In 2018, we pursued studies that encompass three major themes. 1) role of IgG3 in regulating tissue-like memory B cells in HIV infection; 2) identification of a unique memory B-cell population in lymph nodes of HIV-infected individuals with persistent HIV viremia; and 3) studies of non-B cells in HIV disease and B cells in non-HIV diseases. First, in a three-year study recently published in Nature Immunology, we identified IgG3 as a regulator of a population of nonconventional memory B cells, referred to as tissue-like memory B cells, which we first identified in 2008 in HIV-viremic individuals and which arise in response to the immune-activating effects of persistent HIV replication. In the current study, we investigated peripheral blood B cells in a cohort of 108 HIV-infected individuals who were divided into three groups based on HIV disease status: a) early HIV viremia; b) chronic HIV viremia; and c) HIV aviremia (resulting from antiretroviral therapy). We also included HIV-negative healthy controls. We discovered B cells that expressed IgM and bound IgG3 in vivo; these IgG3+IgM+ B cells were almost exclusively found in individuals with chronic HIV viremia and largely absent in those with early HIV viremia, HIV aviremia and in HIV-negative controls. The presence of IgG3+IgM+ B cells was also restricted to certain B-cell populations, enriched on nave and tissue-like memory B cells, the latter previously shown to be over-expressed in chronic HIV viremia. This cell surface bound form of IgG3 was found to co-localize and interact with IgM that was expressed on the cell surface as part of the B-cell receptor (BCR). The interaction with IgG3 restricted the response of these B cells to stimulation through the IgM-BCR, especially among tissue-like memory B cells. The restriction was found to be associated with other cell-bound or expressed proteins, including complement protein C1q and C-reactive protein (CRP), as well as the inhibitory receptor CD32b. The IgG3 that bound to IgM-expressing B cells was also present in the serum of individuals with moderate to high-intensity IgG3+IgM+ B cells. This serum-derived IgG3 was transferable to B cells of healthy controls and involved the same proteins associated with patient-derived IgG3+IgM+ B cells, namely C1q, CRP and CD32b. Collectively, these findings demonstrated a novel regulatory function for IgG3 that may help explain the unresponsiveness of tissue-like memory B cells to stimulation, an observation made by us several years ago when we first identified these cells in HIV-infected individuals, and that have since been described in numerous infectious and non-infectious diseases associated with chronic immune activation. Second, in a large collaborative effort that has finally come to fruition after several years of collecting tissue specimens and developing new imaging techniques, we have almost completed a study identifying a new population of memory B cells in lymph nodes of HIV-infected individuals. We have a longstanding interest in investigating B-cell responses to HIV in infected individuals and how these responses are perturbed in the setting of chronic HIV viremia, both in lymphoid tissues and in the peripheral blood. Several years ago, we identified a unique population of memory B cells called tissue-memory B cells that circulate in the peripheral blood at frequencies that are increased in chronic HIV viremia. These B cells show signs HIV-associated exhaustion, including increased expression of inhibitory receptors and reduced capacity to respond to stimulation, resulting in poor responses to HIV, as measured by levels of affinity maturation and capacity to neutralize the virus by antibodies cloned from these memory B cells. We have long hypothesized that these tissue-like memory B cells are the result of dysregulated responses that develop in the setting of HIV-associated lymphoid tissue hyperplasia. Over the past three years, we have developed and implemented a three-pronged approach to identify and characterize B cells in lymph nodes that are the likely precursors of tissue-like memory B cells that circulate in the peripheral blood. First, we used unique markers associated with tissue-like memory B cells, namely CD19hiCD11c+T-bet+ to identify lymph node B cells with similar profiles. These were first identified by conventional flow cytometry on viable B cells isolated from lymph nodes and then identified in corresponding paraffin-embedded tissue sections using multi-parameter confocal microscopy and histo-cytometry. CD19hiCD11c+T-bet+ B cells were clearly identified in non-germinal center areas of lymph node follicles obtained from chronically HIV-viremic individuals. These cells were positioned at the perimeter but largely excluded from germinal centers. Similar to tissue-like memory B cells in the peripheral blood, their lymph node CD19hiCD11c+T-bet+ counterparts were enriched with HIV-specific B cells. Second, we used low input cell RNA sequencing (RNA-seq) to generate gene expression profiles for various B-cell populations that were sorted from viable mononuclear cells isolated from lymph nodes of HIV-viremic individuals and HIV-negative controls. The analyses revealed that HIV-specific B cells were clearly segregated from other memory B-cell populations and were enriched with gene-expression profiles associated with germinal center and T-bet signatures, the latter confirming the confocal imaging and flow cytometric findings and the former confirmed by phenotypic and functional attributes. Third, we performed bulk and single-cell sorting of B cells isolated from lymph nodes of HIV-viremic individuals to investigate BCR properties associated with affinity maturation and to identify clonal lineages within and between B-cell populations. Similar to features previously reported for tissue-like memory B cells in the peripheral blood, we observed decreased levels of somatic hypermutation among lymph node-derived CD19hi memory B cells and HIV-specific memory B cells enriched within the CD19hi population. We also found phylogenetic links between CD19hi memory B cells and germinal center B cells. Collectively, these findings identify a lymphoid tissue source of non-conventional T-bet-expressing memory B cells that arise in the setting of chronic immune activation, with features related to germinal center B cells, but lacking the CD4+ T-cell help associated with germinal center reactions that is needed to mount an effective antibody response. Under the third major theme, we have contributed to several collaborative studies on HIV and other diseases of the immune system with colleagues in the LIR, as well as with other laboratories at NIAID. In HIV-related studies, we collaborated with our colleagues at the NIAID Vaccine Research Center (VRC) with imaging analyses of lymph nodes biopsied from our HIV infection cohort; we used our B-cell expertise to provide other VRC colleagues with analyses of the role of the Fc receptor CD32a in HIV latency; and, we assisted NIAID colleagues in establishing a role for the integrin receptor CD62L in HIV replication. Finally, we have continued to assist several NIH colleagues in characterizing B cells in different settings, including most recently following the administration of glucocorticoids.
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