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International audience; Human γδ T cells contribute to tissue homeostasis and participate in epithelial stress surveillance through mechanisms that are not well understood. Here, we identified ephrin type-A receptor 2 (EphA2) as a stress antigen recognized by a human Vγ9Vδ1 TCR. EphA2 is recognized coordinately by ephrin A to enable γδ TCR activation. We identified a putative TCR binding site on the ligand-binding domain of EphA2 that was distinct from the ephrin A binding site. Expression of EphA2 was up-regulated upon AMP-activated protein kinase (AMPK)-dependent metabolic reprogramming of cancer cells, and coexpression of EphA2 and active AMPK in tumors was associated with higher CD3 T cell infiltration in human colorectal cancer tissue. These results highlight the potential of the human γδ TCR to cooperate with a co-receptor to recognize non-MHC-encoded proteins as signals of cellular dysregulation, potentially allowing γδ T cells to sense metabolic energy changes associated with either viral infection or cancer.

During Plasmodium falciparum infections, erythrocyte-stage parasites inhibit dendritic cell maturation and function; compromising development of effective anti-malarial adaptive immunity. Human Vγ9Vδ2 T-cells can act in vitro as APCs and induce αβ T-cell activation. However, the relevance of this activity in pathophysiological contexts in vivo has remained elusive. Since Vγ9Vδ2 T-cells are activated during the early immune response against P.falciparum infection, we investigated whether they could contribute to the instruction of adaptive immune responses toward malaria parasites. In P.falciparum-infected patients,Vγ9Vδ2 T-cells presented an increased surface expression of APC-associated markers HLA-DR and CD86. In response to infected red blood cells in vitro, Vγ9Vδ2 T-cells readily up-regulated surface expression of HLA-DR, HLA-ABC, CD40, CD80, CD83 and CD86, induced naive αβ T-cell responses, and cross-presented soluble prototypical protein to antigen-specific CD8+ T-cells. Our findings indicate that P. falciparum parasites induce genuine APC properties in Vγ9Vδ2 T-cells and qualify this subset as an alternative professional APC in malaria patients, which could be harnessed for therapeutic interventions and vaccine design.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

International audience; In this commentary of Koonin's target paper, we defend an extended view of CRISPR-Cas immunity by arguing that CRISPR-Cas includes, but cannot be reduced to, defence against nonself. CRISPR-Cas systems can target endogenous elements (for example in DNA repair) and tolerate exogenous elements (for example some phages). We conclude that the vocabulary of "defence" and "nonself" might be misleading when describing CRISPR-Cas systems. 2

Background Rabbit antithymocyte globulin (rATG) induction is associated with profound immunosuppression, leading to a higher risk of cytomegalovirus (CMV) infection compared with anti–interleukin 2 receptor antibody (anti–IL-2RA). However, this risk, depending on the baseline CMV serological recipient/donor status, is still controversial. Methods The CMV DNAemia-free survival between rATG- and anti–IL-2RA–treated patients was analyzed in donor-positive/recipient-negative (D+R−) and recipient-positive (R+) patients in 1 discovery cohort of 559 kidney transplant recipients (KTRs) and 2 independent cohorts (351 and 135 kidney KTRs). The CMV-specific cell-mediated immunity (CMI) at baseline and at different time points after transplantation was assessed using an interferon γ enzyme-linked immunosorbent spot assay. Results rATG increased the risk of CMV DNAemia in R+ but not in D+R− KTRs. In R+ CMI-positive (CMI+) patients, the CMV DNAemia rate was higher in rATG-treated than in anti–IL-2RA–treated patients; no difference was observed among R+ CMI-negative (CMI−) patients. Longitudinal follow-up demonstrated a deeper depletion of preformed CMV CMI in R+ rATG-treated patients. Conclusions D+R− KTRs have the highest risk of CMV DNAemia, but rATG adds no further risk. Among R+ KTRs, we described 3 groups, the least prone being R+CMI+ KTRs without rATG, then R+CMI+ KTRs with rATG, and finally R+CMI− KTRs. CMV serostatus, baseline CMV-specific CMI, and induction therapy may lead to personalized preventive therapy in further studies.

Anti-HLA donor-specific antibodies are deleterious for organ transplant survival. Class I HLA donor-specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti-denatured HLA antibodies (anti-dHLAs). Anti-dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti-dHLAs to be discriminated from anti-nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid-treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti-dHLAs. However, some anti-dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti-nHLAs conserved significant reactivity toward acid-treated LSAB. After depleting serum anti-nHLA reactivity with HLA-typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid-treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti-nHLAs and anti-dHLAs, or anti-nHLAs recognizing acid-resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti-HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti-HLA antibodies in human serum.

