Germinal Center T follicular helper (GC-Tfh) cell impairment in chronic HIV infection involves c-Maf signaling.

We have recently demonstrated that the function of T follicular helper (Tfh) cells from lymph nodes (LN) of HIV-infected individuals is impaired. We found that these cells were unable to provide proper help to germinal center (GC)-B cells, as observed by altered and inefficient anti-HIV antibody response and premature death of memory B cells. The underlying molecular mechanisms of this dysfunction remain poorly defined. Herein, we have used a unique transcriptional approach to identify these molecular defects. We consequently determined the transcriptional profiles of LN GC-Tfh cells following their interactions with LN GC-B cells from HIV-infected and HIV-uninfected individuals, rather than analyzing resting ex-vivo GC-Tfh cells. We observed that proliferating GC-Tfh cells from HIV-infected subjects were transcriptionally different than their HIV-uninfected counterparts, and displayed a significant downregulation of immune- and GC-Tfh-associated pathways and genes. Our results strongly demonstrated that MAF (coding for the transcription factor c-Maf) and its upstream signaling pathway mediators (IL6R and STAT3) were significantly downregulated in HIV-infected subjects, which could contribute to the impaired GC-Tfh and GC-B cell functions reported during infection. We further showed that c-Maf function was associated with the adenosine pathway and that the signaling upstream c-Maf could be partially restored by adenosine deaminase -1 (ADA-1) supplementation. Overall, we identified a novel mechanism that contributes to GC-Tfh cell impairment during HIV infection. Understanding how GC-Tfh cell function is altered in HIV is crucial and could provide critical information about the mechanisms leading to the development and maintenance of effective anti-HIV antibodies.

A follicular regulatory Innate Lymphoid Cell population impairs interactions between germinal center Tfh and B cells.

Innate Lymphoid Cells (ILCs) are immune cells typically found on mucosal surfaces and in secondary lymphoid organs where they regulate the immune response to pathogens. Despite their key role in the immune response, there are still fundamental gaps in our understanding of ILCs. Here we report a human ILC population present in the follicles of tonsils and lymph nodes termed follicular regulatory ILCs (ILCFR) that to our knowledge has not been previously identified. ILCFR have a distinct phenotype and transcriptional program when compared to other defined ILCs. Surprisingly, ILCFR inhibit the ability of follicular helper T (Tfh) cells to provide B cell help. The localization of ILCFR to the germinal centers suggests these cells may interfere with germinal center B cell (GC-B) and germinal center Tfh cell (GC-Tfh) interactions through the production of transforming growth factor beta (TGF-ß. Intriguingly, under conditions of impaired GC-Tfh-GC-B cell interactions, such as human immunodeficiency virus (HIV) infection, the frequency of these cells is increased. Overall, we predict a role for ILCFR in regulating GC-Tfh-GC-B cell interactions and propose they expand in chronic inflammatory conditions.

Levels of Soluble CD14 and Tumor Necrosis Factor Receptors 1 and 2 May Be Predictive of Death in Severe Coronavirus Disease 2019.

People infected with severe acute respiratory syndrome coronavirus 2 display a wide range of illness, from asymptomatic infection to severe respiratory distress resulting in death. We measured serum biomarkers in uninfected individuals and in individuals with mild, moderate, or critical coronavirus disease 2019 (COVID-19) disease. Levels of monocyte activation (soluble CD14 and fatty acid-binding protein 4) and inflammation (tumor necrosis factor receptors 1 and 2 [TNFR1 and TNFR2]) were increased in COVID-19 individuals, regardless of disease severity. Among patients with critical disease, individuals who recovered from COVID-19 had lower levels of TNFR1 and TNFR2 at hospital admission compared to these levels in patients with critical disease who ultimately died.

Delayed differentiation of potent effector CD8+ T cells reducing viremia and reservoir seeding in acute HIV infection.

CD8+ T cells play a critical role in controlling HIV viremia and could be important in reducing HIV-infected cells in approaches to eradicate HIV. The simian immunodeficiency virus model provided the proof of concept for a CD8+ T cell-mediated reservoir clearance but showed conflicting evidence on the role of these cells to eliminate HIV-infected cells. In humans, HIV-specific CD8+ T cell responses have not been associated with a reduction of the HIV-infected cell pool in vivo. We studied HIV-specific CD8+ T cells in the RV254 cohort of individuals initiating ART in the earliest stages of acute HIV infection (AHI). We showed that the HIV-specific CD8+ T cells generated as early as AHI stages 1 and 2 before peak viremia are delayed in expanding and acquiring effector functions but are endowed with higher memory potential. In contrast, the fully differentiated HIV-specific CD8+ T cells at peak viremia in AHI stage 3 were more prone to apoptosis but were associated with a steeper viral load decrease after ART initiation. Their capacity to persist in vivo after ART initiation correlated with a lower HIV DNA reservoir. These findings demonstrate that HIV-specific CD8+ T cell magnitude and differentiation are delayed in the earliest stages of infection. These results also demonstrate that potent HIV-specific CD8+ T cells contribute to the reduction of the pool of HIV-producing cells and the HIV reservoir seeding in vivo and provide the rationale to design interventions aiming at inducing these potent responses to cure HIV infection.

