Search

Supplementary materials and methods

Supplementary Figures 1-6. Suppl. Fig. 1. Mel-ILP induces release of certain cytokines, however does not affect infiltration of immune cells into tumor bed. Suppl. Fig. 2. Melphalan induces apoptosis of melanoma cells. Suppl. Fig. 3 Melphalan induces exposure of danger signals. Suppl. Fig. 4. Dying human melanoma cells induce danger signaling-independent in vitro stimulation of immune cells. Suppl. Fig. 5. In vitro treated melanoma cells do not strongly activate NK cells. Suppl. Fig. 6. (A) The phenotypic maturation of murine iDCs (B) Effect of antibodybased elimination of CD4+ and CD8+ cells in prophylactic mice vaccination experiment

Supplementary Tables 1-3. Supplementary Table 1. Meta-data analysis of clinical response to melphalan-based isolated limbtreatment procedures in melanoma patients. Supplementary Table 2. Meta-data analysis of clinical response to melphalan/TNF or Mel/TNF/IFNγ combination therapies for isolated limb-treatment procedures in melanoma patients. Supplementary Table 3. Follow up information about the patients whose tumour samples were used for analysis of infiltration of immune cells and expression levels of cytokines.

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.

International audience; Lichen planus (LP) is a cutaneomucosal chronic inflammatory disease characterized by a CD8 + cytotoxic T-lymphocytes (CTL) infiltrate. In erosive oral LP, we found HPV16specific activated CTL in lesions, supporting a pathogenic contribution of HPV16. Here, we investigated whether a similar scenario occurs in other clinical forms of LP and in lichen sclerosus et atrophicus (LSA), another chronic disease also affecting the mucosa and/or the skin. Blood CTL from LP and LSA patients expressed significant higher levels of granzyme B, perforin and CD107a proteins than healthy donors. Expansions of TCRVß3 + CTL, with presence of TCR clonotypes identical to those previously detected in erosive oral LP, were found both in blood and mucosal/skin lesions of LP, and not of LSA patients. These expansions were enriched with HPV16-specific CD8 + T-cells as shown by their recognition of the E7 11-20 immunodominant epitope. In LSA patients, the peripheral repertoire of CTL was oligoclonal for TCRVß6 + CTL. Finally, although patients with LP and LSA have developed antibodies against HPV16

Shigella remains in the top four pathogens responsible for moderate to severe diarrhoea in children below 5 years of age. The shigella O-specific polysaccharide (O-SP) is a promising vaccine target. We developed a conjugate vaccine prototype incorporating a unique well defined synthetic oligosaccharide hapten, chemically designed for optimal antigenic, conformational, structural, and functional mimicry of the O-SP from Shigella flexneri 2a (SF2a). We aimed to assess the safety, tolerability, and immunogenicity of this original synthetic oligosaccharide-based vaccine candidate, SF2a-TT15, conceived to drive the antibody response towards the key protective determinants of the native lipopolysaccharide antigen, in a first-in-human phase 1 study.We did a first-in-human, dose-escalating, single-blind, observer-masked, randomised, placebo-controlled study at the Clinical Research Center of Tel Aviv Sourasky Medical Center (Israel). Participants were healthy adults aged 18-45 years with low titres of serum SF2a-specific IgG antibodies. 64 eligible participants were assigned to one of two cohorts. 32 participants in each of the two cohorts were randomly assigned via computer-generated algorithm in a stepwise manner to receive the 2 μg (cohort 1) and 10 μg oligosaccharide dose (cohort 2) of the SF2a-TT15 vaccine candidate non-adjuvanted or adjuvanted with aluminium hydroxide (alum) or matching placebos. The vaccine was administered as three single intramuscular injections into the arm, 28 days apart. The primary outcome was the incidence and severity of adverse events, which were assessed in the intention-to-treat safety population analysis including all participants who were randomly assigned and received at least one vaccine or placebo injection. The immunogenicity endpoints were secondary outcomes and were analysed in all participants who were randomly assigned, received all of the assigned injections before the time of the immunogenicity assessment, and provided blood samples for immunological follow-up (per-protocol immunogenicity analysis). The study is registered with ClinicalStudies.gov, NCT02797236 and is completed.Of 203 volunteers initially screened, 64 participants were enrolled between Sept 20, 2016, and Sept 26, 2017. In each of the two cohorts, 12 participants received the adjuvanted vaccine, 12 received the non-adjuvanted vaccine and eight received the matching placebo (four each). The SF2a-TT15 glycoconjugate was well tolerated at both doses. No serious or severe adverse events occurred. Overall, seven (88%) of eight to 12 (100%) of 12 in each group of volunteers had one adverse event or more after receiving the study agents with the majority of adverse events, 300 (98%) of 307, considered mild in intensity. Of the seven adverse events defined as moderate in severity, one (nausea) was suspected to be related to the vaccine candidate. At all post-immunisation days and for both oligosaccharide doses, whether adjuvanted or not, SF2a-TT15 induced significantly higher serum IgG anti-SF2a lipopolysaccharide geometric mean titres (GMTs) as compared with baseline or with the corresponding GMTs in placebo recipients (p0·01). After one injection, the non-adjuvanted 10 μg oligosaccharide dose induced a 27-times increase in IgG GMT (5080 vs 189) and the non-adjuvanted 2 μg oligosaccharide dose induced a five-times increase (1411 vs 283), compared with baseline. Alum enhanced the specific IgG response at 2 μg oligosaccharide dose after the third injection (GMTs 3200 vs 1176, p=0.045).SF2a-TT15 was safe and well tolerated and induced high titres of anti-SF2a LPS IgG antibodies. These results support further evaluation of this original synthetic oligosaccharide-protein conjugate vaccine candidate for safety, immunogenicity, and protective efficacy in target populations.The European Union Seventh Framework Programme.

