Evidence of Early B-Cell Dysregulation in Simian Immunodeficiency Virus Infection: Rapid Depletion of Na?ve and Memory B-Cell Subsets with Delayed Reconstitution of the Na?ve B-Cell Population

Despite eliciting a robust antibody response in humans, several studies in human immunodeficiency virus (HIV)-infected patients have demonstrated the presence of B-cell deficiencies during the chronic stage of infection. While several explanations for the HIV-induced B-cell deficit have been proposed, a clear mechanistic understanding of this loss of B-cell functionality is not known. This study utilizes simian immunodeficiency virus (SIV) infection of rhesus macaques to assess B-cell population dynamics beginning at the acute phase and continuing through the chronic phase of infection. Flow cytometric assessment demonstrated a significant early depletion of both naïve and memory B-cell subsets in the peripheral blood, with differential kinetics for recovery of these populations. Furthermore, the altered numbers of naïve and memory B-cell subsets in these animals corresponded with increased B-cell activation and altered proliferation profiles during the acute phase of infection. Finally, all animals produced high titers of antibody, demonstrating that the measurement of virus-specific antibody responses was not an accurate reflection of alterations in the B-cell compartment. These data indicate that dynamic B-cell population changes in SIV-infected macaques arise very early after infection at the precise time when an effective adaptive immune response is needed.Effective B-cell responses result in the generation of memory B-cell populations which are able to proliferate and produce antibodies that can control primary and secondary insults by microbial pathogens (2). Impaired maturation and timing of B-cell-mediated immune responses result in the production of ineffective antibodies, which are unable to control infection and may result in the persistence of the pathogen (36). Although human immunodeficiency virus (HIV) infection generally elicits high-titer antibodies, virus-specific titers do not correlate with delayed clinical progression, suggesting that antibodies produced during HIV infection are not sufficient to provide long-term viral control (6). Ineffective antibody production in the context of HIV infection could be a result of numerous T-cell and B-cell abnormalities induced either directly or indirectly through infection. B-cell perturbations, characterized during chronic infection, include hypergammaglobulinemia (11, 31), a diminished in vitro response to mitogenic stimulation (10, 37), diminished antibody responses to vaccination (15, 23), and loss of memory B-cell subsets (3, 10, 37). It is highly likely that these B-cell abnormalities are linked with the inability of HIV-infected individuals to form effective antibody responses to HIV and opportunistic pathogens.B-cell perturbations during acute HIV infection may lead to dysfunctions observed during chronic infection. Despite numerous reports that hypothesized that B-cell phenotypic and functional abnormalities arise due to the effects of chronic infection, a limited number of acute infection studies have provided evidence that B-cell dysfunctions may be initiated much earlier. Studies by De Milito et al. and others have reported a decrease in CD27⁺ B cells associated with chronic HIV infection (3, 4, 10-12, 15, 30, 31, 36-38, 40). The reduction of this population may explain the diminished antibody responses to non-HIV antigens present in HIV-infected individuals. However, the mechanism for this loss of memory B cells during chronic infection is unclear. One possibility is that B-cell losses are related to reduced T-cell numbers. In a study by Titanji et al., a strong correlation between the number of CD4 T cells and the percentage of memory B cells was reported in chronic HIV infection (37). Conversely, others have reported that no correlation was found between CD4 numbers and memory B-cell numbers (3, 10). Interestingly, reductions in percentages of B cells, increased expression of Fas on B cells, increased total plasma IgG levels, a decreased percentage of IgM memory B cells, and decreased B-cell responses to antigenic stimulation have been shown to occur within 6 months of HIV infection (36, 37). Disruption of germinal centers in the gut during acute HIV infection may also compromise the humoral immune response (20). While these studies provide insight into virus-induced changes in the B-cell compartment during infection, it is difficult to ascertain precisely when these changes occur, due to limitations in sample size and numbers during this early period of infection. The conflicting reports reflect the high amount of variability present in human HIV infection and illuminate the need for a model to study B-cell populations in which experimental parameters can be more rigorously controlled. An understanding of the effects of HIV on the B-cell population during this critical early phase of infection is needed to determine how the initial interactions between virus and host immune system set the stage for long-term disease progression in the infected host. The simian immunodeficiency virus (SIV)/macaque model provides a system in which to ask these questions.Studies in SIV-infected macaques have demonstrated that the number of total B (CD20⁺) cells in the periphery decreases dramatically during the acute phase of infection (13, 24). The loss of these cells coincides with a similar depletion of peripheral CD4 T cells and is associated with primary viremia. Interestingly, the loss of total B cells is greater in magnitude than the loss of CD4⁺ T cells (24). In order to understand how these cells are being depleted, it is necessary to characterize B-cell subsets during SIV infection in the macaque. The present study was designed to assess phenotypic changes in B-cell numbers during the acute phase of SIV infection, both in the total B-cell population as well as in B-cell subsets. Our results identified early, rapid changes in B-cell subsets that were not apparent in analysis of the total B-cell population. Specifically, we identified a significant depletion from the periphery of both the naïve (CD20⁺ CD27⁻) and memory (CD20⁺ CD27⁺) B-cell populations during acute infection and increased total B-cell population activation that may be related to ineffective antibody production commonly associated with SIV infection. Furthermore, the data demonstrate that measurement of envelope-specific antibody responses was not a sensitive reflection of SIV effects on B-cell subsets. These data provide novel information about the timing and dynamics of phenotypic changes in the B-cell compartment during SIV infection that may be associated with functional changes observed later in chronic infection. These results can be used to tailor therapeutic treatments designed to preserve the B-cell compartment early in SIV/HIV infection.     

