IgG3 regulates tissue-like memory B cells in HIV-infected individuals.

Immunoglobulin G3 (IgG3) has an uncertain role in the response to infection with and vaccination against human immunodeficiency virus (HIV). Here we describe a regulatory role for IgG3 in dampening the immune system-activating effects of chronic HIV viremia on B cells. Secreted IgG3 was bound to IgM-expressing B cells in vivo in HIV-infected chronically viremic individuals but not in early-viremic or aviremic individuals. Tissue-like memory (TLM) B cells, a population expanded by persistent HIV viremia, bound large amounts of IgG3. IgG3 induced clustering of B cell antigen receptors (BCRs) on the IgM+ B cells, which was mediated by direct interactions between soluble IgG3 and membrane IgM of the BCR (IgM-BCR). The inhibitory IgG receptor CD32b (FcγRIIb), complement component C1q and inflammatory biomarker CRP contributed to the binding of secreted IgG3 onto IgM-expressing B cells of HIV-infected individuals. Notably, IgG3-bound TLM B cells were refractory to IgM-BCR stimulation, thus demonstrating that IgG3 can regulate B cells during chronic activation of the immune system.

Chronic virus infection enforces demethylation of the locus that encodes PD-1 in antigen-specific CD8(+) T cells.

Functionally exhausted T cells have high expression of the PD-1 inhibitory receptor, and therapies that block PD-1 signaling show promise for resolving chronic viral infections and cancer. By using human and murine systems of acute and chronic viral infections, we analyzed epigenetic regulation of PD-1 expression during CD8(+) T cell differentiation. During acute infection, naive to effector CD8(+) T cell differentiation was accompanied by a transient loss of DNA methylation of the Pdcd1 locus that was directly coupled to the duration and strength of T cell receptor signaling. Further differentiation into functional memory cells coincided with Pdcd1 remethylation, providing an adapted program for regulation of PD-1 expression. In contrast, the Pdcd1 regulatory region was completely demethylated in exhausted CD8(+) T cells and remained unmethylated even when virus titers decreased. This lack of DNA remethylation leaves the Pdcd1 locus poised for rapid expression, potentially providing a signal for premature termination of antiviral functions.

Genetic and Epigenetic Regulation of PD-1 Expression.

The inhibitory immune receptor programmed cell death-1 (PD-1) is intricately regulated. In T cells, PD-1 is expressed in response to most immune challenges, but it is rapidly downregulated in acute settings, allowing for normal immune responses. On chronically stimulated Ag-specific T cells, PD-1 expression remains high, leading to an impaired response to stimuli. Ab blockade of PD-1 interactions during chronic Ag settings partially restores immune function and is now used clinically to treat a variety of devastating cancers. Understanding the regulation of PD-1 expression may be useful for developing novel immune-based therapies. In this review, the molecular mechanisms that drive dynamic PD-1 expression during acute and chronic antigenic stimuli are discussed. An array of cis-DNA elements, transcription factors, and epigenetic components, including DNA methylation and histone modifications, control PD-1 expression. The interplay between these regulators fine-tunes PD-1 expression in different inflammatory environments and across numerous cell types to modulate immune responses.

The molecular signature of murine T cell homeostatic proliferation reveals both inflammatory and immune inhibition patterns.

T lymphocyte homeostatic proliferation, driven by the engagement of T cell antigen receptor with self-peptide/major histocompatibility complexes, and signaling through the common γ-chain-containing cytokine receptors, is critical for the maintenance of the T cell compartment and is regulated by the Fas death receptor (Fas, CD95). In the absence of Fas, Fas-deficient lymphoproliferation spontaneous mutation (lpr) mice accumulate homeostatically expanded T cells. The functional consequences of sequential rounds of homeostatic expansion are not well defined. We thus examined the gene expression profiles of murine wild-type and Fas-deficient lpr CD8+ T cell subsets that have undergone different amounts of homeostatic proliferation as defined by their level of CD44 expression, and the CD4-CD8-TCRαß+ T cell subset that results from extensive homeostatic expansion of CD8+ T cells. Our studies show that recurrent T cell homeostatic proliferation results in global gene expression changes, including the progressive upregulation of both cytolytic proteins such as Fas-Ligand and granzyme B as well as inhibitory proteins such as programmed cell death protein 1 (PD-1) and lymphocyte activating 3 (Lag3). These findings provide an explanation for how augmented T cell homeostatic expansion could lead to the frequently observed clinical paradox of simultaneous autoinflammatory and immunodeficiency syndromes and provide further insight into the regulatory programs that control chronically stimulated T cells.