International audience; We report the safety (primary endpoint) and efficacy (secondary endpoint) of a novel intracerebral gene therapy at 5.5 years of follow-up in children with Sanfilippo B. An uncontrolled, phase 1/2 clinical trial was performed in four patients aged 20, 26, 30, and 53 months. Treatment consisted of 16 intracerebral and cerebellar deposits of a recombinant adeno-associated viral vector encoding human α-N-acetylglucosaminidase (rAAV2/5-hNAGLU) plus immunosuppression. An intermediate report at 30 months was previously published. Thirty treatment-emergent adverse events were reported between 30 and 66 months after surgery, including three classified as severe with no serious drug reactions. At 5.5 years, NAGLU activity was persistently detected in the lumbar cerebrospinal fluid (18% of unaffected control level). Circulating T cells reacting against NAGLU peptides were present, indicating a lack of acquired tolerance. Patients 2, 3, and 4 showed progressive brain atrophy and neurocognitive evolution that did not differ from untreated Sanfilippo A/B children. Patient 1, enrolled at 20 months of age, had a milder disease with normal brain imaging and a significantly better cognitive outcome than the three other patients and untreated patients, although not equivalent to normal children. After 5.5 years, the primary endpoint of this study was achieved with a good safety profile of the proposed treatment. We have also observed sustained enzyme production in the brain and absence of immunological tolerance. Cognitive benefit was not confirmed in the three oldest patients. Milder disease in the youngest patient supports further investigations of adeno-associated vector-mediated intracerebral gene therapy in Sanfilippo B.

International audience; Understanding the viral-host cell interface during HIV-1 infection is a prerequisite for the development of innovative antiviral therapies. Here we show that the suppressor of G2 allele of skp1 (SUGT1) is a permissive factor for human immunodeficiency virus (HIV)-1 infection. Expression of SUGT1 increases in infected cells on human brain sections and in permissive host cells. We found that SUGT1 determines the permissiveness to infection of lymphocytes and macrophages by modulating the nuclear import of the viral genome. More importantly, SUGT1 stabilizes the microtubule plus-ends (+MTs) of host cells (through the modulation of microtubule acetylation and the formation of end-binding protein 1 (EB1) comets). This effect on microtubules favors HIV-1 retrograde trafficking and replication. SUGT1 depletion impairs the replication of HIV-1 patient primary isolates and mutant virus that is resistant to raltegravir antiretroviral agent. Altogether our results identify SUGT1 as a cellular factor involved in the post-entry steps of HIV-1 infection that may be targeted for new therapeutic approaches.

