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Summary: Purinergic receptors and nucleotide-binding domain leucine-rich repeat containing (NLR) proteins have been shown to control viral infection. Here, we show that the NLR family member NLRP3 and the purinergic receptor P2Y2 constitutively interact and regulate susceptibility to HIV-1 infection. We found that NLRP3 acts as an inhibitory factor of viral entry that represses F-actin remodeling. The binding of the HIV-1 envelope to its host cell receptors (CD4, CXCR4, and/or CCR5) overcomes this restriction by stimulating P2Y2. Once activated, P2Y2 enhances its interaction with NLRP3 and stimulates the recruitment of the E3 ubiquitin ligase CBL to NLRP3, ultimately leading to NLRP3 degradation. NLRP3 degradation is permissive for PYK2 phosphorylation (PYK2Y402∗) and subsequent F-actin polymerization, which is required for the entry of HIV-1 into host cells. Taken together, our results uncover a mechanism by which HIV-1 overcomes NLRP3 restriction that appears essential for the accomplishment of the early steps of HIV-1 entry. : Paoletti et al. identified a constitutive interaction between NLRP3 and P2Y2 that regulates HIV-1 entry into target cells. They revealed that NLRP3 represses viral entry by impairing F-actin reorganization. HIV-1 overcomes this host cellular resistance by inducing NLRP3 degradation through the activation of P2Y2-dependent signaling pathway. Keywords: NLRP3, P2Y2, CBL, inflammasome, viral entry, HIV

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ?accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. "Regulated cell death" (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Nomenclature Committee on Cell Death. Equipe 11 Apoptose, Cancer et Immunité. Centre de Recherche des Cordeliers; Francia

Background HIV-associated neurocognitive disorders (HAND) persist in the post-HAART era, characterized by asymptomatic neurocognitive impairment (ANI) and mild neurocognitive disorders (MND). High mobility group box 1 (HMGB1) is a non-histone chromosomal protein widely expressed in the nucleus of all eukaryotic cells, including brain cells, which acts as a potent proinflammatory cytokine when actively secreted from immune cells. Recent reports suggested that HMGB1 acts on microglial cells to promote neuroinflammation. In this study, our aim was to determine whether HMGB1 is involved in HAND, but also to identify early new markers of neurological impairment in HIV-infected patients.

SC599 vaccine is a live Shigella dysenteriae 1 strain attenuated by deletion of invasion [icsA], iron chelation [ent, fep] and shiga toxin A subunit [stxA] genes. In a preliminary Phase 1 single dose prospective study, we showed that SC599 vaccine was well tolerated, and the maximum tolerable dose was greater than 10(8) CFU [Sadorge C, Ndiaye A, Beveridge N, Frazer S, Giemza R, Jolly N, et al. Phase 1 clinical trial of live attenuated Shigella dysenteriae type-1 DeltaicsA Deltaent Deltafep DeltastxA:HgR oral vaccine SC599 in healthy human adult volunteers. Vaccine 2008; 26(7):978-8]. In this Phase 2 trial, three groups of volunteers ingested a single dose of SC599 [10(5) CFU, n=38; 10(7) CFU, n=36] or placebo [n=37]. Both 10(5) and 10(7) CFU doses were immunogenic, inducing significant IgA and IgG LPS-specific ASCs and antibody responses, comparable in magnitude to those of other strains that prevented illness following experimental challenge. In the intention to treat analysis, 34.2% and 44.4% IgA ASC responders were detected in the 10(5) and 10(7) CFU groups respectively (p0001 vs placebo for both groups), as well as 31.6% and 33.3% serum IgA responders (p001 and p0.001 vs placebo for 10(5) and 10(7) CFU groups, respectively). No difference between the two vaccine groups was observed. No stxB-specific antibody response was detected in the vaccines. SC599 excretion occurred in 23.7 and 30.6% of subjects in the 10(5) and 10(7) CFU groups, respectively. SC599 vaccine was well tolerated, and the reported adverse events were mainly digestive. These results indicate that a single oral immunization of SC599 vaccine elicits a significant circulating IgA ASC and serum antibody response that may confer protection against the most severe symptoms of Shigellosis in responders to the vaccine.

Systemic chemotherapy generally has been considered immunosuppressive, but it has become evident that certain chemotherapeutic drugs elicit immunogenic danger signals in dying cancer cells that can incite protective antitumor immunity. In this study, we investigated whether locoregionally applied therapies, such as melphalan, used in limb perfusion for melanoma (Mel-ILP) produce related immunogenic effects. In human melanoma biopsies, Mel-ILP treatment upregulated IL1B, IL8, and IL6 associated with their release in patients' locoregional sera. Although induction of apoptosis in melanoma cells by melphalan in vitro did not elicit threshold levels of endoplasmic reticulum and reactive oxygen species stress associated with danger signals, such as induction of cell-surface calreticulin, prophylactic immunization and T-cell depletion experiments showed that melphalan administration in vivo could stimulate a CD8+ T cell–dependent protective antitumor response. Interestingly, the vaccination effect was potentiated in combination with exogenous calreticulin, but not tumor necrosis factor, a cytokine often combined with Mel-ILP. Our results illustrate how melphalan triggers inflammatory cell death that can be leveraged by immunomodulators such as the danger signal calreticulin. Cancer Res; 75(8); 1603–14. ©2015 AACR.

HIV-1 infection is associated with immune deficiency and metabolic perturbations leading to malnutrition and lipodystrophy. Because immune response and metabolic perturbations (protein and lipid metabolism) are partly regulated by glucocorticoids and DHEA, we determined serum cortisol and DHEA concentrations, and the cortisol/DHEA ratio in HIV-positive men, either untreated or receiving various antiretroviral treatments (ART), including highly active antiretroviral therapy (HAART). Cortisol levels were found increased in all patients, whatever the stage of the disease and independently of the ART treatment. In contrast, serum DHEA was elevated in the asymptomatic stage, and it was below normal values in AIDS patients, either untreated or mono-ART-treated. The DHEA level was low in HAART-treated patients with lipodystrophy (LD+) and highly increased in HAART-treated patients without lipodystrophy (LD-). Consequently, the cortisol/DHEA ratio was similar to controls in asymptomatic untreated or mono-ART-treated patients, but increased in AIDS patients. Interestingly, this ratio was increased in LD+ HAART-treated men, but normalized in LD- HAART-treated patients. Changes in the cortisol/DHEA ratio were negatively correlated with the in vivo CD4 T-cell counts, with the malnutrition markers, such as body-cell mass and fat mass, and with the increased circulating lipids (cholesterol, triglycerides, and apolipoprotein B) associated to the lipodystrophy syndrome. Our observations show that the cortisol/DHEA ratio is dramatically altered in HIV-infected men, particularly during the syndromes of malnutrition and lipodystrophy, and this ratio remains elevated whatever the antiretroviral treatment, including HAART. These findings have practical clinical implications, since manipulation of this ratio could prevent metabolic (protein and lipid) perturbations.