NF-κB regulates PD-1 expression in macrophages.

Programmed cell death-1 (PD-1) is responsible for T cell exhaustion during chronic viral infections and is expressed on a variety of immune cells following activation. Despite its importance, the mechanisms that regulate PD-1 in cell types other than CD8 T cells are poorly defined. In this study, the molecular mechanisms for inducing PD-1 expression in CD4 T cells, macrophages, and B cells were explored. In CD4 T cells, PD-1 induction following TCR stimulation required NFAT, as the calcineurin/NFAT pathway inhibitor cyclosporin A was able to block PD-1 induction in a manner similar to that seen in CD8 T cells. In contrast, LPS but not PMA and ionomycin stimulation was able to induce PD-1 expression in macrophages in a manner insensitive to cyclosporin A-mediated inhibition. B cells could use both pathways, although the levels of PD-1 expression were highest with PMA and ionomycin. An NF-κB binding site located upstream of the gene in conserved region C was required for NF-κB-dependent PD-1 gene activation in macrophages. Chromatin immunoprecipitation showed NF-κB p65 binding to this region following stimulation of macrophages with LPS. PD-1 induction was associated with histone modifications characteristic of accessible chromatin; however, in contrast to CD8 T cells, conserved region B in macrophages did not lose CpG methylation upon stimulation and PD-1 expression. The linkage of TLR/NF-κB signaling to the induction of PD-1 suggests the possibility of an opportunistic advantage to microbial infections in manipulating immune inhibitory responses.

STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells.

Programmed death-1 (PD-1) is a crucial negative regulator of CD8 T cell development and function, yet the mechanisms that control its expression are not fully understood. Through a nonbiased DNase I hypersensitivity assay, four novel regulatory regions within the Pdcd1 locus were identified. Two of these elements flanked the locus, bound the transcriptional insulator protein CCCTC-binding factor, and interacted with each other, creating a potential regulatory compartmentalization of the locus. In response to T cell activation signaling, NFATc1 bound to two of the novel regions that function as independent regulatory elements. STAT binding sites were identified in these elements as well. In splenic CD8 T cells, TCR-induced PD-1 expression was augmented by IL-6 and IL-12, inducers of STAT3 and STAT4 activity, respectively. IL-6 or IL-12 on its own did not induce PD-1. Importantly, STAT3/4 and distinct chromatin modifications were associated with the novel regulatory regions following cytokine stimulation. The NFATc1/STAT regulatory regions were found to interact with the promoter region of the Pdcd1 gene, providing a mechanism for their action. Together these data add multiple novel distal regulatory regions and pathways to the control of PD-1 expression and provide a molecular mechanism by which proinflammatory cytokines, such as IL-6 or IL-12, can augment PD-1 expression.

Cutting edge: Prolonged exposure to HIV reinforces a poised epigenetic program for PD-1 expression in virus-specific CD8 T cells.

Ag-specific CD8 T cells play a critical role in controlling HIV infection but eventually lose antiviral functions in part because of expression and signaling through the inhibitory programmed death-1 (PD-1) receptor. To better understand the impact of prolonged TCR ligation on regulation of PD-1 expression in HIV-specific CD8 T cells, we investigated the capacity of virus-specific CD8 T cells to modify the PD-1 epigenetic program after reduction in viral load. We observed that the transcriptional regulatory region was unmethylated in the PD-1(hi) HIV-specific CD8 T cells, whereas it remained methylated in donor-matched naive cells at acute and chronic stages of infection. Surprisingly, the PD-1 promoter remained unmethylated in HIV-specific CD8 T cells from subjects with a viral load controlled by antiviral therapy for >2 y or from elite controllers. Together, these data demonstrate that the epigenetic program at the PD-1 locus becomes fixed after prolonged exposure to HIV virus.