International audience; Zika virus (ZIKV) dramatically emerged in French Polynesia and subsequently in the Americas where it has been associated with severe neurological complications in adults and newborns, respectively. Although plasmacytoid dendritic cells (pDCs) are a key sensor of viral infection and are critical for initiating an antiviral response, little is known about the impact of ZIKV infection on pDCs. Here, we investigated the susceptibility of human pDCs to infection with multiple strains of ZIKV and further investigated the impact of infection on pDCs functions. We observed that pDCs were refractory to cell-free ZIKV virions but were effectively infected when co-cultured with ZIKV-infected cells. However, exposure of pDCs to ZIKV-infected cells resulted in limited maturation/activation with significant down regulation of CD303 expression, a severe impairment of inflammatory cytokine production, and an inability to mount an IFN-a response. We show that ZIKV developed a strategy to inhibit the IFN-a response in primary human pDCs likely mediated through NS1-dependent CD303 signaling, thus suggesting a new mechanism of immune evasion.

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

For administration of multiple live attenuated vaccines, the Advisory Committee on Immunization Practices recommends either simultaneous immunization or period of at least 28 days between vaccines, due to a possible reduction in the immune response to either vaccine. The main objective of this study was to compare the immune response to measles (alone or combined with mumps and rubella) and yellow fever vaccines among infants aged 6–24 months living in a yellow fever non-endemic country who had received measles and yellow fever vaccines before travelling to a yellow fever endemic area. Subjects and methods: A retrospective, multicenter case-control study was carried out in 7 travel clinics in the Paris area from February 1st 2011 to march 31, 2015. Cases were defined as infants immunized with the yellow fever vaccine and with the measles vaccine, either alone or in combination with mumps and rubella vaccine, with a period of 1–27 days between each immunization. For each case, two controls were matched based on sex and age: a first control group (control 1) was defined as infants having received the measles vaccine and the yellow fever vaccine simultaneously; a second control group (control 2) was defined as infants who had a period of more than 27 days between receiving the measles vaccine and yellow fever vaccine. The primary endpoint of the study was the percentage of infants with protective immunity against yellow fever, measured by the titer of neutralizing antibodies in a venous blood sample. Results: One hundred and thirty-one infants were included in the study (62 cases, 50 infants in control 1 and 19 infants in control 2). Of these, 127 (96%) were shown to have a protective titer of yellow fever antibodies. All 4 infants without a protective titer of yellow fever antibodies were part of control group 1. Discussion: The measles vaccine, alone or combined with mumps and rubella vaccines, appears to have no influence on humoral immune response to the yellow fever vaccine when administered between 1 and 27 days. The absence of protective antibodies against yellow fever was observed only among infants who received both vaccines simultaneously. Conclusion: These results may support a revision of current vaccination recommendations concerning the administration of these two live attenuated vaccines either on the same day or at least 28 days apart. Our findings show no statistically significant difference if the interval between both vaccines is more than 24 h, but the immune response seems to be reduced when the two vaccines are given at the same time.

Summary Background Mucopolysaccharidosis type IIIB syndrome (also known as Sanfilippo type B syndrome) is a lysosomal storage disease resulting in progressive deterioration of cognitive acquisition after age 2–4 years. No treatment is available for the neurological manifestations of the disease. We sought to assess the safety and efficacy of a novel intracerebral gene therapy. Methods Local regulatory authorities in France allowed inclusion of up to four children in this phase 1/2 study. Treatment was 16 intraparenchymal deposits (four in the cerebellum) of a recombinant adenoassociated viral vector serotype 2/5 (rAAV2/5) encoding human α-N-acetylglucosaminidase (NAGLU) plus immunosuppressive therapy. We assessed tolerance, neurocognitive progression, brain growth, NAGLU enzymatic activity in CSF, and specific anti-NAGLU immune response for 30 months after surgery. This trial is registered with EudraCT, number 2012-000856-33, and the International Standard Clinical Trial Registry, number ISRCTN19853672. Findings Of seven eligible children, the four youngest, from France (n=2), Italy (n=1), and Greece (n=1), aged 20, 26, 30, and 53 months, were included between February, 2012, and February, 2014. 125 adverse events were recorded, of which 117 were treatment emergent and included six classified as severe, but no suspected unexpected serious adverse drug reactions were seen. Vector genomes were detected in blood for 2 days after surgery. Compared with the natural history of mucopolysaccharidosis type III syndromes, neurocognitive progression was improved in all patients, with the youngest patient having function close to that in healthy children. Decrease in developmental quotient was −11·0 points in patient one, −23·0 in patient two, −29·0 in patient three, and −17·0 in patient four, compared with −37·7 in the natural history of the disease. NAGLU activity was detected in lumbar CSF and was 15–20% of that in unaffected children. Circulating T lymphocytes that proliferated and produced tumour necrosis factor α upon ex-vivo exposure to NAGLU antigens were detectable at 1–12 months and 3–12 months, respectively, but not at 30 months in three of four patients. Interpretation Intracerebral rAVV2/5 was well tolerated and induced sustained enzyme production in the brain. The initial specific anti-NAGLU immune response that later subsided suggested acquired immunological tolerance. The best results being obtained in the youngest patient implies a potential window of opportunity. Longer follow-up is needed to further assess safety outcomes and persistence of improved cognitive development. Funding Association Francaise Contre les Myopathies, Vaincre les Maladies Lysosomales, Institut Pasteur, and UniQure.