Thimerosal is a preservative used in multidose vials of vaccine formulations to prevent bacterial and fungal contamination. We recently reported that nanomolar concentrations of thimerosal induce cell cycle arrest of human T cells activated via the TCR and inhibition of proinflammatory cytokine production, thus interfering with T-cell functions. Given the essential role of dendritic cells (DCs) in T-cell polarization and vaccine immunity, we studied the influence of non-toxic concentrations of thimerosal on DC maturation and functions. Ex-vivo exposure of human monocyte-derived DCs to nanomolar concentrations of thimerosal prevented LPS-induced DC maturation, as evidenced by the inhibition of morphological changes and a decreased expression of the maturation markers CD86 and HLA-DR. In addition thimerosal dampened their proinflammatory response, in particular the production of the Th1 polarizing cytokine IL-12, as well as TNF-α and IL-6. DC-dependent T helper polarization was altered, leading to a decreased production of IFN-γ IP10 and GM-CSF and increased levels of IL-8, IL-9, and MIP-1α. Although multi-dose vials of vaccines containing thimerosal remain important for vaccine delivery, our results alert about the ex-vivo immunomodulatory effects of thimerosal on DCs, a key player for the induction of an adaptive response.

There is growing interest in the use of innate immune reactions in the therapy and prophylaxis of various diseases. Natural T (NT) lymphocytes that recognize infected cells or microbial compounds without the classical genetic restriction by polymorphic MHC molecules are crucial components of innate immunity. NT cells bearing the Vgamma9Vdelta2 T-cell receptor (TCR) are broadly reactive against intracellular pathogens, can lyse human immunodeficiency virus (HIV) infected cells, and release cytokines capable of regulating HIV replication. The potent antiviral activities of Vgamma9Vdelta2 T cells may help to contain viral spread during acute HIV infection and/or to prevent the establishment of viral persistence. Substantial changes in the composition and function of circulating gammadelta T-cell pools occur in HIV-infected patients. These changes a) may contribute to the etiopathogenesis of opportunistic infections and neoplasms, and b) are partly reversed by highly active anti-retroviral therapy (HAART). In addition to direct antiviral activities, activated gammadelta T cells influence dendritic cell maturation and the adaptive alphabeta T-cell response. Vgamma9Vdelta2 T cells can be stimulated in vivo and in vitro by various nonpeptidic antigens (NpAgs) and recent animal experimental data suggest that activated Vgamma9Vdelta2 T cells may help to control SIV replication. Currently, NpAgs are being assessed as potential therapeutic agents in AIDS, tuberculosis and certain cancers susceptible to Vgamma9Vdelta2 T-cell effector mechanisms.

Background: A syndrome of lipodystrophy, associated with hypertriglyceridaemia, hypercholesterolaemia, hyperinsulinaemia and peripheral insulin resistance has been reported in protease inhibitor (PI)-treated HIV-infected patients. Because lipid metabolism, fat mass distribution and insulin resistance are partly regulated by steroid hormones, we questioned whether lipodystrophy is related to hormonal perturbations. Objective: To evaluate serum lipid and steroid hormone concentrations in HIV-positive men on highly active antiretroviral therapy (HAART) in order to determine whether dyslipidaemia, peripheral loss of fatty tissue and central fat accumulation are related to steroid hormone modifications. Design: A cross-sectional study. Methods: Thirty-seven HIV-1-positive men on HAART, 23 of whom had symptoms of lipodystrophy, according to a subjective clinical score of lipodystrophy (SCSL), were tested. Serum concentrations of cholesterol, triglycerides and their subclasses, apolipoproteins and steroid hormones, including cortisol, dehydroepiandrosterone (DHEA), DHEA sulphate, androstenedione, testosterone and dihydrotestosterone were measured. Results: Serum cholesterol, very low density lipoprotein (VLDL) cholesterol, triglycerides, VLDL triglycerides, high density lipoprotein (HDL) and low density lipoprotein (LDL) triglycerides, apolipoprotein B (ApoB) and atherogenic ratios of cholesterol:HDL cholesterol, LDL cholesterol:HDL cholesterol and ApoB:apolipoprotein A1 (ApoA1) were significantly increased in lipodystrophy-positive compared with lipodystrophy-negative men. The serum cortisol level was similar in lipodystrophy-positive versus lipodystrophy-negative men, but was elevated compared with controls. Serum DHEA was significantly lower in lipodystrophy-positive versus lipodystrophy-negative men and, consequently, the cortisol:DHEA ratio was increased in lipodystrophy-positive patients. A positive correlation was found between the cortisol:DHEA ratio and increased levels of atherogenic lipids. In addition, the SCSL was positively correlated with dyslipidaemia and the cortisol:DHEA ratio. Conclusion: This study demonstrates an association between the cortisol:DHEA ratio, lipid alterations and lipodystrophy. This syndrome might result from an imbalance between peripheral lipolysis and lipogenesis, both regulated by cortisol and DHEA.

Most adult peripheral blood gammadelta T cells express Vgamma9/Vdelta2-encoded TCR that recognize a restricted set of nonpeptidic phosphorylated compounds, referred to as phosphoantigens. They also express various MHC class I-specific inhibitory receptors (IR), in particular CD94/ NKG2-A heterodimers, which participate in the fine tuning of their TCR-mediated activation threshold. Most mature Vgamma9/Vdelta2 T cells express surface CD94 receptors, unlike cord blood or thymus-derived Vgamma9/Vdelta2 clones, thus suggesting a role for the microenvironment in IR expression. In the present study we show that most CD94- Vgamma9Vdelta2 PBL ex vivo express an intracellular pool of CD94/NKG2-A receptors that is translocated to the cell surface upon activation by phosphoantigens or IL-2. In stark contrast, intracellular CD94/NKG2-A complexes are undetectable in CD94- thymus or PBL-derived mature Vdelta2 T cell clones, and no surface induction is observed following phosphoantigen activation of T cell clones. Altogether these results provide new insights into the regulation of CD94/NKG2-A expression on T lymphocytes and suggest the existence of distinct mechanisms controlling in vivo and in vitro induction of IR on these cells.