The Cortical Actin Determines Different Susceptibility of Na?ve and Memory CD4+ T Cells to HIV-1 Cell-to-Cell Transmission and Infection

Memory CD4+ T cells are preferentially infected by HIV-1 compared to naïve cells. HIV-1 fusion and entry is a dynamic process in which the cytoskeleton plays an important role by allowing virion internalization and uncoating. Here, we evaluate the role of the cortical actin in cell-to-cell transfer of virus antigens and infection of target CD4+ T cells. Using different actin remodeling compounds we demonstrate that efficiency of HIV-internalization was proportional to the actin polymerization of the target cell. Naïve (CD45RA+) and memory (CD45RA−) CD4+ T cells could be phenotypically differentiated by the degree of cortical actin density and their capacity to capture virus. Thus, the higher cortical actin density of memory CD4+ T cells was associated to increased efficiency of HIV-antigen internalization and the establishment of a productive infection. Conversely, the lower cortical actin density in naïve CD4+ T cells restricted viral antigen transfer and consequently HIV-1 infection. In conclusion, the cortical actin density differentially affects the susceptibility to HIV-1 infection in naïve and memory CD4+ T cells by modulating the efficiency of HIV antigen internalization.     

Isolation and Th17 Differentiation of Na?ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-β. After incubation for at least 72 hours and for up to five days at 37 °C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.     

Memory CD8+ T Cells: Orchestrators and Key Players of Innate Immunity?

Over the past decades, the dichotomy between innate and adaptive immune responses has largely dominated our understanding of immunology. Upon primary encounter with microbial pathogens, differentiation of adaptive immune cells into functional effectors usually takes several days or even longer, making them contribute to host protection only late during primary infection. However, once generated, antigen-experienced T lymphocytes can persist in the organism and constitute a pool of memory cells that mediate fast and effective protection to a recall infection with the same microbial pathogen. Herein, we challenge this classical paradigm by highlighting the “innate nature” of memory CD8⁺ T cells. First, within the thymus or in the periphery, naïve CD8⁺ T cells may acquire phenotypic and functional characteristics of memory CD8⁺ T cells independently of challenge with foreign antigens. Second, both the “unconventional” and the “conventional” memory cells can rapidly express protective effector functions in response to sets of inflammatory cytokines and chemokines signals, independent of cognate antigen triggering. Third, memory CD8⁺ T cells can act by orchestrating the recruitment, activation, and licensing of innate cells, leading to broad antimicrobial states. Thus, collectively, memory CD8⁺ T cells may represent important actors of innate immune defenses.     