Transcriptional profiles reveal a stepwise developmental program of memory CD8(+) T cell differentiation.

The generation of CD8(+) T-cell memory is a major aim of vaccination. While distinct subsets of CD8(+) T-cells are generated following immunization that differ in their ability to confer long-term immunity against infection, the transcriptional profiles of these subsets within endogenous vaccine-induced CD8(+) T cell responses have not been resolved. Here, we measure global transcriptional profiles of endogenous effector (TEFF), effector memory (TEM) and central memory (TCM) CD8(+) T-cells arising from immunization with three distinct prime-boost vaccine regimens. While a proportion of transcripts were uniquely regulated within distinct CD8(+) T cell populations, we observed progressive up- or down-regulation in the expression of a majority of differentially expressed transcripts when subsets were compared in the order TN>TCM>TEM>TEFF. Strikingly, when we compared global differences in gene expression between TN, TCM, TEM and TEFF cells with known transcriptional changes that result when CD8(+) T cells repetitively encounter antigen, our analysis overwhelmingly favored a model whereby cumulative antigen stimulation drives differentiation specifically from TN>TCM>TEM>TEFF and this was common to all vaccines tested. These findings provide insight into the molecular basis of immunological memory and identify potential biomarkers for characterization of vaccine-induced responses and prediction of vaccine efficacy.

Cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells.

Dengue virus (DENV) is a re-emerging arthropod borne flavivirus that infects more than 300 million people worldwide, leading to 50,000 deaths annually. Because dendritic cells (DC) in the skin and blood are the first target cells for DENV, we sought to investigate the early molecular events involved in the host response to the virus in primary human monocyte-derived dendritic cells (Mo-DC). Using a genome-wide transcriptome analysis of DENV2-infected human Mo-DC, three major responses were identified within hours of infection - the activation of IRF3/7/STAT1 and NF-κB-driven antiviral and inflammatory networks, as well as the stimulation of an oxidative stress response that included the stimulation of an Nrf2-dependent antioxidant gene transcriptional program. DENV2 infection resulted in the intracellular accumulation of reactive oxygen species (ROS) that was dependent on NADPH-oxidase (NOX). A decrease in ROS levels through chemical or genetic inhibition of the NOX-complex dampened the innate immune responses to DENV infection and facilitated DENV replication; ROS were also essential in driving mitochondrial apoptosis in infected Mo-DC. In addition to stimulating innate immune responses to DENV, increased ROS led to the activation of bystander Mo-DC which up-regulated maturation/activation markers and were less susceptible to viral replication. We have identified a critical role for the transcription factor Nrf2 in limiting both antiviral and cell death responses to the virus by feedback modulation of oxidative stress. Silencing of Nrf2 by RNA interference increased DENV-associated immune and apoptotic responses. Taken together, these data demonstrate that the level of oxidative stress is critical to the control of both antiviral and apoptotic programs in DENV-infected human Mo-DC and highlight the importance of redox homeostasis in the outcome of DENV infection.

Rapid temporal changes in the expression of a set of neuromodulatory genes during alcohol withdrawal in the dorsal vagal complex: molecular evidence of homeostatic disturbance.