Riluzole attenuates neuropathic pain and enhances functional recovery in a rodent model of cervical spondylotic myelopathy.

Cervical spondylotic myelopathy (CSM) is the commonest cause of spinal cord impairment worldwide and despite surgical treatment, it is commonly associated with chronic neuropathic pain and neurological impairment. Based on data suggesting a key role of sodium and glutamate mediated cellular injury in models of spinal cord compression, we examined whether riluzole, a sodium channel/glutamate blocker, could improve neurobehavioral outcomes in a rat model of CSM. To produce chronic progressive compression of the cervical spinal cord, we used an established model of graded mechanical cord compromise developed in our laboratory. The chronic (8weeks) mechanical compression of the cervical spinal cord resulted in persistent mechanical allodynia and thermal hyperalgesia at 8weeks. Moreover, we found increased expression of phosphorylated NR1 and NR2B in the dorsal horns as well as astrogliosis and increased microglia expression in the dorsal horns after mechanical compression. Following daily systemic administration for 7weeks after the induction of compression, riluzole (8mg/kg) significantly attenuated forelimb and hindlimb mechanical allodynia and alleviated thermal hyperalgesia in the tail. Importantly, riluzole led to a decrease in swing phase duration, an increase in hind leg swing speed and an increase paw intensity in gait analysis. Riluzole also decreased the number of phosphorylated NR1 and phosphorylated NR2B positive cells in the dorsal horns and the microglia activation in the dorsal horns. Together, our results indicate that systemic riluzole administration during chronic cervical spinal cord compression is effective at protecting spinal cord tissue, preserving neurobehavioral function and alleviating neuropathic pain, possibly by decreasing NMDA receptor phosphorylation in astrocytes and by eliminating microglia activation. As such, riluzole represents a promising clinical treatment for CSM.

Abasic sites and strand breaks in DNA cause transcriptional mutagenesis in Escherichia coli.

DNA damage occurs continuously, and faithful replication and transcription are essential for maintaining cell viability. Cells in nature are not dividing and replicating DNA often; therefore it is important to consider the outcome of RNA polymerase (RNAP) encounters with DNA damage. Base damage in the DNA can affect transcriptional fidelity, leading to production of mutant mRNA and protein in a process termed transcriptional mutagenesis (TM). Abasic (AP) sites and strand breaks are frequently occurring, spontaneous damages that are also base excision repair (BER) intermediates. In vitro studies have demonstrated that these lesions can be bypassed by RNAP; however this has never been assessed in vivo. This study demonstrates that RNAP is capable of bypassing AP sites and strand breaks in Escherichia coli and results in TM through adenine incorporation in nascent mRNA. Elimination of the enzymes that process these lesions further increases TM; however, such mutants can still complete repair by other downstream pathways. These results show that AP sites and strand breaks can result in mutagenic RNAP bypass and have important implications for the biologic endpoints of DNA damage.

Sub-lethal exposure to chlorfenapyr reduces the probability of developing Plasmodium falciparum parasites in surviving Anopheles mosquitoes.

