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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.

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

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.

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.

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.

Severe combined immunodeficiency (SCID) caused by mutations in RAG1 or RAG2 genes is characterized by a complete block in T- and B-cell development. The only curative treatment is allogeneic hematopoietic stem cell transplantation, which gives a high survival rate (90%) when an HLA-genoidentical donor exists but unsatisfactory results when only partially compatible donors are available. We have thus been interested in the development of a potential alternative treatment by using retroviral gene transfer of a normal copy of RAG1 cDNA. We show here that this approach applied to RAG-1-deficient mice restores normal B- and T-cell function even in the presence of a reduced number of mature B cells. The reconstitution is stable over time, attesting to a selective advantage of transduced progenitors. Notably, a high transgene copy number was detected in all lymphoid organs, and this was associated with a risk of lymphoproliferation as observed in one mouse. Altogether, these results demonstrate that correction of RAG-1 deficiency can be achieved by gene therapy in immunodeficient mice but that human application would require the use of self-inactivated vector to decrease the risk of lymphoproliferative diseases.

We report the detection of an interleukin-4 (IL-4) variant whose expression is tightly associated with deprivation apoptosis. It is detected with the 8D4 anti–IL-4 monoclonal antibody (mAb) not only in T helper-2 (Th2) but also in Th1 clones, and primary T cells, and it is a nonsecreted molecule. It is not expressed during primary necrosis. Our data suggest that de novo IL-4 transcription of an alternative IL-4 mRNA (IL-4δ13) is induced during deprivation apoptosis. In HIV-infected patients, increased expression of IL-4 in T cells is highly correlated to increased apoptosis, restricted to 8D4 reactivity (r2 = 0.84 between % 8D4-8+ and % 7- amino-actinomycin D–positive [7-AAD+] peripheral T cells, P

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 We have previously shown that lipid alterations in HIV-1-associated lipodystrophy (LD) are correlated with decreased serum dehydroepiandosterone (DHEA) and increased cortisol:DHEA ratio and IFN-α levels. Objective To evaluate in a longitudinal study whether steroid and cytokine modifications are associated with the evolution of physical changes and lipid alterations associated with LD. Methods Thirty-four HIV-1-positive men were followed during 32·5 ± 4·0 months and tested at four time-points. The patients were subdivided into five groups according to physical changes and anthropometric measurements: LD-negative, initially LD-negative becoming LD-positive, LD-positive unchanged, aggravated or improved. Serum lipids, apolipoproteins, adrenal steroids and cytokines were measured and compared with baseline values. Results (1) LD aggravation is associated with persistent elevated lipids, a decrease in serum DHEA, an increase in cortisol:DHEA ratio and persistent high levels of IFN-α. (2) LD improvement is associated with normalization of serum lipids, an increase in serum DHEA leading to normalization in cortisol:DHEA ratio, and normalization of IFN-α levels. (3) In LD-positive men evolution of VLDL cholesterol is negatively correlated with DHEA (r = −0·56, P

Natural T (NT) lymphocytes recognize infected cells or microbial compounds without the classical genetic restriction of polymorphic major histocompatibility complex (MHC) molecules. NT cells are mainly composed of αβ and γδ T lymphocytes that express natural killer (NK) receptors and recognize preferentially various nonpeptidic antigens. Similar to NK cells, NT lymphocytes can see and kill target cells deficient in the expression of one or more MHC class I molecules. NT cells expressing the αβ TCR can recognize lipid and lipoglycan antigens presented in the context of nonpolymorphic CD1 molecules, whereas phosphocarbohydrates and alkylamines induce constitutive response of Vγ9Vδ2 T cells. The stimulation of Vγ9Vδ2 T cells with phosphocarbohydrates induces the production of cytokines (IFNγ and TNFα) and the release of chemokines with suppressive activity on HIV replication. In addition, stimulated Vγ9Vδ2 T cells exert a cytolytic activity against HIV-infected targets. In HIV-infected patients, a quantitative and qualitative alteration is observed early during the infection. Vγ9Vδ2 T cells are deleted and the remaining γδ cells are anergic. Th1 cytokines (IL-12 and IL-15) positively regulate cytokine production by Vγ9Vδ2 T cells but they are inefficient in restoring normal functions in patients’ γδ T cells. Interestingly, partial restoration of the immune system under highly active antiretroviral therapies (HAART) is associated to the recovery of functional Vγ9Vδ2 T cells. A large panel of phosphocarbohydrates able to selectively stimulate Vγ9Vδ2 T cells is currently available, and preliminary experiments in monkeys suggest their in vivo efficacy in helping to control SIV replication. These observations prompt the question of new immune intervention involving molecules that stimulate NT cells.