Vγ9Vδ2-encoded T-cell receptors crossreact with a variety of nonpeptidic phosphoantigens derived from numerous infectious agents. The reactivities of Vγ9Vδ2 T cells induced by different stimuli are controlled by receptors for major histocompatibility complex class I molecules. These and other important areas of natural anti-infectious immunity were discussed at a recent workshop1The International Workshop on Innate Immunity in AIDS and Tuberculosis was held at Valle d'Aosta, Italy, on 1–3 December 1997. 1 .

The present article compares the reliability of four previously described cytofluorometric methods of apoptosis quantification for phenotyping apoptotic human lymphocytes. Each of these assays detects distinct cellular alterations of the apoptotic process. Alteration in plasma membrane integrity can be evaluated following 7-AAD incorporation and the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane can be detected through the FITC–annexin V staining. DNA strand breaks in apoptotic nuclei can be evidenced by the ISNT assay and finally morphological modifications can be followed with FSC/SSC criteria. Comparative analysis of apoptosis in cultured PBMC from HIV-infected patients considering the FSC/SSC parameters, 7-AAD stainability and annexin V fixation revealed that the latter identifies early apoptotic cells, also characterized as 7-AADlow with a reduced FSC. Moreover these three methods proved to be reliable and gave statistically similar results when combined with cell surface detection of antigens such as CD4, CD8 and CD19 by specific mAbs. Importantly, the 7-AAD assay easily allowed the identification of debris/apoptotic bodies, which were still stained by anti-cell surface mAbs and might therefore significantly distort the apoptosis percentage in a given lymphocyte subset. In the present report we also point out that the ISNT assay is not appropriate for phenotyping apoptotic lymphocytes in PBMC. Indeed it can particularly underestimate the rate of apoptosis in the B-cell subset. This was found to be related to the apoptosis-associated decrease in cell surface antigen expression, which is dramatically exacerbated in the ISNT assay because of the stripper effect of ethanol used for cell permeabilization. Finally, we propose a three step analytical strategy to accurately phenotype apoptotic peripheral human lymphocytes. It includes two gating steps performed on FSC/SSC criteria and 7-AAD/FSC parameters to eliminate monocytes, granulocytes and debris-apoptotic bodies, the third step being the phenotyping step itself, performed in dual or triple staining experiments. Altogether these observations emphasize that it is essential to assess critically the ability of a cytofluorometric method to phenotype apoptotic cells in complex lymphoid populations and that inaccurate identification of cell subsets undergoing apoptosis can be readily overcome by gating properly the lymphoid population, and using assays which preserve cell surface structure.

Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover, Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands, such as HIV and CpG respectively, turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions, and pDC production of IFN-α as well as cell–cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection, but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here, we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α, TNF-α, IFN-γ and IL-12, and CCR5-interacting chemokines (MIP-1α and MIP-1β) in NK-pDCs co-cultures. At high HIV-1BaL concentrations, the addition of NK cells did not promote the release of these mediators, suggesting that once efficiently triggered by the virus, pDCs could not integrate new activating signals delivered by NK cells. However, high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly, we identified the alarmin HMGB1, released at pDC-NK cell synapse, as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover, HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1, HMGB1-specific antibodies, sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether, these findings identify HMGB1 as a trigger for IFN-α-mediated TRAIL expression at the surface of pDCs and NK cells, and they suggest a novel mechanism of innate control of HIV-1 infection., Author Summary Plasmacytoid dendritic cells (pDC) are the most potent IFN-α-producing cells and serve as an essential link between innate and adaptive immunity. Exposure of pDCs to HIV-1 triggers IFN-α production, which in turn upregulates TNF-related apoptosis-inducing ligand (TRAIL), turning pDCs into killer pDCs, able to kill infected CD4+ T cells. At sites of infection, pDCs might activate or get activated by Natural killer (NK) cells, and pDC-NK cell-cell contact is required to promote the cytolytic potential of NK cells. Functional defects in the pDC and NK cell compartments were reported in the setting of HIV-1 infection, but the precise mechanisms by which HIV impairs NK cell and pDC crosstalk remain to be fully elucidated. To address this question, we developed an ex-vivo model of NK-pDC interaction, based on a short-term contact between sorted peripheral NK cells and purified pDCs exposed to HIV-1BaL. We found that the concentration of HIV-1 is critical to sustain the functional activation of both pDCs and NK cells. Moreover, we identified the alarmin HMGB1 as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1-exposed pDCs and NK cells. HMGB1 was also found crucial for HIV-1-induced translocation of TRAIL on both pDC and NK cell membrane. The in vivo relevance of the interdependency between HMGB1, IFN- and TRAIL is suggested by the strong positive correlations between circulating levels of these mediators in a cohort of 67 HIV-1 infected patients. Altogether these findings highlight a new function for HMGB1 and they suggest a novel mechanism of innate control of HIV infection.

Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France* Corresponding author: M-L Gougeon, Institut Pasteur, Molecular Medicine Department, 28 rue du Dr Roux, 75724 Paris Cedex 15, France. Tel: + 33145688907; fax:+ 33145688909; E-mail: mlgougeo@pasteur.fr

Dendritic cells (DCs) initiate immune responses by transporting antigens and migrating to lymphoid tissues to initiate T-cell responses. DCs are located in the mucosal surfaces that are involved in human immunodeficiency virus (HIV) transmission and they are probably among the earliest targets of HIV-1 infection. DCs have an important role in viral transmission and dissemination, and HIV-1 has evolved different strategies to evade DC antiviral activity. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can act as an alarmin, a danger signal to alert the innate immune system for the initiation of host defense. It is the prototypic damage-associated molecular pattern molecule, and it can be secreted by innate cells, including DCs and natural killer (NK) cells. The fate of DCs is dependent on a cognate interaction with NK cells, which involves HMGB1 expressed at NK–DC synapse. HMGB1 is essential for DC maturation, migration to lymphoid tissues and functional type-1 polarization of naive T cells. This review highlights the latest advances in our understanding of the impact of HIV on the interactions between HMGB1 and DCs, focusing on the mechanisms of HMGB1-dependent viral dissemination and persistence in DCs, and discussing the consequences on antiviral innate immunity, immune activation and HIV pathogenesis.

Human CD4+ αβ T cells are activated via T-cell receptor recognition of peptide epitopes presented by major histocompatibility complex (MHC) class II (MHC-II). The open ends of the MHC-II binding groove allow peptide epitopes to extend beyond a central nonamer core region at both the amino- and carboxy-terminus. We have previously found that these non-bound C-terminal residues can alter T cell activation in an MHC allele-transcending fashion, although the mechanism for this effect remained unclear. Here we show that modification of the C-terminal peptide-flanking region of an influenza hemagglutinin (HA305−320) epitope can alter T-cell receptor binding affinity, T-cell activation and repertoire selection of influenza-specific CD4+ T cells expanded from peripheral blood. These data provide the first demonstration that changes in the C-terminus of the peptide-flanking region can substantially alter T-cell receptor binding affinity, and indicate a mechanism through which peptide flanking residues could influence repertoire selection., Epitopes presented by MHC-II molecules bind to T-cell receptors to activate CD4+ T cells. In this study, changes in the carboxy-terminal region of the influenza hemagglutinin epitope HA305-320 alters the strength of binding to the T-cell receptor, thus modulating T-cell receptor usage and activation.