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.

Introduction Des etudes epidemiologiques ont montre un lien entre infection par HPV16 et survenue d’un lichen plan (LP) erosif (LPE). L’existence de lymphocytes T (LT) CD8+ clonaux specifiques du peptide immunodominant d’HPV16 a ete montree en peripherie et au site lesionnel dans cette forme de LP. Nous avons souhaite tester cette meme hypothese dans les formes non erosives de LP (LPNE), et dans une dermatose proche affectant aussi les muqueuses genitales, le lichen sclero-atrophique (LSA). Patients et methodes Apres consentement eclaire, 20 patients ont ete inclus : 10 avec LPNE, 10 avec LSA, avec recueil sang, serum, brossage et biopsie des lesions. Les temoins etaient des donneurs de sang. Les etudes de cytometrie de flux se faisaient sur sang total. La recherche d’HPV16 se faisait par PCR a partir de l’ADN des brossages et indirectement en serologie (Ac anti-L1 et anti-E6). Les etudes de repertoire s’effectuaient a partir de l’ARN des LT CD8+ tries, par PCR, spectratyping, clonage et sequencage. Resultats Une augmentation significative de l’expression de granzyme, perforine et CD107a par les LT CD8+ peripheriques etait trouvee dans le LPNE et le LSA, par rapport aux temoins. Dans le LPNE, le repertoire de ces LT CD8+ etait clonal avec usage preferentiel du rearrangement Vb3-Jb2.7 (50 %), avec un motif consensuel de 4 acides amines sur les 8 qui composaient le CDR3b. Dans le LSA, le repertoire des CD8+ etait egalement clonal mais utilisait preferentiellement Vb6, avec des clonotypes differents. L’ADN d’HPV16 n’etait jamais trouve dans les lesions des deux dermatoses. Des Ac anti-L1 etaient trouves chez 9/10 LPNE et 7/10 LSA et des Ac anti-E6 chez 3/10 LPNE et chez aucun LSA. Une population CD8+ Vb3+ E711–20 etait identifiee en peripherie chez les LPNE HLA-A2+. Les etudes in situ sont en cours. Discussion Nos resultats montrent une tres forte expression des marqueurs de cytotoxicite par les LT CD8+ peripheriques dans le LPNE et le LSA, proche de ce qui s’observe dans les infections virales chroniques (VIH par exemple). Si un tres grand nombre des LPNE et LSA a ete en contact avec HPV16 (serologie HPV16 L1 positive), l’integration du virus dans le genome n’a ete observee que chez les patients atteints de LPNE. Les biais de repertoire des LT CD8+ dans le LPNE sont strictement superposables a ceux identifies dans le LPE, retrouvant le meme rearrangement Vb3-Jb2.7 et des sequences communes du CDR3. Cette utilisation du Vb3, associee a la reactivite contre HPV16, n’est pas retrouvee dans le LSA mais le caractere clonal observe dans cette pathologie suggere aussi une etiologie virale, qui ne serait pas HPV16. Conclusion L’implication physiopathologique d’HPV16 n’est pas restreinte a la forme erosive du LP et se retrouve aussi dans les formes non erosives de LP, ouvrant ainsi des perspectives therapeutiques dans le LP, comme la vaccination therapeutique contre HPV16 ou une immunotherapie ciblee anti-CD8-Vb3.