Background A key factor leading to impaired immunity in HIV infection is an alteration of the pattern of cytokine response, although its precise nature remains controversial, particularly the in vivo influence of HIV on interleukin (IL)-12 synthesis. Design A cross-sectional study in 73 HIV-infected persons (28 of them receiving highly active antiretroviral therapy) and 18 HIV-seronegative healthy donors. Methods The frequency of monocytes/macrophages (M/M) synthesizing IL-12, IL-10 and tumour necrosis factor alpha (TNF-alpha) was determined in peripheral blood mononuclear cells. The cells were cultured in medium or were stimulated with lipopolysaccharide; proportions of CD64 M/M producing IL-12, TNF-alpha or IL-10 was determined by cytofluorometric analysis. The influence of exogenous interferon gamma (IFN-gamma), IL-10 or IL-15 on IL-12 synthesis was tested. Results Chronic HIV disease is associated with increased priming of M/M for IL-12 (involving both p40 and p70 molecules) and TNF-alpha synthesis; this was associated with cosynthesis of both cytokines by a fraction of M/M. Priming for IL-12 was physiologically enhanced by IFN-gamma and decreased by IL-10; IL-15 had no effect. The proportion of IL-10-producing CD64 M/M was not altered in patients compared with controls but there was an inverse correlation between IL-10-producing M/M and viral load. IL-12 production was not correlated with viral load but was increased following antiretroviral therapy. Following LPS stimulation, IL-12 and TNF-alpha responses were not altered in HIV-positive patients; however, the IL-10 response was decreased but restored by antiretroviral therapy. Conclusion These observations argue for a preserved intrinsic CD64 M/M of IL-12 production in HIV pathogenesis.

Cell-mediated cytotoxicity is a crucial mechanism involved in several fundamental immunological processes such as protection against intracellular pathogens or termination of an immune response. This phenomenon is classically evaluated by the 51Cr release assay, which requires a radioactive isotope and does not permit the characterization of cells involved in the cytotoxic reaction. We describe a new flow cytometry method, developed in the context of CD95-mediated cell death, which allows the precise quantitation of cell-mediated cytotoxicity and the detection of intracellular events involved in the cytotoxic process. This assay uses a combination of two dyes, i.e. 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE) to label effector cells and 7-amino actinomycin D (7-AAD) to stain apoptotic target cells. We show that this assay is more sensitive than the 51Cr release assay and makes it possible to quantitate the percentage of cell lysis and, concomitantly, to immunophenotype target cells. It also facilitates the analysis of some events of the apoptotic pathway such as caspase activation or the expression of mitochondrial molecules. This new assay should contribute to a better understanding of the mechanisms involved in cell-mediated cytotoxicity in normal and pathological situations.

Background Following a lethal injury, two modes of cell death can be distinguished, apoptosis and primary necrosis. Cells pass through a prelethal stage characterized by a preservation of membrane integrity, in which they shrink (apoptosis) or swell (oncosis, the early phase of primary necrosis). During apoptosis, a loss of phospholipid asymmetry leads to exposure of phosphatidylserine (PS) residues on the outer leaflet of the plasma membrane. We examined whether the external PS exposure, initially supposed to be specific for apoptosis, was also observed in oncotic cells. Methods Human peripheral lymphocytes, Jurkat T cells, U937 cells, or HeLa cells were submitted to either apoptotic or oncotic stimuli. PS external exposure was assessed after binding of FITC-conjugated annexin V as was the loss of membrane integrity after propidium iodide (PI) uptake. Morphological examination was performed by optical or electron microscopy. Results Similarly to apoptotic cells, oncotic cells expose external PS residues while preserving membrane integrity. Consequently, oncotic cells exhibit the annexin V+ PI- phenotype, previously considered to be specific for apoptotic cells. Conclusions This study concludes that the annexin V/PI assay does not discriminate between apoptosis and oncosis and that it can be a useful tool to study oncosis by flow cytometry. Cytometry 44:65–72, 2001. © 2001 Wiley-Liss, Inc.