Contact with HIV-1 envelope protein elicits release of ATP through pannexin-1 channels on target cells; by activating purinergic receptors and Pyk2 kinase in target cells, this extracellular ATP boosts HIV-1 infectivity., Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches.

PURPOSE OF REVIEW Natural killer (NK) cells promote antiviral immunity by producing proinflammatory cytokines and by lysing infected cells. In addition, NK cells can modulate dendritic cell functions. NK-dendritic cell crosstalk results in activation of both cell types, with dendritic cells promoting NK-cell activity and NK cells inducing further maturation of dendritic cells. Here we review the recent evidence suggesting that NK-dendritic cell crosstalk is disrupted during HIV-1 infection and we discuss the consequences on HIV persistence in dendritic cells. RECENT FINDINGS NK cell-mediated dendritic cell editing is compromised during HIV-1 infection, and NK cells from viremic individuals show a decreased ability to kill immature dendritic cells. The defect is associated with impaired NKp30 function. Moreover, the resistance of HIV-1-infected dendritic cells to NK-mediated lysis is associated with the upregulation of apoptosis inhibitors, thus protecting infected dendritic cells from TRAIL-dependent apoptosis. These inhibitors are upregulated by the high-mobility group box 1 protein (HMGB1), an alarmin produced at NK-dendritic cell synapse that is essential for NK-dependent dendritic cell maturation, but also promotes viral replication in infected dendritic cells. SUMMARY HIV-1-induced impairment of NK-dendritic cell crosstalk may significantly alter both innate and adaptive immunity. It may also contribute to HIV persistence in dendritic cells through an HMGB1-dependent mechanism.

Early stages of Human Immunodeficiency Virus-1 (HIV-1) infection are associated with local recruitment and activation of important effectors of innate immunity, i.e. natural killer (NK) cells and dendritic cells (DCs). Immature DCs (iDCs) capture HIV-1 through specific receptors and can disseminate the infection to lymphoid tissues following their migration, which is associated to a maturation process. This process is dependent on NK cells, whose role is to keep in check the quality and the quantity of DCs undergoing maturation. If DC maturation is inappropriate, NK cells will kill them (“editing process”) at sites of tissue inflammation, thus optimizing the adaptive immunity. In the context of a viral infection, NK-dependent killing of infected-DCs is a crucial event required for early elimination of infected target cells. Here, we report that NK-mediated editing of iDCs is impaired if DCs are infected with HIV-1. We first addressed the question of the mechanisms involved in iDC editing, and we show that cognate NK-iDC interaction triggers apoptosis via the TNF-related apoptosis-inducing ligand (TRAIL)-Death Receptor 4 (DR4) pathway and not via the perforin pathway. Nevertheless, once infected with HIV-1, DCHIV become resistant to NK-induced TRAIL-mediated apoptosis. This resistance occurs despite normal amounts of TRAIL released by NK cells and comparable DR4 expression on DCHIV. The escape of DCHIV from NK killing is due to the upregulation of two anti-apoptotic molecules, the cellular-Flice like inhibitory protein (c-FLIP) and the cellular inhibitor of apoptosis 2 (c-IAP2), induced by NK-DCHIV cognate interaction. High-mobility group box 1 (HMGB1), an alarmin and a key mediator of NK-DC cross-talk, was found to play a pivotal role in NK-dependent upregulation of c-FLIP and c-IAP2 in DCHIV. Finally, we demonstrate that restoration of DCHIV susceptibility to NK-induced TRAIL killing can be obtained either by silencing c-FLIP and c-IAP2 by specific siRNA, or by inhibiting HMGB1 with blocking antibodies or glycyrrhizin, arguing for a key role of HMGB1 in TRAIL resistance and DCHIV survival. These findings provide evidence for a new strategy developed by HIV to escape immune attack, they challenge the question of the involvement of HMGB1 in the establishment of viral reservoirs in DCs, and they identify potential therapeutic targets to eliminate infected DCs., Author Summary Dendritic cells (DCs), the professional antigen presenting cells, are critical for host immunity by inducing specific immune responses against a broad variety of pathogens. Human Immunodeficiency Virus-1 (HIV-1) has evolved ways to exploit DCs, thereby facilitating viral dissemination and allowing evasion of antiviral immunity. In particular, infected DCs may function as cellular reservoirs for HIV-1, thus contributing to viral persistence in lymphoid tissues. The mechanisms involved in the constitution of HIV reservoirs in DCs are poorly understood. In this study, we reveal that DCs infected with HIV-1 (DCHIV) become resistant to killing by natural killer (NK) cells, early effectors of innate immunity involved in the destruction of virus infected cells or cancer cells. This protection of DCHIV from NK cytotoxicity is induced through a cross-talk between NK cells and DCHIV, which induces the upregulation in DCHIV of two inhibitors of cell death, i.e. cellular-Flice like inhibitory protein (c-FLIP) and cellular inhibitor of apoptosis 2 (c-IAP2). The molecule responsible for the induction of these inhibitors is High-mobility group box 1 (HMGB1), an alarmin involved in the functional maturation of DCs. Blocking HMGB1 restores DCHIV susceptibility to NK cell killing, arguing for a key role of HMGB1 in the persistence of DCHIV. These findings provide evidence of the crucial role of NK-DC cross-talk in promoting viral persistence, and they identify potential therapeutic targets to eliminate infected DCs.