BACKGROUND Single antigen flow beads assays may overestimate sensitization because of the detection of supposedly irrelevant antibodies recognizing denatured class I human leukocyte antigens (HLAs). METHODS Sera of 323 HLA-sensitized kidney transplant candidates positive with a class I HLA single antigen flow beads assay were retested after acid treatment of the beads. Denatured HLA antibodies were identified according to ratio between the measured fluorescence intensity for treated and nontreated beads. T-lymphocyte flow cytometry crossmatches were performed to characterize the ability of these antibodies to recognize HLA on normal cells as a surrogate of their potential clinical relevance. Their impact on organ allocation was evaluated through a calculated panel reactive antibody. The utility of single antigen flow beads largely devoid of denatured HLA (iBeads) was also evaluated. RESULTS Denatured HLA antibodies were detected in 39% of the patients. They provided much less positive flow cytometry crossmatches than anti-native HLA antibodies (16% vs. 83%, P

Screening studies using high-sensitivity and specificity markers indicate a prevalence of celiac disease of up to 1% in European and North-American populations. Celiac disease is a frequent condition that has become an important public health issue. Yet the majority of cases remain undiagnosed due to the polymorphism of its clinical manifestations. The new insight in the pathogenesis of celiac disease has lead to the development of new diagnostic tools. Early screening of symptomatic patients and pre-identified at-risk groups significantly improves the quality of life while reducing morbidity and mortality. However, prophylactic benefits of early diagnosis by assessing the general population have not been shown in any study. French and Northern American scientific societies have introduced serological testing in their newly revised strategies to diagnose celiac disease. Older markers judged insufficiently accurate like anti-gliadin and anti-reticulin antibodies have recently been withdrawn from the list of reimbursed medical expenses in France. Anti-endomysium and tissue transglutaminase IgA antibodies have proven to be at this day the most sensitive and specific markers for the diagnosis and follow-up of patients on gluten-free diet, at the exception of IgA-deficient patients. Assays testing for IgG antibodies are recommended upon IgA-deficiency. Although very accurate, a better standardisation of current assays may enable serological testing to replace in a near future histological confirmation brought by small bowel biopsies which remains today the gold standard test to diagnose celiac disease. Indeed, serological testing represents and attractive alternative as it is less invasive, less expansive, laboursaving and more objective in interpretation.

Celiac disease is an auto-immune enteropathy involving genetic factors. It is associated in almost all the patients, to specific susceptibility alleles encoding histocompatibility antigens (HLA for human leucocyte antigen), specifically certain variants of the HLA-DQ2, and the HLA-DQ8 HLA class II molecules. Its estimated prevalence is 1% in the european and north-american populations. However, although these alleles represent the main genetic factor for this disease, they do not explain it on their own, as they are expressed by up to 30% of the population. Recent immunological advances allowed identifying the immunodominant epitopes of gluten, to establish the role of tissue transglutaminase in the disease and to define at the atomic level the presentation of these antigens by the HLA-DQ molecule. It is noteworthy that the HLA susceptibility alleles only account for 40% of the whole genetic risk, and the challenge is now to explain the remaining 60%. Genome-wide association studies using the DNA arrays technology to screen single nucleotide polymorphisms to pinpoint candidate regions and genes, have started to provide answers, but contradictory results sometimes still persist. Most of the genes emerging as statistically significantly associated with celiac disease are involved in the immune response, and suggest that the situation is complex.

Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N4-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.

International audience; Apoptosis has emerged as a fundamental process important in tissue homeostasis, immune response, and during development. CD95 (also known as Fas), a member of the tumor necrosis factor receptor (TNF-R) superfamily, has been initially cloned as a death receptor. Its cognate ligand, CD95L, is mainly found at the plasma membrane of activated T-lymphocytes and natural killer cells where it contributes to the elimination of transformed and infected cells. According to its implication in the immune homeostasis and immune surveillance, and since several malignant cells of various histological origins exhibit loss-of-function mutations, which cause resistance towards the CD95-mediated apoptotic signal, CD95 has been classified as a tumor suppressor gene. Nevertheless, this assumption has been recently challenged, as in certain pathophysiological contexts, CD95 engagement transmits non-apoptotic signals that promote inflammation, carcinogenesis or liver/peripheral nerve regeneration. The focus of this review is to discuss these apparent contradictions of the known function(s) of CD95.

International audience; The immune system eliminates infected or transformed cells through the activation of the death receptor CD95. CD95 engagement drives the recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates and activates initiator caspases-8 and -10. The CD95-mediated apoptotic signal relies on the capacity to form the CD95/FADD/caspases complex termed the death-inducing signalling complex (DISC). Cells are classified according to the magnitude of DISC formation as either type I (efficient DISC formation) or type II (inefficient). CD95 localised to lipid rafts in type I cells, whereas the death receptor was excluded from these domains in type II cells. Here, we show that inhibition of both PI3K class IA and serine-threonine kinase Akt in type II cells promoted the redistribution of CD95 into lipid rafts, DISC formation and the initiation of the apoptotic signal. Strikingly, these molecular events took place independently of CD95L and the actin cytoskeleton. Overall, these findings highlight that the oncogenic PI3K/Akt signalling pathway participates in maintaining cells in a type II phenotype by excluding CD95 from lipid rafts.