BACKGROUND: Pyrethroid resistance in the key malaria vectors threatens the success of pyrethroid-treated nets. To overcome pyrethroid resistance, Interceptor® G2 (IG2), a 'first-in-class' dual insecticidal net that combines alpha-cypermethrin with chlorfenapyr, was developed. Chlorfenapyr is a pro-insecticide, requiring bio-activation by oxidative metabolism within the insect's mitochondria, constituting a mode of action preventing cross-resistance to pyrethroids. Recent epidemiological trials conducted in Benin and Tanzania confirm IG2's public health value in areas with pyrethroid-resistant Anopheles mosquitoes. As chlorfenapyr might also interfere with the metabolic mechanism of the Plasmodium parasite, we hypothesised that chlorfenapyr may provide additional transmission-reducing effects even if a mosquito survives a sub-lethal dose. METHODS: We tested the effect of chlorfenapyr netting to reduce Plasmodium falciparum transmission using a modified WHO tunnel test with a dose yielding sub-lethal effects. Pyrethroid-resistant Anopheles gambiae s.s. with L1014F and L1014S knockdown resistance alleles and expression levels of pyrethroid metabolisers CYP6P3, CYP6M2, CYP4G16 and CYP6P1 confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) prior to conducting experiments were exposed to untreated netting and netting treated with 200 mg/m3 chlorfenapyr for 8 h overnight and then fed on gametocytemic blood meals from naturally infected individuals. Prevalence and intensity of oocysts and sporozoites were determined on day 8 and day 16 after feeding. RESULTS: Both prevalence and intensity of P. falciparum infection in the surviving mosquitoes were substantially reduced in the chlorfenapyr-exposed mosquitoes compared to untreated nets. The odds ratios in the prevalence of oocysts and sporozoites were 0.33 (95% confidence interval; 95% CI 0.23-0.46) and 0.43 (95% CI 0.25-0.73), respectively, while only the incidence rate ratio for oocysts was 0.30 (95% CI 0.22-0.41). CONCLUSION: We demonstrated that sub-lethal exposure of pyrethroid-resistant mosquitoes to chlorfenapyr substantially reduces the proportion of infected mosquitoes and the intensity of the P. falciparum infection. This will likely also contribute to the reduction of malaria in communities beyond the direct killing of mosquitoes.

MultipleXed Population Selection and Enrichment single nucleus RNA sequencing (XPoSE-seq) enables sample identity retention during transcriptional profiling of rare populations.

Single nucleus RNA-sequencing is critical in deciphering tissue heterogeneity and identifying rare populations. However, current high throughput techniques are not optimized for rare target populations and require tradeoffs in design due to feasibility. We provide a novel snRNA pipeline, MulipleXed Population Selection and Enrichment snRNA-sequencing (XPoSE-seq), to enable targeted snRNA-seq experiments and in-depth transcriptomic characterization of rare target populations while retaining individual sample identity.

High molecular weight hyaluronan reduces lipopolysaccharide mediated microglial activation.

Toll-like receptor-4 (TLR4) signaling has been implicated in microglial activation and propagation of inflammation following spinal cord injury (SCI). As such, modulating microglial activation through TLR4 represents an attractive therapeutic approach to treat SCI. High molecular weight hyaluronan (HMW-HA), a polymer with multiple therapeutic uses, has been previously shown to modulate TLR4 activation in macrophages and has shown early promise as a therapeutic agent in SCI. However, the mechanism associated with HMW-HA has not been fully elucidated or tested in microglia, a similar cell type. In the current study, we sought to determine the effects of HMW-HA on TLR4 activation in microglia and to gain insights into the mechanism of action. Rat primary microglial cultures were exposed to lipopolysaccharides (LPS) and HMW-HA, and the extent and mechanisms of inflammation were studied. HMW-HA decreased LPS mediated IL-1ß, IL-6, and Tumor necrosis factor-α gene expression and IL-6 and nitric oxide production. This decrease was associated with a reduction in ERK 1/2 and p38 phosphorylation, was dependent on the continued presence of HMW-HA, and activation of Akt and A20 protein expression was reduced by HMW-HA. Together, our results show that HMW-HA can reduce LPS-mediated inflammatory signaling in microglia. We suggest that HA possibly mediates its effects by blocking the induction of inflammatory signaling through an extracellular mechanism.

Immunoglobulin G (IgG) attenuates neuroinflammation and improves neurobehavioral recovery after cervical spinal cord injury.

