In 2017, we pursued studies that can be broken down into 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 lymphoid tissues of HIV-infected individuals with chronic HIV viremia; and 3) studies of B cells in non-HIV immunodeficiencies. First, in a two-year study that is almost completed, with a manuscript in final stages of preparation, we identified IgG3 as a regulator of a population of exhausted B cells that we first identified in 2008 in chronically infected HIV-viremic individuals, namely tissue-like memory B cells. 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: early HIV viremia, chronic HIV viremia and HIV aviremia (resulting from antiretroviral therapy). We also included HIV-negative healthy controls. We discovered the presence of B cells in HIV-infected individuals that expressed IgM and bound IgG3 in vivo; these IgG3+IgM+ B cells were almost exclusively restricted to individuals with chronic HIV viremia and largely absent in 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 also found to co-localize with IgM on the surface of B cells, especially tissue-like memory B cells and restricted the response of these B cells to stimulation through the B-cell receptor (BCR). While the origin and form of the IgG3 remains unclear, it was present in the serum of individuals with high- but not low-intensity IgG3+IgM+ B cells; it could be transferred to B cells of healthy controls; and, it had properties that suggested it was in an aggregated form similar to that of immune complexes, thus accounting for its ability to bind B cells. Additional findings suggested that the aggregates contained the complement protein C1q and that these IgG3 complexes bound to B cells through the inhibitory receptor CD32b. Collectively, these findings suggest that we have identified a novel regulatory function for IgG3 that may help explain the unresponsiveness of tissue-like memory B cells to stimulation, an observation we made several years ago when we first identified these cells in HIV-infected individuals, B cells that have since been described in numerous infectious and non-infectious disease settings of chronic immune activation. Second, in a large multi-faceted effort that has finally come to fruition after several years of gathering rare lymphoid tissue specimens and developing new imaging techniques, we are in the final phases of completed a large comprehensive study describing a new population of memory B cells in lymphoid tissues. We have had a longstanding interest in investigating B-cell responses to HIV in infected individuals and how these are perturbed in the setting of chronic HIV viremia, both in lymphoid tissues and 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 by 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-prong approach to identify and characterize B cells in lymph nodes that are the precursors of tissue-like memory B cells found in the peripheral blood. First, we used unique markers associated with tissue-like memory B cells, namely CD20hiCD11c+T-bet+ to identify lymph node B cells with similar profiles. These were first identified by flow cytometry on viable B cells recovered from lymph nodes and then identified in corresponding paraffin-embedded tissue sections by multi-parameter confocal microscopy and histo-cytometry. CD20hiCD11c+T-bet+ B cells were clearly identified in the extra-follicular areas of lymph nodes of chronically HIV-viremic individuals, positioned at the perimeter but not within germinal centers. Similar to tissue-like memory B cells in the peripheral blood, their lymph node CD20hiCD11c+T-bet+ counterparts were enriched with HIV-specific B cells. Second, we used 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 and HIV-negative controls. The analyses thus far show a clear segregation of populations and enrichment of a T-bet-associated signature among HIV-specific memory but not germinal center B cells, consistent with the confocal imaging and flow cytometric findings. Third, we used single-cell sorting of HIV-specific B cells isolated from lymph nodes of HIV-viremic individuals to clone monoclonal antibodies (mAbs) corresponding to their BCR specificity. Similar to the observations we reported last year for tissue-like memory B cells, the HIV-specific mAbs recovered from CD20hiCD11c+T-bet+ lymph node B cells had lower levels of somatic hypermutation and lower capacity to neutralize HIV compared to memory and GC B cells that did not express these markers. Collectively, we believe that we have identified the lymphoid tissue precursors of tissue-like memory B cells that circulate in the blood of chronically HIV-viremic individuals. Third, we have contributed to several collaborative studies on HIV and other diseases of the immune system with colleagues in the LIR, as well as other laboratories at NIAID, other NIH institutes, and abroad. In HIV-related studies, we helped our colleagues at the NIAID Vaccine Research Center with imaging analyses of lymph nodes biopsied from our HIV infection cohort; we used our B-cell expertise to help LIR colleagues with interpretation of a clinical trial testing the effect on the potent HIV-neutralizing antibody VRC01 to prevent viral rebound following treatment interruption in HIV-infected individuals whose viremia had been suppressed by antiretroviral therapy for several years; and, we used our B-cell expertise in HIV infection to help collaborators at the University of Pennsylvania to identify and characterize T-bet expressing B cells in chronic infections such as HIV. Finally, we have continued to assist NIH colleagues in characterizing B cells of patients with X-linked chronic granulomatous disease and patients with acute myeloid leukemia.
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