L'apoptose des lymphocytes T joue un role essentiel dans les processus de developpement et de maturation du systeme immunitaire. En effet, la mort par apoptose est impliquee dans l'etape de maturation intrathymique qui conduit a la deletion clonale de lymphocytes T auroreactifs et a l'etablissement de la tolerance au soi, elle joue un role important dans l'homeostase des lymphocytes T matures repondeurs a une stimulation antigenique, en limitant leur expansion clonale, et elle contribue a l'elimination de cellules infectees par un virus tels que le VIH (virus de l'immunodeficience humaine). Le VIH a la particularite de s'attaquer a une cellule clef du systeme immunitaire, le lymphocyte T auxiliaire, qui exprime a sa surface la molecule CD4, recepteur specifique du VIH. Au travers des cytokines qu'ils synthetisent, les lymphocytes T CD4 aident les effecteurs de l'immunite naturelle, tels que les cellules NK (natural killer), les lymphocytes T γδ et les macrophages, a detruire les cibles infectees par le VIH. Par ailleurs, ils sont essentiels a l'activation et la maturation des lymphocytes B en cellules productrices d'anticorps, ils sont requis pour la differenciation des lymphocytes T CD8 en cellule cytotoxique (CTL) specifique du VIH, et ils representent une source de chimiokines, facteurs inhibiteurs de l'entree du VIH dans la cible T CD4 [32]. En consequence, la disparition progressive de ces lymphocytes T auxiliaires, dependante du VIH, conduit a un defaut de controle par le systeme immunitaire de la replication virale, a la destruction des organes lymphoides ou a lieu la reponse immunitaire, et a l'apparition du syndrome d'immunodeficience acquise (sida), caracterise par le developpement d'infections opportunistes severes pouvant conduire au deces du patient. Les mecanismes responsables de la disparition des lymphocytes T CD4 au cours de l'infection par le VIH sont complexes, et ils ne sont que partiellement elucides. La destruction des lymphocytes T CD4 peut etre la consequence directe de l'infection virale, ou indirecte au travers de l'activation par les proteines du VIH de certaines voies pro-apoptotiques dans des lymphocytes non infectes. Par ailleurs, le systeme immunitaire peut aussi contribuer a cette destruction lymphocytaire, puisque les cellules infectees peuvent etre detruites par les CTL ou par un mecanisme de cytotoxicite dependante des anticorps (ADCC).

L'infection par le virus de l'immunodeficience humaine (VIH) est classiquement caracterisee par la reduction progressive dans le sang du nombre de lymphocytes T exprimant le recepteur CD4 pour le VIH. Ces lymphocytes sont la dible du virus du fait de l'expression de la molecule CD4 et des molecules CCR5 et CXCR4, recepteurs de chimiokines et corecepteurs du VIH. Pendant la phase aigue de l'infection retrovirale, la reponse initiale specifique du VIH met en jeu des effectuers de l'immunite cellulaire tels que les lymphocytes T CD4 auxillaires, producteurs de cytokines et de chimiokines et les lymphocytes T CD8 cytotoxiques, capables de reconnaitre specifiquement et de detruire les cellules infectees par le VIH. Cette reponse immunitaire, qui aboutit dans la plupart des infections a l'elimination de l'agent pathogene, ne permet pas d'eliminer le VIH ni meme de controler suffisamment sa replication, ce qui conduit a la destruction des tissus lymphoides ou generes les effecteurs de l'immunite et ou a lieu la reponse immunitaire [36] . L'image de la pathogenese du sida a beaucoup evolue ces dernieres annees, notamment grâce a une meilleure comprehension de la relation entre la dynamique de la replication virale et la progression de l'infection, et aussi grâce au developpement de nouvelles therapies antiretrovirales qui inhibent efficacement le VIH et ameliorent le statut clinique des patients. Pourtant, la question de la nature des mecanismes cellulaires et moleculaires qui contribuent a la disparition des lymphocytes T CD4 au cours de cette infection n'est toujours pas resolue [17] . Initialement, la decouverte que la molecule CD4 etait le recepteur pour le VIH a suggere que la dispartition des lymphocytes exprimant ce recepteur etait la consequence directe de leur destruction pendant la phase productive du cycle viral. Cependant, l'observation dans les ganglions de patients infectes par le VIH d'un taux important de mort cellulaire par apoptose touchant surtout les lymphocytes T CD4 non infectes mais aussi les lymphocytes T CD8, les lymphocytes B, les cellules NK (natural killer) et les cellules dendritiques [3] , [13] , [40] , suggere que cette infection est associee a une destruction lymphocytaire massive, non limitee aux lymphocytes T CD4, et qui met en jeus des mecanismes indirects de destruction cellulaire. L'alteration progressive du systeme immunitaire chez les personnes infectees par le VIH serait donc en partie la consequence du declenchement d'un programme de mort cellulaire par apoptose qui toucherait non seulement les cellules directement infectees par le virus mais aussi les cellules non infectees [2] , [15] , [16] .