BACKGROUND: Evidence suggests that the inflammatory events in the acute phase of spinal cord injury (SCI) exacerbate the initial trauma to the cord leading to poor functional recovery. As a result, minimizing the detrimental aspects of the inflammatory response after SCI is a promising treatment strategy. In this regard, immunoglobulin G (IgG) from pooled human serum is a promising treatment candidate. Due to its putative, though poorly characterized immuno-modulatory effects, IgG has been used clinically to treat neuroinflammatory disorders such as Guillain-Barré syndrome, but its effects in neurotrauma remain largely unexplored. METHODS: This study examines the potential neuroprotective effects of IgG in a well-characterized cervical model of SCI. Female Wistar rats were subject to moderate-severe clip compression injury at the C7-T1 level. IgG (0.4 g/kg) or saline was injected intravenously to randomly selected animals at 15 min post SCI. At several time points post SCI, biochemical assays, histology and immunohistochemistry analyses, and neurobehavioral assessments were used to examine the neuroprotective effects of IgG at the molecular, cellular, and neurobehavioral levels. RESULTS: We found that intravenous treatment of IgG following acute clip-compression SCI at C7-T1 significantly reduced two important inflammatory cytokines: interleukin (IL)-1ß and IL-6. This early reduction in pro-inflammatory signaling was associated with significant reductions in neutrophils in the spinal cord and reductions in the expression of myeloperoxidase and matrix metalloproteinase-9 in the injured spinal cord at 24 h after SCI. These beneficial effects of IgG were associated with enhanced tissue preservation, improved neurobehavioral recovery as measured by the BBB and inclined plane tests, and enhanced electrophysiological evidence of central axonal conduction as determined by motor-evoked potentials. CONCLUSION: The findings from this study indicate that IgG is a novel immuno-modulatory therapy which shows promise as a potential treatment for SCI.

Influence of testing modality on bioefficacy for the evaluation of Interceptor® G2 mosquito nets to combat malaria mosquitoes in Tanzania.

BACKGROUND: Insecticide-treated net (ITN) durability is evaluated using longitudinal bioefficacy and fabric integrity sampling post-distribution. Interceptor® G2 was developed for resistance management and contains two adulticides: alpha-cypermethrin and chlorfenapyr; it is a pro-insecticide that is metabolized into its active form by mosquito-detoxifying enzymes and may be enhanced when the mosquito is physiologically active. To elucidate the impact of bioassay modality, mosquito exposures of the alphacypermethrin ITN Interceptor® and dual adulticide Interceptor® G2 were investigated. METHODS: This study evaluated the performance of Interceptor® G2 compared to Interceptor® against local strains of mosquitoes in Tanzania. Unwashed and 20× times washed nets were tested. Efficacy of ITNs was measured by four bioassay types: (1) World Health Organisation (WHO) cone test (cone), (2) WHO tunnel test (tunnel), (3) Ifakara ambient chamber test (I-ACT) and (4) the WHO gold standard experimental hut test (hut). Hut tests were conducted against free-flying wild pyrethroid metabolically resistant Anopheles arabiensis and Culex quinquefasciatus. Cone, tunnel and I-ACT bioassays used laboratory-reared metabolically resistant An. arabiensis and Cx. quinquefasciatus and pyrethroid susceptible Anopheles gambiae sensu stricto and Aedes aegypti. RESULTS: Against resistant strains, superiority of Interceptor® G2 over Interceptor® was observed in all "free-flying bioassays". In cone tests (which restrict mosquito flight), superiority of Interceptor® over Interceptor® G2 was recorded. Mortality of unwashed Interceptor® G2 among An. arabiensis was lowest in hut tests at 42.9% (95% CI: 37.3-48.5), although this increased to 66.7% (95% CI: 47.1-86.3) by blocking hut exit traps so mosquitoes presumably increased frequencies of contact with ITNs. Higher odds of mortality were consistently observed in Interceptor® G2 compared to Interceptor® in "free-flying" bioassays using An. arabiensis: tunnel (OR = 1.42 [95% CI:1.19-1.70], p < 0.001), I-ACT (OR = 1.61 [95% CI: 1.05-2.49], p = 0.031) and hut (OR = 2.53 [95% CI: 1.96-3.26], p < 0.001). Interceptor® and Interceptor® G2 showed high blood-feeding inhibition against all strains. CONCLUSION: Both free-flying laboratory bioassays (WHO Tunnel and I-ACT) consistently measured similarly, and both predicted the results of the experimental hut test. For bioefficacy monitoring and upstream product evaluation of ITNs in situ, the I-ACT may provide an alternative bioassay modality with improved statistical power. Interceptor G2® outperformed Interceptor ® against pyrethroid-resistant strains, demonstrating the usefulness of chlorfenapyr in mitigation of malaria.

