap junction channels shutdown under inflammatory conditions, including viral diseases. However, HIV infection upregulates Connexin43 (Cx43) expression and maintains gap junctional communication by unknown mechanism(s).MethodsHuman primary astrocytes were exposed to several HIV proteins as well as to HIV, and expression and function of Connexin43- and Connexin30-containing channels were determined by western blot, immunofluorescence, microinjection of a fluorescent tracer and chromatin immunoprecipitation (ChIP).ResultsHere, we demonstrate that HIV infection increases Cx43 expression in vivo. HIV-tat, the transactivator of the virus, and no other HIV proteins tested, increases Cx43 expression and maintains functional gap junctional communication in human astrocytes. Cx43 upregulation is mediated by binding of the HIV-tat protein to the Cx43 promoter, but not to the Cx30 promoter, resulting in increased Cx43 messenger RNA (mRNA) and protein as well as gap junctional communication.ConclusionsWe propose that HIV-tat contributes to the spread of intracellular toxic signals generated in a few HIV-infected cells into surrounding uninfected cells by upregulating gap junctional communication. In the current antiretroviral era, where HIV replication is often completely suppressed, viral factors such as HIV-tat are still produced and released from infected cells. Thus, blocking the effects of HIV-tat could result in new strategies to reduce the damaging consequences of HIV infection of the CNS.

Maintenance of HIV-Specific Memory B-Cell Responses in Elite Controllers Despite Low Viral Burdens.

Human immunodeficiency virus (HIV)-specific B-cell responses in infected individuals are maintained by active HIV replication. Suppression of viremia by antiretroviral therapy (ART) leads to quantitative and qualitative changes that remain unclear. Accordingly, B-cell responses were investigated in elite controllers (ECs), who maintain undetectable HIV levels without ART, and in individuals whose viremia was suppressed by ART. Despite a higher HIV burden in the ART group, compared with the EC group, frequencies of HIV-specific B cells were higher in the EC group, compared with those in the ART group. However, the initiation of ART in several ECs was associated with reduced frequencies of HIV-specific B cells, suggesting that responses are at least in part sustained by HIV replication. Furthermore, B-cell responses to tetanus toxin but not influenza hemagglutinin in the ART group were lower than those in the EC group. Thus, the superior HIV-specific humoral response in ECs versus ART-treated individuals is likely due to a more intact humoral immune response in ECs and/or distinct responses to residual HIV replication.

Bone Marrow Plasma Cells Are a Primary Source of Serum HIV-1–Specific Antibodies in Chronically Infected Individuals

Several potent and broadly neutralizing Abs to HIV-1 have been isolated recently from peripheral blood B cells of infected individuals, based on prescreening of Ab activity in the serum. However, little is known regarding the cells that make the Abs that circulate in the blood. Accordingly, we investigated the most likely source, the bone marrow, of chronically HIV-1–infected individuals who were not receiving antiretroviral therapy. Increased frequencies of plasma cells, as well as B cell precursors, namely preB-I and preB-II, and decreased frequencies of mature B cells were observed in bone marrow aspirates of these individuals compared with HIV-negative counterparts. Increased frequencies of bone marrow plasma cells are consistent with known hallmarks of HIV-1 infection, namely hypergammaglobulinemia and increased frequencies of peripheral blood plasmablasts. Levels of HIV-1 envelope (Env)-binding and HIV-1–neutralizing Abs were measured in serum, and corresponding frequencies of Ab-secreting or Env-binding cells were measured in the blood (plasmablasts and memory B cells) and in the bone marrow (plasma cells). A strong correlation was observed between serum HIV-1–specific Abs and Env-specific bone marrow–derived plasma cells, but not circulating plasmablasts or memory B cells. These findings demonstrate that, despite HIV-1–induced phenotypic and functional B cell dysregulation in the peripheral blood and secondary lymphoid tissues, bone marrow plasma cells remain a primary source for circulating HIV-1–specific Abs in HIV-1–infected individuals.