BACKGROUND: Chronic alcohol exposure produces neuroadaptation, which increases the risk of cellular excitotoxicity and autonomic dysfunction during withdrawal. The temporal progression and regulation of the gene expression that contributes to this physiologic and behavioral phenotype is poorly understood early in the withdrawal period. Further, it is unexplored in the dorsal vagal complex (DVC), a brainstem autonomic regulatory structure. METHODS: We use a quantitative polymerase chain reaction platform to precisely and simultaneously measure the expression of 145 neuromodulatory genes in more than 100 rat DVC samples from control, chronically alcohol-exposed, and withdrawn rats. To gain insight into the dynamic progression and regulation of withdrawal, we focus on the expression of a subset of functionally relevant genes during the first 48 hours, when behavioral symptoms are most severe. RESULTS: In the DVC, expression of this gene subset is essentially normal in chronically alcohol-exposed rats. However, withdrawal results in rapid, large-magnitude expression changes in this group. We observed differential regulation in 86 of the 145 genes measured (59%), some as early as 4 hours into withdrawal. Time series measurements (4, 8, 18, 32, and 48 hours after alcohol removal) revealed dynamic expression responses in immediate early genes, γ-aminobutyric acid type A, ionotropic glutamate, and G-protein coupled receptors and the Ras/Raf signaling pathway. Together, these changes elucidate a complex, temporally coordinated response that involves correlated expression of many functionally related groups. In particular, the expression patterns of Gabra1, Grin2a, Grin3a, and Grik3 were tightly correlated. These receptor subunits share overrepresented transcription factor binding sites for Pax-8 and other transcription factors, suggesting a common regulatory mechanism and a role for these transcription factors in the regulation of neurotransmission within the first 48 hours of alcohol withdrawal. CONCLUSIONS: Expression in this gene set is essentially normal in the alcohol-adapted DVC, but withdrawal results in immediate, large-magnitude, and dynamic changes. These data support both increased research focus on the biological ramifications of alcohol withdrawal and enable novel insights into the dynamic withdrawal expression response in this understudied homeostatic control center.

Production of SV40-derived vectors.

Recombinant simian virus 40 (rSV40)-derived vectors are particularly useful for gene delivery to bone marrow progenitor cells and their differentiated derivatives, certain types of epithelial cells (e.g., hepatocytes), and central nervous system neurons and microglia. They integrate rapidly into cellular DNA to provide long-term gene expression in vitro and in vivo in both resting and dividing cells. Here we describe a protocol for production and purification of these vectors. These procedures require only packaging cells (e.g., COS-7) and circular vector genome DNA. Amplification involves repeated infection of packaging cells with vector produced by transfection. Cotransfection is not required in any step. Viruses are purified by centrifugation using discontinuous sucrose or cesium chloride (CsCl) gradients and resulting vectors are replication-incompetent and contain no detectable wild-type SV40 revertants. These approaches are simple, give reproducible results, and may be used to generate vectors that are deleted only for large T antigen (Tag), or for all SV40-coding sequences capable of carrying up to 5 kb of foreign DNA. These vectors are best applied to long-term expression of proteins normally encoded by mammalian cells or by viruses that infect mammalian cells, or of untranslated RNAs (e.g., RNA interference). The preparative approaches described facilitate application of these vectors and allow almost any laboratory to exploit their strengths for diverse gene delivery applications.

Titering replication-defective rSV40 vectors.

Recombinant simian virus 40 (rSV40)-derived vectors are particularly useful for gene delivery to bone marrow progenitor cells and their differentiated derivatives, certain types of epithelial cells (e.g., hepatocytes), and central nervous system neurons and microglia. They integrate rapidly into cellular DNA to provide long-term gene expression in vitro and in vivo in both resting and dividing cells. Techniques used to produce, purify, and quantitate these vectors are simple, give reproducible results, and may be used to generate vectors that are deleted only for large T antigen (Tag), or for all SV40-coding sequences capable of carrying up to 5 kb of foreign DNA. Viruses are purified by centrifugation using discontinuous sucrose or cesium chloride (CsCl) gradients. Resulting vectors are replication-incompetent and contain no detectable wild-type SV40 revertants. Viruses are titered by quantitative polymerase chain reaction (qPCR), described here. qPCR measures the number of rSV40 genomes in purified viral stocks using primers specific for the rSV40, coupled with SYBR Green detection of PCR products. Sample purity is assessed using qPCR via melt (dissociation) curve analysis. The only specialized equipment necessary is a quantitative real-time PCR machine.

In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors.

Hematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by cytokine-induced loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Here, we attempted to circumvent such limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)-derived gene delivery vectors were used because they transduce resting CD34+ cells very efficiently. Rats received SV-(Nef-FLAG), carrying FLAG marker epitope--or a control recombinant SV40 (rSV40)--directly into both femoral marrow cavities. Intracellular transgene expression by peripheral blood (PB) or BM cells was detected by cytofluorimetry. An average of 5.3% PB leukocytes expressed FLAG for the entire study--56 weeks. Transgene expression was sustained in multiple cell lineages, including granulocytes (average, 3.3% of leukocytes, 20.4% of granulocytes), CD3+ T lymphocytes (average, 0.53% of leukocytes, 1% of total T cells), and CD45R+ B lymphocytes, indicating gene transfer to long-lived progenitor cells with multilineage capacity. An average of 15% of femoral marrow cells expressed FLAG up to 16.5 months after transduction. Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells and may be worthy of further investigation.

