Jason Ho

Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals

Human immunodeficiency virus (HIV) disease leads to impaired B cell and antibody responses through mechanisms that remain poorly defined. A unique memory B cell subpopulation (CD20hi/CD27lo/CD21lo) in human tonsillar tissues was recently defined by the expression of the inhibitory receptor Fc-receptor-like-4 (FCRL4). In this study, we describe a similar B cell subpopulation in the blood of HIV-viremic individuals. FCRL4 expression was increased on B cells of HIV-viremic compared with HIV-aviremic and HIV-negative individuals. It was enriched on B cells with a tissuelike memory phenotype (CD20hi/CD27−/CD21lo) when compared with B cells with a classical memory (CD27+) or naive (CD27−/CD21hi) B cell phenotype. Tissuelike memory B cells expressed patterns of homing and inhibitory receptors similar to those described for antigen-specific T cell exhaustion. The tissuelike memory B cells proliferated poorly in response to B cell stimuli, which is consistent with high-level expression of multiple inhibitory receptors. Immunoglobulin diversities and replication histories were lower in tissuelike, compared with classical, memory B cells, which is consistent with premature exhaustion. Strikingly, HIV-specific responses were enriched in these exhausted tissuelike memory B cells, whereas total immunoglobulin and influenza-specific responses were enriched in classical memory B cells. These data suggest that HIV-associated premature exhaustion of B cells may contribute to poor antibody responses against HIV in infected individuals.

Cold Urticaria, Immunodeficiency, and Autoimmunity Related to PLCG2 Deletions

BACKGROUND Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. METHODS We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. RESULTS Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. CONCLUSIONS Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).

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.

Normalization of B Cell Counts and Subpopulations after Antiretroviral Therapy in Chronic HIV Disease

BACKGROUND Untreated human immunodeficiency virus (HIV) disease leads to abnormalities in all major lymphocyte populations, including CD4(+) T cells, CD8(+) T cells, and B cells. However, little is known regarding the effect of antiretroviral therapy (ART)-induced decrease in HIV viremia on B cell numbers and subpopulations. METHODS We conducted a longitudinal study to evaluate changes in B cell numbers and subpopulations that occur during the course of 12 months of effective ART in a group of individuals with chronic HIV infection. RESULTS ART-induced decrease in HIV viremia was associated with a significant increase in B cell counts, similar to increases in CD4(+) T cell counts yet distinct from the lack of increase in CD8(+) T cells. The increase in B cell counts was accompanied by a significant decrease in the frequency of apoptosis-prone B cell subpopulations, namely mature activated and immature transitional B cells, which are overrepresented in untreated HIV disease. The increase in B cell counts was reflected by a significant increase in naive and resting memory B cells, both of which represent populations that are essential for generating adequate humoral immunity. CONCLUSIONS Normalization of B cell counts and subpopulations may help to explain the improvement in humoral immunity reported to occur after an ART-induced decrease in HIV viremia.

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.

Two overrepresented B cell populations in HIV-infected individuals undergo apoptosis by different mechanisms

Perturbations of B cells in HIV-infected individuals are associated with the overrepresentation of distinct B cell populations. Here we describe high extrinsic CD95 ligand (CD95L)-mediated apoptosis in CD10−/CD21lo mature/activated B cells that likely arise from HIV-induced immune activation. In addition, high intrinsic apoptosis was observed in CD10+ immature/transitional B cells that likely arise as a result of HIV-induced lymphopenia. CD10+ B cells expressed low levels of Bcl-2 and Bcl-xL, consistent with their high susceptibility to intrinsic apoptosis. Higher levels of activated Bax and Bak were induced in CD10+ B cells compared with CD95L-treated CD10− B cells, consistent with the greater involvement of mitochondria in intrinsic vs. extrinsic apoptosis. Of interest, both extrinsic apoptosis in CD95L-treated CD10− B cells and intrinsic apoptosis in CD10+ B cells were associated with caspase-8 activation. Our data suggest that two distinct mechanisms of apoptosis are associated with B cells of HIV-infected individuals, and both may contribute to the depletion and dysfunction of B cells in these individuals.