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. Methods: CSF and serum were collected from 103 HIV-1-infected patients enrolled in Neuradapt, a prospective study of the prevalence of HAND in HIV-1 infected patients at Nice University Hospital. Stored fluids were assessed for immunological, virological, and brain metabolite parameters. In addition to HIV RNA and DNA measurements, expression of T-cell surface markers of activation (CD38 and HLA-DR) was analyzed on whole blood. Concentration of 27 cytokines and chemokines was measured using multiplex bead assays on serum and CSF. Concentration of HMGB1 and anti-HMGB1 IgG autoantibodies were also measured on the same samples. Changes in cerebral metabolites N-acetyl aspartate (NAA), Choline (Cho) and creatinine (Cr) were assessed by magnetic resonance microscopy (MRS). Results: Clinical, virological and immunological characteristics were comparable between HAND (n = 30) and no HAND (n = 73) patients, except the absolute numbers of CD8+ T cells, which were higher in patients with HAND. Among the 29 molecules tested, only 4 of them were significantly upregulated in the CSF from HAND patients as compared to healthy donors i.e. HMGB1, anti-HMGB1 IgG antibodies, IP-10 and MCP1. CSF HMGB1 levels were positively correlated with HIV-1 DNA in aviremic HAND patients, suggesting a positive impact of HMGB1 on HIV reservoirs. Moreover, in contrast to NAA/Cr and Cho/NAA ratios, circulating anti-HMGB1 IgG antibody levels could discriminate patients with no HAND from patients with no HAND and a single deficit (average ROC-AUC = 0.744, p = 0.03 for viremic patients), thus enabling the identification of a very early stage of neurocognitive impairment, Conclusion: We report that brain injury in chronically HIV-infected patients on stable HAART is strongly associated with persistent CNS inflammation, which is correlated with increased levels of HMGB1 and anti-HMGB1 IgG in the CSF. Moreover, we identified circulating anti-HMGB1 IgG as a very early biomarker of neurological impairment in patients without HAND. These results might have important implication for the identification of patients who are at high risk of developing neurological disorders.

Monitoring T cells in combination with humoral response may be of value to predict clinical protection and cross-protective immunity after influenza vaccination. Elispot technique which measures cytokine produced after antigen-specific T cell stimulation is used routinely to detect and characterize anti-viral T cells. We found that the preservative thimerosal present in most H1N1 pandemic vaccines, induced in vitro abortive activation of T cells followed by cell death leading to false-positive results with the Elispot technique. The size of the spots, usually not measured in routine analysis, appears to be a discriminative criterion to detect this bias. Multi-dose vials of vaccine containing thimerosal remain important for vaccine delivery and our results alert about false-positive results of Elispot to monitor the clinical efficacy of these vaccines. We showed that this finding extends for other T cell monitoring techniques based on cytokine production such as ELISA. Although measuring in vitro immune response using the whole vaccine used for human immunization directly reflects in vivo global host response to the vaccine, the present study strongly supports the use of individual vaccine components for immune monitoring due to the presence of contaminants, such as thimerosal, leading to a bias in interpretation of the results.

BACKGROUND Interleukin (IL)-2 therapy impacts T-cell homeostasis. Whether IL-2 expanded CD4(+) T cells may persist following viral rebound has not been fully investigated. METHODS Patients with CD4(+) T cells 500/μl or more and HIV RNA less than 50 copies/ml were randomized to continue antiretroviral therapy (ART) either alone (n = 67) or combined with three IL-2 cycles (n = 81; 6 million units) twice daily for 5 days at weeks 0, 8, and 16 before stopping ART (week 24). Patients were followed up to 168 weeks. RESULTS At week 24, median CD4(+) T-cell counts were 1198 and 703 cells/μl in the IL-2 and control groups, respectively (P