Hierarchy Low CD4+/CD8+ T-Cell Counts and IFN-γ Responses in HIV-1+ Individuals Correlate with Active TB and/or M.tb Co-Infection

Objective

Detailed studies of correlation between HIV-M.tb co-infection and hierarchy declines of CD8+/CD4+ T-cell counts and IFN-γ responses have not been done. We conducted case-control studies to address this issue.

Methods

164 HIV-1-infected individuals comprised of HIV-1⁺ATB, HIV-1⁺LTB and HIV-1⁺TB^- groups were evaluated. Immune phenotyping and complete blood count (CBC) were employed to measure CD4+ and CD8+ T-cell counts; T.SPOT.TB and intracellular cytokine staining (ICS) were utilized to detect ESAT6, CFP10 or PPD-specific IFN-γ responses.

Results

There were significant differences in median CD4+ T-cell counts between HIV-1⁺ATB (164/μL), HIV-1⁺LTB (447/μL) and HIV-1⁺TB^- (329/μL) groups. Hierarchy low CD4+ T-cell counts (<200/μL, 200-500/μL, >500/μL) were correlated significantly with active TB but not M.tb co-infection. Interestingly, hierarchy low CD8+ T-cell counts were not only associated significantly with active TB but also with M.tb co-infection (P<0.001). Immunologically, HIV-1⁺ATB group showed significantly lower numbers of ESAT-6-/CFP-10-specific IFN-γ+ T cells than HIV-1⁺LTB group. Consistently, PPD-specific IFN-γ+CD4+/CD8+ T effector cells in HIV-1⁺ATB group were significantly lower than those in HIV-1⁺LTB group (P<0.001).

Conclusions

Hierarchy low CD8+ T-cell counts and effector function in HIV-1-infected individuals are correlated with both M.tb co-infection and active TB. Hierarchy low CD4+ T-cell counts and Th1 effector function in HIV-1+ individuals are associated with increased frequencies of active TB, but not M.tb co-infection.     

Regulation of the Na+/K+-ATPase by insulin: Why and how?

The sodium-potassium ATPase (Na⁺/K⁺-ATPase or Na⁺/K⁺-pump) is an enzyme present at the surface of all eukaryotic cells, which actively extrudes Na⁺ from cells in exchange for K⁺ at a ratio of 3:2, respectively. Its activity also provides the driving force for secondary active transport of solutes such as amino acids, phosphate, vitamins and, in epithelial cells, glucose. The enzyme consists of two subunits ( and ) each expressed in several isoforms. Many hormones regulate Na⁺/K⁺ -ATPase activity and in this review we will focus on the effects of insulin. The possible mechanisms whereby insulin controls Na⁺/K⁺-ATPase activity are discussed. These are tissue- and isoform-specific, and include reversible covalent modification of catalytic subunits, activation by a rise in intracellular Na⁺ concentration, altered Na⁺ sensitivity and changes in subunit gene or protein expression. Given the recent escalation in knowledge of insulin-stimulated signal transduction systems, it is pertinent to ask which intracellular signalling pathways are utilized by insulin in controlling Na⁺/K⁺-ATPase activity. Evidence for and against a role for the phosphatidylinositol-3-kinase and mitogen activated protein kinase arms of the insulin-stimulated intracellular signalling networks is suggested. Finally, the clinical relevance of Na⁺/K⁺-ATPase control by insulin in diabetes and related disorders is addressed.     