Background HIV-1 has evolved ways to exploit DCs, thereby facilitating viral dissemination and allowing evasion of antiviral immunity. Recently, the fate of DCs has been found to be extremely dependent on the interaction with autologous NK cells, but the mechanisms by which NK-DC interaction controls viral infections remain unclear. Here, we investigate the impact of NK-DC cross-talk on maturation and functions of HIV-infected immature DCs. Methodology/Principal Findings Immature DCs were derived from primary monocytes, cultured in the presence of IL-4 and GM-CSF. In some experiments, DCs were infected with R5-HIV-1BaL or X4-HIV-1NDK, and viral replication, proviral HIV-DNA and the frequency of infected DCs were measured. Autologous NK cells were sorted and either kept unstimulated in the presence of suboptimal concentration of IL-2, or activated by a combination of PHA and IL-2. The impact of 24 h NK-DC cross-talk on the fate of HIV-1-infected DCs was analyzed. We report that activated NK cells were required for the induction of maturation of DCs, whether uninfected or HIV-1-infected, and this process involved HMGB1. However, the cross-talk between HIV-1-infected DCs and activated NK cells was functionally defective, as demonstrated by the strong impairment of DCs to induce Th1 polarization of naive CD4 T cells. This was associated with the defective production of IL-12 and IL-18 by infected DCs. Moreover, the crosstalk between activated NK cells and HIV-infected DCs resulted in a dramatic increase in viral replication and proviral DNA expression in DCs. HMGB1, produced both by NK cells and DCs, was found to play a pivotal role in this process, and inhibition of HMGB1 activity by glycyrrhizin, known to bind specifically to HMGB1, or blocking anti-HMGB1 antibodies, abrogated NK-dependent HIV-1 replication in DCs. Conclusion These observations provide evidence for the crucial role of NK-DC cross-talk in promoting viral dissemination, and challenge the question of the in vivo involvement of HMGB1 in the triggering of HIV-1 replication and replenishment of viral reservoirs in AIDS.

International audience; DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.

Comparison of TCRαβ repertoires of myelin oligodendrocyte glycoprotein (MOG)-specific T lymphocytes in C57BL/6 and TdT-deficient littermates (TdT−/−) generated during experimental autoimmune encephalomyelitis (EAE) highlights a link between a diversified TCRαβ repertoire and EAE relapses. At the onset of the disease, the EAE-severity is identical in TdT+/− and TdT−/− mice and the neuropathologic public MOG-specific T cell repertoires express closely similar public Vα-Jα and Vβ-Jβ rearrangements in both strains. However, whereas TdT+/+ and TdT+/− mice undergo successive EAE relapses, TdT−/− mice recover definitively and the lack of relapses does not stem from dominant regulatory mechanisms. During the first relapse of the disease in TdT+/− mice, new public Vα-Jα and Vβ-Jβ rearrangements emerge that are distinct from those detected at the onset of the disease. Most of these rearrangements contain N additions and are found in CNS-infiltrating T lymphocytes. Furthermore, CD4+ T splenocytes bearing these rearrangements proliferate to the immunodominant epitope of MOG and not to other immunodominant epitopes of proteolipid protein and myelin basic protein autoantigens, excluding epitope spreading to these myelin proteins. Thus, in addition to epitope spreading, a novel mechanism involving TCRαβ repertoire diversification contributes to autoimmune progression.

The role of B cells in autoimmune diseases has not been fully elucidated. It is also unclear whether breaking of B cell tolerance in patients with autoimmune diseases is due to underlying defects in the molecular mechanisms involved in the arrangement of antibody genes or deficiencies in the subsequent selective influences that shape the antibody repertoire. Analysis of immunoglobulin (Ig) variable (V) gene usage is beginning to provide answers to some of these questions. Such analyses have identified some differences in the basic Ig V gene repertoire of patients with autoimmune diseases compared to healthy controls, even though none of these differences can be considered major. Defects in positive and negative selection, mutational targeting and, in some cases, receptor editing have also been detected. In addition, analysis of Ig V gene usage in target organs and tissues of patients with autoimmune diseases have clearly demonstrated that there is a highly compartmentalized clonal expansion of B cells driven by a limited number of antigens in these tissues. Great progress has been made in the structural and functional characterization of disease-associated antibodies, largely because of the development of the combinatorial library technique. Use of antibodies generated by this technique offers great promise in identifying B cell epitopes on known target antigens and in gaining greater insights into the pathogenic role of B cells in both B- and T-cell-mediated autoimmune diseases.

International audience; Thimerosal is a preservative used widely in vaccine formulations to prevent bacterial and fungal contamination in multidose vials of vaccine. Thimerosal was included in the multidose non-adjuvanted pandemic 2009 H1N1 vaccine Panenza. In the context of the analysis of the ex-vivo T cell responses directed against influenza vaccine, we discovered the in vitro toxicity Panenza, due to its content in thimerosal. Because thimerosal may skew the immune response to vaccines, we investigated in detail the ex-vivo effects of thimerosal on the fate and functions of T cells in response to TCR ligation. We report that ex-vivo exposure of quiescent or TCR-activated primary human T cells to thimerosal induced a dose-dependent apoptotic cell death associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, cytochrome c release from the mitochondria and caspase-3 activation. Moreover, exposure to non-toxic concentrations of thimerosal induced cell cycle arrest in G0/G1 phase of TCR-activated T cells, and inhibition of the release of proinflammatory cytokines such as IFN gamma, IL-1 beta, TNF alpha, IL-2, as well as the chemokine MCP1. No shift towards Th2 or Th17 cells was detected. Overall these results underline the proapoptotic effect of thimerosal on primary human lymphocytes at concentrations 100 times less to those contained in the multidose vaccine, and they reveal the inhibitory effect of this preservative on T-cell proliferation and functions at nanomolar concentrations.

Vgamma9Vdelta2 T lymphocytes are broadly reactive against various intracellular pathogens and display both lytic and proliferative responses to human immunodeficiency virus (HIV)-infected cells. HIV infection of peripheral blood mononuclear cell cultures led to absolute increases in Vgamma9Vdelta2 T cells accompanied by decreased p24 levels. Strong gammadelta T cell activation with nonpeptidic mycobacterial phosphoantigens (TUBAg1 extract or synthetic isopentenyl pyrophosphate) resulted in potent inhibition of HIV replication through soluble released factors. Subsequent analyses showed that phosphoantigen-activated gammadelta T cells produced substantial amounts of beta-chemokines (macrophage inflammatory protein [MIP]-1alpha, MIP-1beta, and regulated-on-activation, normal T-cell-expressed and -secreted beta-chemokine [RANTES]), which represent the natural ligand for the CCR5 HIV coreceptor. Accordingly, anti-beta-chemokine antibodies neutralized the inhibition of monocytotropic HIV strains by gammadelta T cell-released factors. Moreover, a T-tropic HIV strain using the CXCR4 coreceptor for virus entry was potently inhibited. Together, these data reveal that phosphoantigen-activated gammadelta T cells are an important source of CC chemokines and may suppress HIV replication through cell-released antiviral factors.