Highly-active antiretroviral therapy (HAART) has lead to a dramatic decrease in the morbidity of patients infected with the human immunodeficiency virus (HIV). However, metabolic side effects, including lipodystrophy-associated (LD-associated) dyslipidemia, have been reported in patients treated with antiretroviral therapy. This study was designed to determine whether successful HAART was responsible for a dysregulation in the homeostasis of tumor necrosis factor- (TNF-), a cytokine involved in lipid metabolism. Cytokine production was assessed at the single cell level by flow cytometry after a short-term stimulation of peripheral blood T cells from HIV-infected (HIV+) patients who were followed during 18 months of HAART. A dramatic polarization to TNF- synthesis of both CD4 and CD8 T cells was observed in all patients. Because it was previously shown that TNF- synthesis by T cells was highly controlled by apoptosis, concomitant synthesis of TNF- and priming for apoptosis were also analyzed. The accumulation of T cells primed for TNF- synthesis is related to their escape from activation-induced apoptosis, partly due to the cosynthesis of interleukin-2 (IL-2) and TNF-. Interestingly, we observed that LD is associated with a more dramatic TNF- dysregulation, and positive correlations were found between the absolute number of TNF- CD8 T-cell precursors and lipid parameters usually altered in LD including cholesterol, triglycerides, and the atherogenic ratio apolipoprotein B (apoB)/apoA1. Observations from the study indicate that HAART dysregulates homeostasis of TNF- synthesis and suggest that this proinflammatory response induced by efficient antiretroviral therapy is a risk factor of LD development in HIV+ patients.

This study analyzes the degree of immune activation and characterizes apoptosis in lymphocytes from healthy West African donors or patients infected with human immunodeficiency virus (HIV)-1 or -2. The lower decline of CD4 T cells in HIV-2- compared with HIV-1-infected donors is associated with lower levels of immune activation, evaluated by HLA-DR expression on lymphocytes and sera concentrations of IgG and beta2 microglobulin (beta2m). Ex vivo apoptosis was found in both infections in all lymphocyte subsets, including CD4 and CD8 T cells, as well as B cells, but was lower in HIV-2 than in HIV-1 infection. Interestingly, high correlations were found in HIV-2- and HIV-1-infected donors between the level of CD4 T cell apoptosis and beta2m concentration and progression of the disease. These observations support the hypothesis that long-term activation of the immune system, weaker in HIV-2 infection, significantly contributes to T cell deletion and disease evolution.

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.

The CD56+CD16+ natural killer (NK) cell population plays a crucial role in eliminating virus-infected cells and is diminished in HIV-infected individuals. This study examined the effects of exogenous interleukin (IL)-15 on proliferation and survival of CD56+ and CD16+ cells of HIV-infected individuals. When used at equivalent concentrations in vitro, IL-15 was more potent than IL-2 as a growth factor for CD56+ cells, as well as for CD16+ cells and also CD4+ and CD8+ T cells. Analysis of cell survival in etoposide-treated cultures indicated that IL-15 was also more potent than IL-2 as a survival factor for CD56+ cells by virtue of its greater ability to up-regulate bcl-2 expression. Although IL-15-induced proliferation of CD56+ cells was accompanied by increased apoptosis, IL-15 was more effective than IL-2 in increasing the representation of viable CD56+ cells in the peripheral blood mononuclear cell population, but less effective in increasing T cell representation. The immunotherapeutic potential of IL-15 appears superior to IL-2 in regard to expanding NK cell populations in HIV-infected individuals, but needs to be weighed against poorer increases in T cell populations.

HIV infection is associated with increased representation of T cells bearing an activated, memory (CD45RO+) phenotype. Although administration of antiretroviral agents and interleukin-2 (IL-2) augment depleted CD4+ T-cell numbers, such therapies have been preferentially beneficial for CD45RO+ T cells. Interleukin-15 (IL-15) exhibits many biological activities in common with IL-2, including promoting T-cell survival and proliferation. The present study found that these two cytokines differed in their ability to induce proliferation, enhance survival, and control apoptosis of CD45RO+ and CD45RO- T-cell populations of human immunodeficiency- (HIV) infected individuals. When used at equivalent concentrations in vitro, IL-15 was more potent than IL-2 in activating and stimulating proliferation of CD4+CD45RO+, CD8+CD45RO+ and CD8+CD45RO- cells, but failed to be more effective than IL-2 in reducing apoptosis. Poor activation of CD4+CD45RO- cells by IL-15 and to IL-2 appeared to be attributable to low expression of the beta receptor chain utilized by both cytokines. However, IL-15 was more effective than IL-2 in enhancing survival of the CD4+CD45RO- population, suggesting a greater protective effect of IL-15 for naive CD4+ T cells, which are preferentially lost in HIV-infected individuals.