Introduction Lorsque une personne doit recevoir plusieurs vaccins vivants attenues, il est recommande de les administrer soit simultanement, soit en respectant un delai minimal de 28 jours entre les deux injections, en raison d’une possible interference negative sur la reponse immune a chacun de ces vaccins. L’objectif principal de cette etude etait de comparer la reponse humorale au vaccin amaril et au vaccin rougeole-oreillons-rubeole chez des enfants âges de 6 a 24 mois, vaccines avant un voyage en zone d’endemie amarile. Materiels et methodes Une etude retrospective cas-temoins multicentrique a ete menee dans 7 centres de vaccinations internationales de la region parisienne. Le statut immunologique d’enfants ayant recu le vaccin amaril et le vaccin rougeoleux, seul ou combine aux vaccins rubeole et oreillons, a un intervalle compris entre 1 et 27 jours (cas) a ete compare a celui d’enfants vaccines simultanement (temoins no 1) ou a plus de 27 jours d’intervalle (temoins no 2) contre la fievre jaune et la rougeole (la rubeole et les oreillons). Le critere d’evaluation principal etait le pourcentage d’enfants presentant une immunite protectrice vis-a-vis de la fievre jaune (titre neutralisant ≥ 10). Resultats Cent trente et un enfants ont ete inclus, 62 cas, 50 temoins no 1 et 19 temoins no 2. Parmi eux, 127 (96 %) avaient un titre protecteur d’anticorps antiamarils ; les quatre enfants non immunises vis-a-vis de la fievre jaune appartenaient tous au groupe temoins no 1. Conclusion Des etudes complementaires seraient necessaires pour confirmer ou infirmer ces resultats et modifier les recommandations actuelles, en supprimant l’intervalle minimum de 27 jours a respecter entre les vaccins rougeole-oreillons-rubeole et amaril. Nous pensons cependant qu’il est des a present possible de conseiller aux centres de vaccinations internationales de ne pas administrer ces deux vaccins le meme jour.

Viruses manipulate host cells to ensure their own survival and, at late stages of the viral life cycle, they kill the infected target cell to ensure their propagation. In addition, some viruses induce a bystander killing, a viral strategy to escape from the host's innate and cognate defense systems. In HIV-infection, the disabling of the immune system is initially due to the preferential depletion by apoptosis of virus-specific CD4(+) T cells in lymphoid tissues, followed by the destruction of non-infected bystander cells. Both the extrinsic and the intrinsic pathways are activated, and this is the consequence of systemic immune activation. This review presents recent developments showing that the gastrointestinal tract is the major reservoir of infected cells and the site of rapid and profound loss of CD4 T cells, and that microbial translocation from the gastrointestinal tract is the cause of immune activation. Furthermore, apoptosis mechanisms involved in HIV-induced neuropathological disorders are discussed, including the role of syncytia that involve the sequential activation of ATM, p38MAPK and p53. Finally, HIV-associated dementia (HAD) was recently found in monkey models to be linked to inhibition of autophagy in neurons, suggesting that homeostasis of autophagy is a reliable security factor for neurons, and challenging the development of new therapeutics aimed at boosting neuronal autophagy to prevent HAD.

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.

The peripheral expansion of α/β+-CD4−CD8− double negative (DN) T cells in patients with autoimmune lymphoproliferative syndrome (ALPS) is a consistent feature of this disease, and part of the diagnostic criteria of ALPS. The origin of these cells remains undetermined. They could derive from mature T cells that have lost coreceptor expression, or represent a special minor cell lineage. To investigate relationship of DN and single positive (SP) T cells in ALPS, we used Immunoscope technology to analyze the TCRVβ repertoire diversity of sorted DN and SP T cells, and we performed CDR3 sequence analyses of matching clonotypes. We show that DN T cells express all the Vβ gene families that are used by their SP counterparts, though they dominantly use some Vβ genes. Analysis of CDR3 length distribution revealed a diverse polyclonal TCR repertoire for sorted CD4+ T cells, whereas both DN and CD8+ T cells showed a skewed TCR repertoire with oligoclonal expansions throughout most of the Vβ families. CDR3 sequencing of matching clonotypes revealed a significant sharing of CDR3 sequences from selected Vβ-Jβ transcripts between DN and CD8+ T cells. Altogether, these data strongly argue for a CD8 origin of DN T cells in ALPS.