The aim of this study was to define a simple and reliable method to detect simultaneously surface and intracellular antigens in apoptotic peripheral human lymphocytes. This approach requires a permeabilizing procedure for intracellular access of mAbs, which raises the important question of the influence of this procedure on parameters which identify apoptotic cells and on the surface expression of antigens. We compared the effects of three currently used permeabilizing methods (saponin quillaia bark 0.05%, Triton X-100 0.1, ethanol 70%) on the quantification of apoptotic lymphocytes, defined according to FSC/SSC criteria or following 7-AAD staining, and on the detection of surface CD3, CD4, CD8, Fas, CD45R0 molecules. The combined detection of these surface antigens with intracellular molecules, including Bcl-2 and cytokines (IFN γ , TNF α , IL-2) was also analysed in the context of these three permeabilizing procedures. All the experiments were performed on PBMC from HIV-infected donors, known to undergo excessive apoptosis following short-term culture. We report that permeabilization with saponin is the only procedure which allows: (1) the preservation of lymphocyte morphology determined by the FSC/SSC parameters; (2) the quantification of apoptotic lymphocytes following 7-AAD staining; (3) a reliable surface immunophenotyping, maintaining a good antibody binding capacity (ABC); (4) the proper detection of intracellular membrane bound antigens (Bcl-2) and intracellular cytokines (IFN γ , TNF α , IL-2); (5) the combined detection of apoptotic nuclei, surface antigens and intracellular molecules. Altogether these observations demonstrate that the simultaneous analysis of extracellular and intracellular antigens in apoptotic cells belonging to a complex lymphoid populations such as PBMC can be readily overcome provided the detergent used for cell permeabilization is appropriate and the successive staining procedures performed in a defined order.

The potential deleterious effect through a CD95-based pathway of anti-viral cytotoxic lymphocyte (CTL) during HIV-infection was studied. The present paper reports that a Nef specific CTL line derived from an HIV-infected person is able to kill not only Nef-expressing target cells but also CD95+ compliant Jurkat cells. The two mechanisms of cytotoxicity, i.e. perforin-vs-CD95-dependent were differentiated according to their respective Ca(2+)-dependence. The existence of the dual killing machinery in the anti-HIV CTL line was correlated with the coexpression in these cells of perforin and CD95-L molecules. A model of AIDS pathogenesis involving the deleterious effect through the CD95 pathway of the viral specific CTL response is discussed.

A 38-year-old woman resident of Ivory Coast died of AIDS, while remaining human immunodeficiency virus (HIV)-seronegative. She had been regularly tested because her husband was HIV-seropositive. The subject's lack of specific antibodies was assessed using commercial tests and confirmed by a radioimmunoprecipitation assay of the patient's virus. She was unquestionably HIVI-infected, with a high plasma virus load, and her virus could be isolated. Molecular analyses indicated this retrovirus was clade A, which is common in Africa, and it was highly homologous to the virus isolated from her husband. The subject's seronegative status was thought to be due to rapid depletion of specific CD4 + helper T cells, resulting from accelerated disease progression, and was host-related rather than due to a specific HIV strain.

Mucosal associated invariant T cells (MAIT) are innate T lymphocytes that detect a large variety of bacteria and yeasts. This recognition depends on the detection of microbial compounds presented by the evolutionarily conserved major-histocompatibility-complex (MHC) class I molecule, MR1. Here we show that MAIT cells display cytotoxic activity towards MR1 overexpressing non-hematopoietic cells cocultured with bacteria. The NK receptor, CD161, highly expressed by MAIT cells, modulated the cytokine but not the cytotoxic response triggered by bacteria infected cells. MAIT cells are also activated by and kill epithelial cells expressing endogenous levels of MRI after infection with the invasive bacteria Shigella flexneri. In contrast, MAIT cells were not activated by epithelial cells infected by Salmonella enterica Typhimurium. Finally, MAIT cells are activated in human volunteers receiving an attenuated strain of Shigella dysenteriae-1 tested as a potential vaccine. Thus, in humans, MAIT cells are the most abundant T cell subset able to detect and kill bacteria infected cells., Author Summary Human Mucosa-Associated Invariant T cells (MAIT) detect microbe-derived compounds presented by the MHC-like molecule, MR1. These foreign antigens are produced by a wide variety of microbes, including commensal and pathogenic bacteria or yeasts. MAIT cells expend shortly after birth and constitute the major antibacterial T cell subset described and, hence, could play important roles in infectious diseases. Here we show that MAIT cells recognize epithelial cells infected by the intestinal pathogen Shigella flexneri in a process requiring endogenous MR1, while the closely related bacterium Salmonella Tyhpimurium is not. Upon recognition, infected epithelial cells are efficiently lysed by MAIT cells. We also show that the triggering of CD161, a natural killer receptor highly expressed by MAIT cells, can modulate the cytokine but not the cytotoxic function of these cells. Finally, we provide evidence that MAIT cells are activated during the course of an experimental enteric infection in humans. Our study provides important insight on the antibacterial function of MAIT cells and their interaction with pathogenic bacterial species.

Peripheral blood mononuclear cells and lymphoid tissues from HIV-infected individuals display high levels of "tissue" transglutaminase (tTG) with respect to seronegative persons. In asymptomatic individuals, > 80% of the circulating CD4+ T cells synthesize tTG protein and the number of these cells matches the level of apoptosis detected in the peripheral blood mononuclear cells from the same patients. In HIV-infected lymph nodes tTG protein is localized in large number of cells (macrophages, follicular dendritic cells, and endothelial cells), showing distinctive morphological and biochemical features of apoptosis as well as in lymphocytes and syncytia. These findings demonstrate that during the course of HIV infection, high levels of apoptosis also occur in the accessory cells of lymphoid organs. The increased concentration of epsilon(gamma-glutamyl)lysine isodipeptide, the degradation product of tTG cross-linked proteins, observed in the blood of HIV-infected individuals demonstrates that the enzyme accumulated in the dying cells actively cross-links intracellular proteins. The enhanced levels of epsilon(gamma-glutamyl)lysine in the blood parallels the progression of HIV disease, suggesting that the isodipeptide determination might be a useful method to monitor the in vivo rate of apoptosis.

Mycoplasma penetrans is a mycoplasma species newly isolated from the urine of human immunodeficiency virus (HIV)-infected individuals and presents the only case in which an association has been found between antibodies against a mycoplasma and HIV infection. To further explore the effects of M. penetrans on the immune system, we studied the influence of this mycoplasma on peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-infected individuals. M. penetrans induced, in addition to blastogenesis of PBMCs, a significant proliferative response associated with the expression of some activation markers such as CD69, HLA-DR, and CD25. This M. penetrans-dependent lymphocyte activation was observed not only in healthy donors but also in HIV-infected persons at different stages of the disease. In addition, our study revealed that both CD4+ and CD8+ T lymphocytes were responsive to M. penetrans. Interestingly, the mitogenic activity of M. penetrans was associated with mycoplasma cells but not with the supernatants of mycoplasma culture. The potent stimulating activity of M. penetrans on T lymphocytes from HIV-infected individuals is of particular interest in view of the supposed contribution of immune activation to HIV replication and disease progression.