Multiplex polymerase chain reaction diagnostics of bed bug (Hemiptera: Cimicidae).

Cimex lectularius L. (Hemiptera: Cimicidae) is a widespread blood feeding pest of humans around the world, including North America, and has recently undergone a resurgence. A molecular diagnostic technique applying multiplex polymerase chain reaction (PCR) was developed to distinguish bed bug eggs, leg fragments, and degraded samples from other arthropods that frequently occur in human dwellings. A 410-428-bp region of the mitochondrial DNA 16S rRNA gene was used. To design C. lectularius-specific PCR primers, DNA sequences of various bed bug samples from the United States, Canada, and Australia, along with sequences of other Cimicidae and arthropods that often occur in dwellings, were considered. Based on DNA sequence variation, one reverse PCR primer specific for C. lectularius was identified. Multiplex PCR using three primers will yield a 417- and 140-bp amplicon for C. lectularius and a single 410-428-bp amplicon for other taxa. This assay was successful in identifying C. lectularius eggs, leg fragments, and degraded samples. This technique should provide a reliable, quick, and economical technique for identifying C. lectularius, when morphological identification is not possible.

Shared transcriptional profiles of atypical B cells suggest common drivers of expansion and function in malaria, HIV, and autoimmunity.

Chronic infectious diseases have a substantial impact on the human B cell compartment including a notable expansion of B cells here termed atypical B cells (ABCs). Using unbiased single-cell RNA sequencing (scRNA-seq), we uncovered and characterized heterogeneities in naïve B cell, classical memory B cells, and ABC subsets. We showed remarkably similar transcriptional profiles for ABC clusters in malaria, HIV, and autoimmune diseases and demonstrated that interferon-γ drove the expansion of ABCs in malaria. These observations suggest that ABCs represent a separate B cell lineage with a common inducer that further diversifies and acquires disease-specific characteristics and functions. In malaria, we identified ABC subsets based on isotype expression that differed in expansion in African children and in B cell receptor repertoire characteristics. Of particular interest, IgD+IgMlo and IgD-IgG+ ABCs acquired a high antigen affinity threshold for activation, suggesting that ABCs may limit autoimmune responses to low-affinity self-antigens in chronic malaria.

A quantitative ELISA for bioactive anti-Nogo-A, a promising regenerative molecule for spinal cord injury repair.

The detection and quantification of bioactive anti-Nogo-A mAbs, which is of interest for the treatment of spinal cord injury, has previously been accomplished using cellular or indirect immunoassays. In one such assay the presence of Nogo-A inhibits neurite outgrowth from the PC12 neuronal cell line: pre-treatment with anti-Nogo-A overcomes this inhibition and the concentration of anti-Nogo-A is correlated with the reduction in growth inhibition. In the current work we demonstrate the first anti-Nogo-A sandwich ELISA utilizing a Nogo-A fragment in the role of capture agent and the anti-Nogo-A mAb 11c7 as the soluble analyte. Because the Nogo-A fragment contains the amino acid sequence against which 11c7 was raised, we postulate this combination reproduces the native binding mechanism and results in the detection of bioactive anti-Nogo-A. In support of this hypothesis, we have found good agreement between the inhibitory action of the Nogo-A fragment and myelin proteins used in existing PC12 cell assays. Importantly, unlike the several days required for cellular assays the ELISA is a fast and easy to use method for the detection and quantification of bioactive 11c7 in the range of 500-6000 pg/mL.

Systemic polyethylene glycol promotes neurological recovery and tissue sparing in rats after cervical spinal cord injury.