Gag-Specific CD4 and CD8 T-Cell Proliferation in Adolescents and Young Adults with Perinatally Acquired HIV-1 Infection Is Associated with Ethnicity — The ANRS-EP38-IMMIP Study

The ANRS-EP38-IMMIP study aimed to provide a detailed assessment of the immune status of perinatally infected youths living in France. We studied Gag-specific CD4 and CD8 T-cell proliferation and the association between the proliferation of these cells, demographic factors and HIV disease history. We included 93 youths aged between 15 and 24 years who had been perinatally infected with HIV. Sixty-nine had undergone valid CFSE-based T-cell proliferation assays. Gag-specific proliferation of CD4 and CD8 T cells was detected in 12 (16%) and 30 (38%) patients, respectively. The Gag-specific proliferation of CD4 and CD8 T cells was more frequently observed in black patients than in patients from other ethnic groups (CD4: 32% vs. 4%, P = 0.001; CD8: 55% vs. 26%, P = 0.02). Among aviremic patients, the duration of viral suppression was shorter in CD8 responders than in CD8 nonresponders (medians: 54 vs. 20 months, P = 0.04). Among viremic patients, CD8 responders had significantly lower plasma HIV RNA levels than CD8 nonresponders (2.7 vs. 3.7 log₁₀ HIV-RNA copies/ml, P = 0.02). In multivariate analyses including sex and HIV-1 subtype as covariables, Gag-specific CD4 T-cell proliferation was associated only with ethnicity, whereas Gag-specific CD8 T-cell proliferation was associated with both ethnicity and the duration of viral suppression. Both CD4 and CD8 responders reached their nadir CD4 T-cell percentages at younger ages than their nonresponder counterparts (6 vs. 8 years, P = 0.04 for both CD4 and CD8 T-cell proliferation). However, these associations were not significant in multivariate analysis. In conclusion, after at least 15 years of HIV infection, Gag-specific T-cell proliferation was found to be more frequent in black youths than in patients of other ethnic groups, despite all the patients being born in the same country, with similar access to care.     

Increased Bone Marrow (BM) Plasma Level of Soluble CD30 and Correlations with BM Plasma Level of Interferon (IFN)-γ, CD4/CD8 T-Cell Ratio and Disease Severity in Aplastic Anemia

Idiopathic aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome. Immune abnormalities such as decreased lymphocyte counts, inverted CD4/CD8 T-cell ratio and increased IFN-γ-producing T cells have been found in AA. CD30, a surface protein belonging to the tumor necrosis factor receptor family and releasing from cell surface as a soluble form (sCD30) after activation, marks a subset of activated T cells secreting IFN-γ when exposed to allogeneic antigens. Our study found elevated BM plasma levels of sCD30 in patients with SAA, which were closely correlated with disease severity, including absolute lymphocyte count (ALC) and absolute netrophil count (ANC). We also noted that sCD30 levels were positively correlated with plasma IFN-γ levels and CD4/CD8 T-cell ratio in patients with SAA. In order to explain these phenomena, we stimulated T cells with alloantigen in vitro and found that CD30⁺ T cells were the major source of IFN-γ, and induced CD30⁺ T cells from patients with SAA produced significantly more IFN-γ than that from healthy individuals. In addition, increased proportion of CD8⁺ T cells in AA showed enhanced allogeneic response by the fact that they expressed more CD30 during allogeneic stimulation. sCD30 levels decreased in patients responded to immunosuppressive therapy. In conclusion, elevated BM plasma levels of sCD30 reflected the enhanced CD30⁺ T cell-mediated immune response in SAA. CD30 as a molecular marker that transiently expresses on IFN-γ-producing T cells, may participate in mediating bone marrow failure in AA, which also can facilitate our understanding of AA pathogenesis to identify new therapeutic targets.     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

Enhanced Activation of Memory,but Not Na?ve,B Cells in Chronic Hepatitis C Virus-Infected Patients with Cryoglobulinemia and Advanced Liver Fibrosis