Resume Ces dernieres annees, la recherche sur le sida a progresse de facon considerable dans la connaissance de la physiopathologie de l'infection par le VIH et dans le developpement de nouvelles therapeutiques. Cependant, les mecanismes moleculaires qui contribuent a la destruction des lymphocytes T CD4, cibles du virus, ne sont toujours pas elucides. Comme cela est resume dans cette revue, des etudes recentes ont revele l'existence dans les lymphocytes T de personnes infectees par le VIH d'une deregulation importante du controle du processus d'apoptose. La stimulation persistante du systeme immunitaire infecte par le VIH est responsable de l'activation du systeme de mort cellulaire dependant de Fas et de l'inhibition du systeme de survie dependant de Bcl-2, induisant l'apoptose des lymphocytes de patients. La perte selective des lymphocytes T helper CD4 tout au long de l'infection par le VIH peut etre la consequence d'un renouvellement plus rapide de ces lymphocytes, ou de l'action selective de certains genes viraux ou de certaines cytokines, qui programmeraient plus specifiquement ces lymphoytes T pour l'apoptose. La depletion lymphocytaire touche aussi les cellules T CD8, qui ne sont pas les cibles du VIH, et l'activation de programmes de mort cellulaire par l'infection virale dans les cellules non infectees peut contribuer a leur destruction. Cette revue resume les differentes voies apoptotiques induites par le VIH dans les lymphocytes de personnes infectees par le VIH et en discute les consequences sur la pathogenese du sida.

Resume L'apoptose est caracterisee par de nombreuses alterations morphologiques, membranaires, mitochondriales et nucleaires, analysables par cytofluorimetrie en flux. La perte de l'integrite de la membrane plasmique est detectable par l'incorporation d'agents fluorescents tels que la 7-AAD ou le BET. L'exposition de la phosphatidylserine sur le feuillet externe de la membrane de la cellule apoptotique peut etre revelee par la fixation d'annexine-V. Les modifications nucleaires telles que la condensation chromatinienne sont analysables apres incorporation d'AO ou d'IP, et la fragmentation internucleosomale peut etre mise en evidence par la nick translation in situ. L'expression de la proteine mitochondriale 7A6 est detectee par la fixation de l'anticorps Apo2.7, et les alterations structurales et fonctionnelles des mitochondries par l'utilisation de NAO et de DiOC 6 (3). Dans cet article, nous evaluons ces tests de detection de l'apoptose sur des thymocytes murins, des lymphocytes humains et des lignees tumorales. Nous presentons une analyse multiparametrique combinant la detection de molecules de surface et de molecules intracellulaires sur les cellules apoptotiques. Enfin, nous proposons des strategies d'analyse de populations complexes telles que les cellules mononucleees du sang.

Apoptosis markers and the rate of lymphocyte apoptosis were followed in the peripheral blood of HIV infected persons at various stages of disease. Our study suggests that the early increase in memory cells following therapy may also be due to a significant decrease in apoptosis in this subset. The intrinsic resistance to apoptosis in the naive subset appears to be maintained following HIV infection and is not modified following highly active anti-retroviral treatment (HAART).

Human Vgamma9Vdelta2 T cells contribute to immunity against intracellular pathogens and recognize nonpeptidic antigens, such as the mycobacterial phosphoantigen TUBAg. HIV infection is associated with a polyclonal decrease of peripheral Vgamma9Vdelta2 T cells and we previously reported that the remaining cells show a proliferative anergy to stimulation with Mycobacterium tuberculosis in 60% of patients. Because of alterations in the Th1/Th2 cytokine balance reported in HIV infection, we analyzed, at the single-cell level, the influence of exogenous IL-4, IL-10, IL-12 and IL-15 on the response to mycobacterial phosphoantigens of gammadelta T cells from HIV-infected patients and healthy donors. We report that the strong gammadelta T cell response to TUBAg is characterized by the rapid and selective production of the Th1/proinflammatory cytokines IFN-gamma and TNF-alpha in responder HIV-infected donors. In addition, a positive regulation by IL-12 and IL-15 of the production of these cytokines by Vgamma9Vdelta2 T cells in response to nonpeptidic ligands was observed, whereas IL-4 and IL-10 had no effect. In contrast, Vgamma9Vdelta2 T cells from the anergic HIV-infected donors had lost the ability to produce Th1 cytokines and were not shifted towards a Th2 profile. Furthermore, neither IL-12 nor IL-15 could reverse this functional anergy. The consequences of these observations are discussed in the context of HIV pathogenesis.

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.