SIV infection of rhesus macaques results in dysfunctional T- and B-cell responses to neo and recall Leishmania major vaccination

HIV infection is characterized by immune system dysregulation, including depletion of CD4+ T cells, immune activation, and abnormal B- and T-cell responses. However, the immunologic mechanisms underlying lymphocytic dysfunctionality and whether it is restricted to immune responses against neo antigens, recall antigens, or both is unclear. Here, we immunized SIV-infected and uninfected rhesus macaques to induce immune responses against neo and recall antigens using a Leishmania major polyprotein (MML) vaccine given with poly-ICLC adjuvant. We found that vaccinated SIVuninfected animals induced high frequencies of polyfunctional MML-specific CD4+ T cells. However, in SIV-infected animals, CD4+ T-cell functionality decreased after both neo (P = .0025) and recall (P = .0080) MML vaccination. Furthermore, after SIV infection, the frequency of MML-specific antibody-secreting classic memory B cells was decreased compared with vaccinated, SIV-uninfected animals. Specifically, antibody-secreting classic memory B cells that produced IgA in response to either neo (P = .0221) or recall (P = .0356) MML vaccinations were decreased. Furthermore, we found that T-follicular helper cells, which are essential for priming B cells, are preferentially infected with SIV. These data indicate that SIV infection results in dysfunctional T-cell responses to neo and recall vaccinations, and direct SIV infection of T-follicular helper cells, both of which probably contribute to deficient B-cell responses and, presumably, susceptibility to certain opportunistic infections.

CpG Oligonucleotides Enhance Proliferative and Effector Responses of B Cells in HIV-Infected Individuals

Stimulation through TLR represents a new therapeutic approach for enhancing Ab responses to vaccination. Considering that Ab responses are decreased in HIV disease and that B cells express TLR9 and respond to TLR9 agonists, we investigated the responsiveness of B cell subpopulations from HIV-infected and uninfected individuals to the TLR9 agonist CpG oligonucleotide type B (CpG-B) in the presence and absence of BCR ligation and T cell help (CD40L). CpG-B was equally effective in stimulating the proliferation of naive B cells of HIV-infected individuals and HIV-negative individuals, and, when combined with BCR and CD40 ligation, cytokine secretion by naive B cells was also comparable in HIV-infected and uninfected individuals. In contrast, CD27+ memory/activated B cells of HIV-infected individuals with active disease were less responsive to CpG-B in terms of proliferation and cytokine secretion when compared with CD27+ B cells of HIV-negative and HIV-infected individuals whose viremia was controlled by antiretroviral therapy. These findings suggest that despite abnormalities in memory B cells of HIV-infected individuals with active disease, naive B cells of HIV-infected individuals, irrespective of disease status, can respond to TLR9 agonists and that the incorporation of such agents in vaccine formulations may enhance their Ab responses to vaccination.

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.

Role for CD21 in the establishment of an extracellular HIV reservoir in lymphoid tissues.

Follicular dendritic cells (FDC) represent a major extracellular reservoir for HIV. A better understanding of the mechanisms of virion attachment to FDC may offer new avenues for reducing viral burdens in infected individuals. We used a murine model to investigate the establishment of extracellular HIV reservoirs in lymph nodes (LN). Consistent with findings in human tissues, CD21 was required for trapping of HIV to LN cells, as evidenced by significantly reduced virion binding when mice were pretreated with a C3 ligand-blocking anti-CD21 mAb and absence of virion trapping in CD21 knockout mice. Also consistent with findings in human tissues, the majority of HIV virions were associated with the FDC-enriched fraction of LN cell preparations. Somewhat surprisingly, HIV-specific Abs were not essential for HIV binding to LN cells, indicating that seeding of the FDC reservoir may begin shortly after infection and before the development of HIV-specific Abs. Finally, the virion-displacing potential for anti-CD21 mAbs was investigated. Treatment of mice with anti-CD21 mAbs several days after injection of HIV significantly reduced HIV bound to LN cells. Our findings demonstrate a critical role for CD21 in HIV trapping by LN cells and suggest a new therapeutic avenue for reducing HIV reservoirs.