What they are, how they work and why they do what they do? The story of SV40-derived gene therapy vectors and what they have to offer.

The natural function of viruses is to deliver their genetic material to cells. Among the most effective of viruses in doing that is Simian Virus-40 (SV40). The properties that make SV40 a successful virus make it an attractive candidate for use as a gene delivery vehicle: high titer replication, infectivity for almost all nucleated cell types whether the cells are dividing or resting, potential for integration into cellular DNA, a peculiar pathway for entering cells that bypasses the cells' antigen processing apparatus, very high stability, and the apparent ability to activate expression of its own capsid genes in trans. Exploiting these and other characteristics of wild type (wt) SV40, increasing numbers of laboratories are studying recombinant (r) SV40-derived vectors. Among the uses to which these vectors have been applied are: delivering therapy to inhibit HIV, hepatitis C virus (HCV) and other viruses; correction of inherited hepatic and other protein deficiencies; immunizing against lentiviral and other antigens; treatment of inherited and acquired diseases of the central nervous system; protecting the lung and other organs from free radical-induced injury; and many others. The effectiveness of these vectors is a reflection of the adaptive evolution that produced their parent virus, wt SV40. This article explores how and why these vectors work, their strengths and their limitations, and provides a functional model for their exploitation for experimental and clinical applications.

Durability of transgene expression and vector integration: recombinant SV40-derived gene therapy vectors.

Many applications of gene delivery require long-term transgene expression. In dividing cells, this result necessitates vector genome persistence, usually by integrating into cellular DNA. Since recombinant gene delivery vectors derived from tag-deleted, replication-incompetent simian virus-40 (SV40) provide for long-term transgene expression in resting and dividing cells, we tested whether such enduring transgene expression reflected integration into cellular genomes. Several lines of evidence suggested this likelihood. After transduction in vitro, continuously dividing cell lines and continuously stimulated primary cells uniformly showed transgene expression for many months. Mice whose livers were transduced in vivo, partially resected, and allowed to regenerate showed comparable levels of transgene expression in regenerated and preoperative livers. Thus, replicationincompetent SV40 vectors (rSV40) persist in vitro and in vivo despite extensive cell division. We tested the possibility that this persistence reflected integration directly. Southern blot analyses of genomic DNA from transduced 293 cells showed that vector genome incorporation into cell DNA happened within days of transduction. Episomal vector DNA was barely detectable 96 hours post-transduction. Inverted PCR, used to characterize vector integration points, showed vector DNA integrated randomly into the cell genome. The circular rSV40 genome opened at different points in each integrand. A significant proportion of the integrands did not contain the entire vector sequence, but rather only portions thereof. Quantitative Southern blot analysis showed approximately 3.05 transgene copies per cell. Therefore, recombinant SV40 gene delivery vectors integrate into the cellular DNA of both resting and dividing cells, and do so randomly and within days of transduction. This integration may explain long-term transgene expression.

Comparison of two different preparations of HIV immune globulin for efficiency of neutralization of HIV type 1 primary isolates.

The objective of this study was to compare the virus-neutralizing ability of two different preparations of HIV immune globulin (HIVIG) isolated from human plasma units that were selected according to two different criteria. The first preparation, designated NYBC-HIVIG, was isolated from plasmas with high neutralizing antibody titers against HIV-1. The second preparation, designated NABI-HIVIG, was isolated from plasma with high titers of antibody to the HIV-1 p24 antigen. A panel of primary HIV-1 isolates was phenotypically characterized by their ability to induce syncytia in CEM-SS cells. Neutralization of this panel of primary isolates by the two HIVIG preparations was assessed in HeLa-MAGI-CCR5 cells, utilizing a luminescence-based assay. In addition, the reactivities of these two preparations with a panel of HIV-1 gp120 proteins, V3 loop peptides, and HIV-1 p24 antigen were determined. Both HIVIG preparations were shown to neutralize all virus isolates tested. However, doses of NABI-HIVIG required for 50% virus neutralization were 2.2- to 4.4- fold (mean, 3.2-fold) higher than the required doses of NYBC-HIVIG. Comparative antigen-binding assays showed that, although NABI-HIVIG possessed higher titers of antibody to HIV-1 p24, NYBC-HIVIG generally contained higher titers of antibody to HIV-1 gp120 and V3 peptides. These experiments show that the criteria used for selection of source plasmas for isolation of HIVIG can influence the effective concentration of virus-neutralizing antibody present in the final immunoglobulin preparation, and may determine the doses required for clinical efficacy.