Background Plasmacytoid dendritic cells (pDCs) mainly contribute to antiviral immunity through recognition of viral components resulting in the production of type-I interferon (IFN), a powerful innate antiviral cytokine. IFN-alpha production by pDCs is promoted by a cross-talk with NK cells, that triggers in return the cytotoxicity of NK cells. Given the essential role of pDCs and NK cells in viral control, we addressed the question of the impact of HIV on NK-pDC cross-talk, and the consequences on viral innate immunity.

Stiff person syndrome (SPS) is a rare, neurological disorder characterized by sudden cramps and spasms. High titers of enzyme-inhibiting IgG autoantibodies against the 65 kD isoform of glutamic acid decarboxylase (GAD65) are a hallmark of SPS, implicating an autoimmune component in the pathology of the syndrome. Studying the B cell compartment and the anti-GAD65 B cell response in two monozygotic twins suffering from SPS, who were treated with the B cell-depleting monoclonal anti-CD20 antibody rituximab, we found that the humoral autoimmune response in SPS is composed of a rituximab-sensitive part that is rapidly cleared after treatment, and a rituximab-resistant component, which persists and acts as a reservoir for autoantibodies inhibiting GAD65 enzyme activity. Our data show that these potentially pathogenic anti-GAD65 autoantibodies are secreted by long-lived plasma cells, which may either be persistent or develop from rituximab-resistant memory B lymphocytes. Both subsets represent only a fraction of anti-GAD65 autoantibody secreting cells. Therefore, the identification and targeting of this compartment is a key factor for successful treatment planning of SPS and of similar autoimmune diseases.

Background Dendritic cells (DCs) are professional antigen-presenting cells that form cellular networks surveying for pathogens and providing the first immunological barrier to the external environment. The fate of DCs is dependent on a cross-talk with NK cells that may lead to DC’s killing (editing) which is believed to keep in check their quality and quantity. Considering that HIV-1-infected DCs may become persistent viral reservoirs, we addressed the question of NK’ sr ole in infected DC’s elimination as well as the mechanisms involved. Methods Immature DCs (iDCs) were derived from CD14+ monocytes cultured for 6 days in the presence of IL-4 and GM-CSF. iDCs were infected with R5-HIV-1BAL (DCHIV). 24 h cocultures with autologous purified activated NK cells (aNK) were performed and DC’s apoptosis was analyzed by multiparametric flow cytometry, combining 7-AAD staining with the detection of death/survival molecules. Gene array analyses were performed to detect variations in gene expression between different coculture conditions. siRNA magnetofection was performed to silence c-FLIP and c-IAP2 anti-apoptotic genes’ expression in DCs. Live video microscopy was used to dissect apoptotic events during aNK-iDC contact. Results We show that, while iDCs were susceptible to NK editing, involving TRAIL/DR4 and not the perforin-pathway, DCHIV were resistant to NK-dependent cytotoxicity. We report that NK cells induce in DCHIV a dramatic increase in the expression of two anti-apoptotic molecules, c-FLIP and c-IAP2, responsible for the resistance of DCHIV to TRAIL-induced apoptosis. Moreover, we found that HMGB1, a key mediator of NK-DC crosstalk, is responsible for the upregulation of these two inhibitors. The consequence of the escape of DCHIV from NK cytotoxicity is an HMGB1-dependent increase in HIV replication in DCs, which is mediated by HMGB1. Discussion These observations show that under physiological conditions, the editing process of iDCs by NK cells occurs through rapid induction of TRAIL apoptosis in iDCs. Following HIV infection of DCs, NK cells increase DCs’ survival through an HMGB1-dependant mechanism inducing c-IAP2 and c-FLIP upregulation. This study provides new insights into how HIV hijacks DCs and uses the NK-DC crosstalk to maintain viability of longterm reservoirs, and it identifies potential therapeutic targets to eliminate infected DCs.

We described in this paper the characteristics of a syngeneic anti-idiotypic serum made in BALB/c against BALB/c anti-poly (Glu 60 Ala 30 Tyr 10 ) (GAT) antibodies. This serum recognizes idiotypic determinants present in all anti-GAT sera whatever the allotypic markers of the mice used to prepare the sera. The functional effect of this serum on two helper cell lines is also described. Cell line BDF 1 /52 was obtained from GAT immunized lymph node cells (LNC). Cell line BDF 1 /E 3 was selected from splenic T-cells educated in vitro on GAT-pulsed adherent cells. Both lines were propagated in presence of filler cells, antigen, and medium containing T-cell growth factor(s) from splenic cells activated with concanavalin A. Both cell lines exhibit a helper activity as measured by the plaque-forming cell (PFC) response they induce in vitro in the presence of DNP-GAT and DNP sensitized B cells. Their helper activity is specific and they require a hapten-carrier bridge to activate B cells. These lines are able to induce IgG 1 , IgG 2a and IgG 2b anti-TNP PFC. Syngeneic anti-idiotypic serum B 658 inhibits specifically the function of these two lines but does not affect the helper activity of an OVA-specific T-cell line. The blocking activity of the serum can be adsorbed on a hybridoma protein with anti-GAT activity. This inhibition affects more dramatically the IgG 1 response than the IgG 2a and IgG 2b responses.

The supernatant of the T helper cell clone 52.3 (52.3-SN) was shown to induce polyclonal activation of resting B cells. 52.3-SN acts on most small B cells and through the allogeneic barrier. This supernatant induces cell size increase, RNA and DNA synthesis, and appearance of interleukin-2 and transferrin receptor. These results are interpreted as indicating the existence of a B Cell Activating Factor (BCAF) acting on resting B cells in an MHC-unrestricted way.TH cells can be obtained in an intermediate state of activation where they secrete lymphokines leading to B cell proliferation and not the biological activities leading to plasmocyte development. TH cell clones can induce sIgG− and sIgA− unprimed B cells to switch and express all classes and subclasses of immunoglobulin. The bulk of the response consists of IgM. Among the non IgM isotypes, IgG1 and IgA predominate. The supernatants prepared from TH cells reproduce these effects.