We have analyzed the IgM, IgG and IgA BCR repertoire in PBC patients by means of quantitative RT-PCR and CDR3-spectratyping with immunoscope technology. PBMC from 35 PBC patients and 18 normal controls were analyzed. Quantitative B cell repertoire analysis of IgM from healthy donors showed the preferential usage of VH3a, VH3b and VH4 families. Very similar VH family usage was observed in IgM B cells from PBC patients. CDR3- spectratyping of IgM BCR rearrangements showed a Gaussian distribution for dominant VH families in control donors, and similar diversity was found for the VH3b family in PBC patients. In contrast, VH3a and VH4 families showed oligoclonal expansions in some patients. Quantitative B cell repertoire analysis of IgG and IgA did not reveal any difference in VH chain distribution in PBC patients as compared to the control donors. Immunoscope profiles of CDR3 length distribution showed several peak expansions in B cells from control donors, particularly for the VH3a and VH4 families. CDR3 length distribution profiles of IgG and IgA from PBC patients were oligoclonal too, with expansions throughout the various VH chains. However, no common expansions within the CDR3 region were found intraindividually between IgG, IgA and IgM, and between patients. In conclusion, immunoscope technology does provide, for the first time, a sensitive and rapid method for detailed immunoglobulin gene usage analysis in peripheral B cells from PBC patients. This study failed to demonstrate preferential B cell rearrangements in the blood of patients with PBC, but this technology may be more successful if applied to the analysis of compartmental B cells (i.e. liver infiltrating B cells).

Both differentiation and function of CD4+CD25high naturally arising regulatory T cells (Treg), which play a key role in the control of autoimmunity, are thought to depend on TCR specificity. In the present study, we comparatively measured the αβTCR repertoire sizes of human peripheral blood Treg and CD4+CD25− T cells by using a methodology based on PCR amplification and sequencing analysis. We show that Treg use a large unrestricted αβ TCR repertoire, the size and diversity of which are closely similar to those of CD4+CD25− T cells, with a mean estimated size of 3.5 × 106 distinct αβ TCR vs 4.7 × 106 distinct αβTCR for CD4+CD25− T cells. In addition, a 24% overlap between the repertoires of these two CD4+ subsets in the periphery is found. These data emphasize the intersection between naturally occurring Treg and effector T cell peripheral repertoires and provide new insights into the ontogeny of Treg in humans.

International audience; Background: Obtaining a comprehensive quantitative figure of the determinants of influenza infection will help identify priority targets for future influenza mitigation interventions. We developed an original causal model integrating highly diverse factors and their dependencies, to identify the most critical determinants of pandemic influenza infection (H1N1pdm09) during the 2010-2011 influenza season.Methods: We used data from 601 households (1450 participants) included in a dedicated cohort. Structural equations were used to model direct and indirect relationships between infection and risk perception, compliance with preventive behaviours, social contacts, indoor and outdoor environment, sociodemographic factors and pre-epidemic host susceptibility. Standardised estimates (βstd) were used to assess the strength of associations (ranging from -1 for a completely negative association to 1 for a completely positive association).Results: Host susceptibility to H1N1pdm09 and compliance with preventive behaviours were the only two factors directly associated with the infection risk (βstd=0.31 and βstd=-0.21). Compliance with preventive behaviours was influenced by risk perception and preventive measures perception (βstd=0.14 and βstd=0.27). The number and duration of social contacts were not associated with H1N1pdm09 infection.Conclusions: Our findings suggest that influenza vaccination in addition to public health communication campaigns focusing on personal preventive measures should be prioritised as potentially efficient interventions to mitigate influenza epidemics.

International audience; Erosive oral lichen planus (OLP) is a chronic, disabling mucocutaneous dysimmune rare disease characterized by mucosal inflammatory erosive lesions with pathological evidence for a marked CD8+ cytotoxic T-lymphocyte (CTL) infiltration. However, the specificity of lesional CTL in OLP has never been analyzed. To investigate the molecular mechanisms underlying dysregulation of T-cell immune responses in patients with OLP, we studied the diversity and antigen specificity of the TCR expressed by CD8+ T cells using dextramer staining, spectratyping, and TCR sequencing in 10 OLP patients undergoing extracorporeal photochemotherapy. Expansions of TCRVβ3-bearing CD8+ T cells were found in peripheral blood and in lesional tissues of OLP patients. Spectratyping and sequencing studies identified specific clonotypes in each patient. These expansions were enriched with human papillomavirus 16 (HPV16)-specific CD8+ T cells in HLA-A*0201+ patients as shown by their immune recognition of the E711-20 immunodominant epitope. Under treatment with extracorporeal photochemotherapy, clonotypic CD8+ T-cell expansions decreased in parallel with clinical remission. Altogether, these data establish a link between HPV infection and OLP pathogenesis by identifying a massive clonal expansion of CD8+ T cells with increased frequency of HPV 16-specific CD8+ T cells in OLP patients.

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.