Mixed cryoglobulinemia is the most common extrahepatic disease manifestation of chronic hepatitis C virus (HCV) infection, where immunoglobulins precipitate at low temperatures and cause symptoms such as vasculitis, glomerulonephritis and arthralgia. HCV-associated cryoglobulinemia is also strongly linked with the development of B cell non-Hodgkin lymphoma. Abnormal B cell function in HCV infections can lead to the formation of HCV cryoglobulin complexes that usually comprise monoclonal rheumatoid factor and HCV-specific immune complexes. The aim of this study was to characterize the activation phenotype of B cells from patients with chronic HCV infection in comparison to healthy controls using flow cytometry. In addition, we determined how the activation status varies depending on the presence of cryoglobulinemia and advanced liver fibrosis. We found that only memory B cells, not naïve cells, were significantly activated in chronic HCV infection when compared with healthy controls. We also identified markers of memory B cell activation that were specific for HCV patients with cryoglobulinemia (CD86, CD71, HLA-DR) and advanced liver disease (CD86). Our results demonstrate that HCV infection has differential effects on B cells depending on the severity of hepatic and extrahepatic disease.     

Duration of Untreated Psychosis Is Associated with Temporal and Occipitotemporal Gray Matter Volume Decrease in Treatment Na?ve Schizophrenia

Background

Long duration of untreated psychosis (DUP) is associated with poor treatment outcome. Whether or not DUP is related to brain gray matter volume abnormalities in antipsychotic medication treatment naïve schizophrenia remains unclear at this time.

Methods

Patients with treatment-naïve schizophrenia and healthy controls went through brain scan using high resolution Magnetic Resonance Imaging. DUP was evaluated using the Nottingham Onset Schedule (NOS), and dichotomized as short DUP (≤ 26 weeks) or long DUP (>26 weeks). Voxel-based methods were used for volumetric measure in the brain.

Results

Fifty-seven patients (27 short DUP and 30 long DUP) and 30 healthy controls were included in the analysis. There were significant gray matter volumetric differences among the 3 groups in bilateral parahippocampus gyri, right superior temporal gyrus, left fusiform gyrus, left middle temporal gyrus, and right superior frontal gyrus (p''s<0.01). Compared with healthy controls, the long DUP group had significantly smaller volume in all these regions (p''s <0.05). Compared with the short-DUP group, the long-DUP group had significantly smaller volume in right superior temporal gyrus, left fusiform gyrus, and left middle temporal gyrus (p''s<0.01).

Conclusion

Our findings suggest that DUP is associated with temporal and occipitotemporal gray matter volume decrease in treatment naïve schizophrenia. The brain structural changes in untreated psychosis might contribute to poor treatment response and long-term prognosis in this patient population.     

Liver Is Able to Activate Na?ve CD8+ T Cells with Dysfunctional Anti-Viral Activity in the Murine System

The liver possesses distinct tolerogenic properties because of continuous exposure to bacterial constituents and nonpathogenic food antigen. The central immune mediators required for the generation of effective immune responses in the liver environment have not been fully elucidated. In this report, we demonstrate that the liver can indeed support effector CD8⁺ T cells during adenovirus infection when the T cells are primed in secondary lymphoid tissues. In contrast, when viral antigen is delivered predominantly to the liver via intravenous (IV) adenovirus infection, intrahepatic CD8⁺ T cells are significantly impaired in their ability to produce inflammatory cytokines and lyse target cells. Additionally, intrahepatic CD8⁺ T cells generated during IV adenovirus infection express elevated levels of PD-1. Notably, lower doses of adenovirus infection do not rescue the impaired effector function of intrahepatic CD8⁺ T cell responses. Instead, intrahepatic antigen recognition limits the generation of potent anti-viral responses at both priming and effector stages of the CD8⁺ T cell response and accounts for the dysfunctional CD8⁺ T cell response observed during IV adenovirus infection. These results also implicate that manipulation of antigen delivery will facilitate the design of improved vaccination strategies to persistent viral infection.