Polyethylene glycol (PEG) has been reported to possess fusogenic properties that may confer neuroprotection after spinal cord injury (SCI), but there is uncertainty regarding the mechanisms of PEG in vivo and the robustness of its protective effects. We hypothesized that PEG promotes preservation of cytoskeletal proteins associated with white matter protection and neurobehavioral recovery after SCI. In proof-of-principle experiments using a pin-drop organotypic culture model of SCI, PEG attenuated neural cell death. Adult rats underwent 35-g clip compression SCI at C8 and were randomized postinjury to receive intravenous 30% PEG or sterile Ringer's lactate solution. Confocal microscopy and high-performance liquid chromatography of fluorescein-conjugated PEG permitted in vivo quantification of PEG concentrations in the injured and uninjured spinal cord. Western blot, immunohistochemistry, and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining demonstrated that PEG reduced 200-kd neurofilament degradation and apoptotic cell death. Polyethylene glycol also promoted spinal cord tissue sparing based on retrograde axonal Fluoro-Gold tracing and morphometric histological assessment. Polyethylene glycol also promoted significant, although modest, neurobehavioral recovery after SCI. Collectively, these results indicate that PEG protects key axonal cytoskeletal proteins after SCI, and that the protection is associated with axonal preservation. The modest extent of locomotor recovery after treatment with PEG suggests, however, that this compound may notconfer sufficient neuroprotection to be used clinically as a single treatment.

Overexpression of T-bet in HIV infection is associated with accumulation of B cells outside germinal centers and poor affinity maturation.

Nearly all chronic human infections are associated with alterations in the memory B cell (MBC) compartment, including a large expansion of CD19hiT-bethi MBC in the peripheral blood of HIV-infected individuals with chronic viremia. Despite their prevalence, it is unclear how these B cells arise and whether they contribute to the inefficiency of antibody-mediated immunity in chronic infectious diseases. We addressed these questions by characterizing T-bet-expressing B cells in lymph nodes (LN) and identifying a strong T-bet signature among HIV-specific MBC associated with poor immunologic outcome. Confocal microscopy and quantitative imaging revealed that T-bethi B cells in LN of HIV-infected chronically viremic individuals distinctly accumulated outside germinal centers (GC), which are critical for optimal antibody responses. In single-cell analyses, LN T-bethi B cells of HIV-infected individuals were almost exclusively found among CD19hi MBC and expressed reduced GC-homing receptors. Furthermore, HIV-specific B cells of infected individuals were enriched among LN CD19hiT-bethi MBC and displayed a distinct transcriptome, with features similar to CD19hiT-bethi MBC in blood and LN GC B cells (GCBC). LN CD19hiT-bethi MBC were also related to GCBC by B cell receptor (BCR)-based phylogenetic linkage but had lower BCR mutation frequencies and reduced HIV-neutralizing capacity, consistent with diminished participation in GC-mediated affinity selection. Thus, in the setting of chronic immune activation associated with HIV viremia, failure of HIV-specific B cells to enter or remain in GC may help explain the rarity of high-affinity protective antibodies.

Molecular mechanisms of Fas-mediated cell death in oligodendrocytes.

Oligodendrocyte cell death is a significant component of the secondary damage following spinal cord injury (SCI) and other neurodegenerative disorders. However, the mechanisms underlying oligodendroglial apoptotic cell death and the potential relationship to Fas receptor (FasR) activation require further clarification. Here, using MO3.13, a human oligodendroglial cell line, we show clear evidence of apoptosis upon exposure to soluble Fas ligand (sFasL). Apoptosis was linked to caspase-8, -9, and -3 activity and resulted in DNA fragmentation detected by deoxynucleotide transferase dUTP nick end-labeling (TUNEL). Dissipation of mitochondrial membrane potential (DeltaPsim) was an early event and temporally coincided with mitochondrial outer membrane permeability (MOMP), demonstrated by the presence of cytochrome c and apoptosis inducing factor (AIF) in cytosolic fractions. Pretreatment with 100 microM of the caspase inhibitor zVAD-fmk prior to sFasL exposure reduced caspase activation, the dissipation of DeltaPsim, MOMP, and apoptotic cell death. These data provide clear evidence that Fas activation induces apoptosis in oligodendrocytes signaling through intrinsic and extrinsic events. Moreover, we provide evidence for the first time that AIF may play a role in caspase-independent apoptotic execution following Fas activation of oligodendrocytes. These data also add to an emerging body of evidence, which strongly implicates Fas-mediated apoptosis of oligodendrocytes as a potential mediator in the pathobiology of a variety of neurological disorders, including SCI.