Two different monoclonal anti-idiotypic (Id) antibodies, HP-Id20 and HP-Id22, recognizing two discrete idiotopes characteristic of the anti-poly(Glu60 Ala30 Tyr10) (GAT) response were used to immunize BALB/c (GAT-responder) and DBA/1 (GAT-nonresponder) mice. The monoclonals were injected either copolymerized with keyhole limpet haemocyanin or polymerized with glutaraldehyde. The specific response was studied by two assays: (a) inhibition of binding of monoclonal anti-GAT antibody G5Bb2-2 to HP-Id20 and HP-Id22 and (b) GAT binding assays. In BALB/c GAT-responder mice, HP-Id20 and HP-Id22 immunization led to the preferential stimulation of immunoglobulin idiotypically related to anti-GAT antibodies (Ab1') and expressing anti-GAT activity. The results obtained with BALB/c nu/nu mice indicated that this response is T-cell-dependent. By means of the same experimental protocol GAT-nonresponder animals could be induced to produce anti-GAT antibodies after HP-Id immunization. This last result indicates that anti-Id immunization can bypass Ir gene control and does not preferentially stimulate the induction of GAT-specific T suppressor cells.

CD8+ T cells may play a beneficial role in human immunodeficiency virus (HIV)-infected patients by two mechanisms: HIV-specific cytotoxic activity and secretion of a soluble mediator(s) that inhibits HIV replication in vitro. Here we characterized both activities mediated by an HIV p24 gag -specific cytotoxic T lymphocyte (CTL) CD8+ clone derived from an HIV-infected patient. When the CTL clone was mixed with HIV-infected autologous CD4+ T cells, viral replication was suppressed. This viral inhibition was observed in heterologous CD4+ T cells and when CD8+ and CD4+ populations were separated by a semipermeable membrane, demonstrating the involvement of a diffusible factor(s). The lysis of autologous HIV-infected T cells was also detected. However, HIV suppression was more efficient when CD4+ and CD8+ T cells shared major histocompatibility complex alleles and were in direct contact. Thus, one and the same CD8+ T cell population can mediate both lysis of HIV-infected targets and nonlytic suppression of HIV replication. These results underline the multiple roles of CD8+ T lymphocytes in the suppression of HIV-infected cells.

The fine specificity of anti-Glu60Ala30Tyr10 (GAT) and anti-Glu60Ala40 (GA) proliferating cells was studied. T cells primed with GAT proliferate both to GAT and GA and GA-primed T cells proliferate also to GA and GAT. This cross-reactivity was unexpected given the results previously reported on the fine specificity of anti-GAT antibodies. The effect on the proliferation of BALB/c lymph node cells (LNC) of a syngeneic anti-idiotypic serum, prepared in BALB/c against anti-GAT antibodies, was studied. Two major points are made in this paper: (i) the in vitro addition of the anti-idiotypic serum in cultures containing GAT-primed LNC and GAT enhances the proliferation of GAT-specific T cells; (ii) the anti-idiotypic serum is effective in priming in vivo LNC which then acquire the capacity to proliferate specifically with GAT in vitro. These results further confirm the existence of idiotype-like determinants on T cells.

Eight monoclonal anti-idiotypic antibodies directed against public idiotopes have been further characterized: (a) they bind to public idiotopes with a high affinity; (b) they recognize all anti-poly(Glu60, Ala30, Tyr10) (GAT) antibodies as measured by inhibition of the anti-GAT plaque-forming cell response. This has been verified in three strains of mice. These reagents were not able to detect idiotope expression on eight GAT-specific helper T cell lines and clones. This result was obtained by two techniques: (a) idiotope expression at the T cell surface was measured by indirect immunofluorescence using a cell sorter with surface antigens H-2D, Thy-1.2, Lyt-1 and L3T4 as positive controls; (b) after immunoadsorption of [35S]methionine-labeled cellular extracts from two lines, no unique molecule was retained by the HP-idp22 monoclonal anti-idiotypic antibody coupled to Sepharose. Despite these negative results, this antibody was found to prime lymph node cells in vivo, which were able to proliferate specifically in response to GAT. Two T cell lines derived from this lymphocyte population do not express any of the idiotopes tested. These results suggest that monoclonal anti-idiotypic antibodies may be influencing T lymphocyte activity indirectly.

Recognition by specific T helper (T h ) cells of antigen presented by antigen-presenting cells (APC) involves, in addition to the antigen-specific receptor, non-antigen-specific molecules such as L3T4 and LFA1. In the present study, we analyzed the relationship between the avidity for antigen presented by APC of three T h cell lines and the participation of L3T4 and LFA1 cell surface antigens. We found a correlation between the avidity of T h cells for the complex GAT/Ia on APC measured by two independent assays and the participation of the cell-adhesion molecules L3T4 as measured by the ability of corresponding monoclonal antibody (MAb) to block the antigen-induced proliferation of T h cells. In contrast to the situation found with cytolytic T-lymphocyte (CTL) clones, we also found a differential inhibiting effect of anti-LFA1 MAb on the GAT-specific proliferation of the three T h clones. The results indicate a direct correlation between the inhibitory effects of anti-LFA1 and anti-L3T4 MAb and the affinity of T h cells for the complex formed by antigen and Ia.

A substantial amount of work has been accumulated trying to understand the cellular basis for the Ir gene control of the immune response to the synthetic polymer (Glu60 Ala30 Tyr10) (GAT). Conflicting data has been published. Yano et al. (1) suggested that the defect of nonresponder (NR) animals bearing H-2q and H-2S haplotype was the incapacity of their antigen presenting cells (APC) to present GAT to T cells. On the contrary Araneo et al.(2) showed that NR APC can present antigen to lymph nodes (LN) T cells from (responder x nonresponder) animals (R x NR) F1. Using a different approach Kapp et al. (3) have shown the selective induction of T suppressor (TS) cells in GAT NR animals. These TS cells inhibit the anti-GAT response induced by immunizing NR animals with GAT complexed to methylated serum albumin (GAT-MBSA). The mechanism by which GAT-MBSA induces an anti-GAT response in NR animals is not understood : some GAT specific T helper (TH) cells are induced but contrary to GAT specific TH cells from R animals these cells cannot generate anti-GAT response in presence of soluble GAT (4). Nothing is known about the capacity of soluble GAT to induce helper T cells in nonresponder animals.

At an early stage of HIV-1 transmission, plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells are recruited into mucosal tissues. pDCs are the major source of type I interferon (IFN-α), a powerful innate antiviral cytokine, and a strong pDC response is associated with spontaneous virus control. The contribution of pDCs to licensing NK cells and inducing antiviral immunity make them a key player in the early phase of HIV-1 infection. In contrast, the chronic expression of IFN-α was found to be a key mediator for HIV pathogenesis. We addressed herein the question of the molecular mechanisms involved in the generation of Interferon-producing Killer pDCs (IKpDCs), and the consequences on viral control.