Resume Les facteurs de regulation de l’interferon (interferon regulatory factor [IRF]) ont initialement ete identifies dans le cadre de la recherche sur les interferons de type 1. Depuis, de nombreux travaux ont permis de demontrer leur role dans la regulation de la reponse immunitaire innee, du developpement des differentes cellules du systeme immunitaire et de l’oncogenese. Plusieurs etudes genetiques ont aussi permis d’identifier des variants genetiques, principalement d’IRF5, associes a un risque accru de developper des pathologies auto-immunes. Le role biologique des IRF dans ces pathologies a ete etudie in vitro mais aussi in vivo dans differents modeles murins. Nous resumons dans cette revue les roles des differents IRF et, notamment dans les voies de signalisation des Toll-like recepteurs. Nous rapportons aussi les differents travaux impliquant les IRF dans les pathologies dysimmunitaires.

SummaryBackgroundIt is documented that omalizumab treatment reduces the cell surface expression of immunoglobulin E high-affinity receptor (FcɛRI) on several cell types. This has not been investigated in patients with uncontrolled severe persistent allergic asthma.MethodsIn a double-blind, randomized, placebo-controlled study, patients with severe allergic asthma uncontrolled by high dose inhaled corticosteroids and long-acting β2-agonist received either omalizumab (n = 20) or placebo (n = 11) over 16 weeks at appropriate doses and frequencies. Baseline and end of study (week 16) FcɛRI expression on basophils and plasmacytoid dendritic cells was determined by flow cytometry for the primary endpoint. Secondary efficacy endpoints included asthma control and lung function as part of an initial investigation into the use of FcɛRI expression as a marker of response.ResultsIn the omalizumab group, and with respect to placebo, FcɛRI expression was significantly reduced at end of study on basophils (−82.6%, p

Background Kidney transplant recipients infected with cytomegalovirus (CMV) undergo a persistent gammadelta T cell expansion in their peripheral blood. The anti-CMV function of these cells was previously demonstrated by their ability to kill CMV-infected cells in vitro. Methods To gain insight into the role of gammadelta T cells within the antiviral immune network, we compared the expansion kinetics of these T cells with that of CMV pp65-specific CD8(+) alphabeta T cells in the peripheral blood of twenty-one kidney transplant recipients. Results Both the percentage and the absolute number of pp65-specific CD8(+) T cells and gammadelta T cells showed a concomitant increase and persistence in most of the kidney transplant recipients with CMV infection. Both cell subsets exhibited an effector/memory phenotype (CD28(-), CD27(-), and CD45RA(+)) that predominated for the entire follow-up period. Conclusions In conclusion, CMV-specific CD8(+) alphabeta T cells and gammadelta T cells share common expansion kinetics and a common effector phenotype, suggesting that these cell types act similarly in response to CMV infection.

gammadelta T cells recognize stress-induced autoantigens and contribute to immunity against infections and cancer. Our previous study revealed that Vdelta2-negative ((neg)) gammadelta T lymphocytes isolated from transplant recipients infected by cytomegalovirus (CMV) killed both CMV-infected cells and HT29 colon cancer cells in vitro. To investigate the antitumor effects of Vdelta2(neg) clones in vivo, we generated hypodermal HT29 tumors in immunodeficient mice. Concomitant injections of Vdelta2(neg)clones, in contrast to Vdelta2(+) cells, prevented the development of HT29 tumors. Vdelta2(neg) clones expressed chemokine C-C motif receptor 3 (CCR3) and migrated in vitro in response to chemokines secreted by HT29 cells, among which were the CCR3 ligands macrophage inflammatory protein-1delta and monocyte chemoattractant protein-4. More importantly, a systemic i.p. treatment with Vdelta2(neg) clones delayed the growth of HT29 s.c. tumors. The effect of in vivo gammadelta T-cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-CCR3 antibody. gammadelta T-cell passive immunotherapy was dependent on the cytotoxic activity of the gammadelta effectors toward their targets because Vdelta2(neg) clones were not able to inhibit the growth of A431 hypodermal tumors. Our findings suggest that CMV-specific Vdelta2(neg) cells could target in vivo cancer cells, making them an attractive candidate for antitumor immunotherapy.

Background The short-term results of kidney transplantation are mainly attributed to the use of calcineurin inhibitors (CNI). However, opportunistic infections and cytomegalovirus (CMV) infections remain frequent and occur in the case of overimmunosuppression. Measurement of the biological effects of CNI could provide clues to identify overimmunosuppressed kidney transplant recipients (KTR) who would subsequently develop CMV infection. Methods Forty-one KTR given cyclosporine (n=18) or tacrolimus (n=23) were followed up every week during 1 month, then every month during 11 months, by measuring the patient's whole blood ability to inhibit interleukin-2 (IL-2) gene transcription and by measuring the patient's intracellular T-cell IL-2, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) production in response to polyclonal activation. Results Cytomegalovirus infection or disease occurred in 19 patients and was significantly associated with CMV serological status D+R- and D+R+ (HR=19.6 and 6.6, respectively, P=0.0001). Immunosuppressive treatment was associated with a long-lasting decrease of all cytokines produced by the patient's T-cells. However, none of these assays taken separately at any of the time points of the follow-up allowed to predict the occurrence of a subsequent CMV infection. We only observed a weak association between cumulative low levels of the percentage of TNF-alpha producing CD8+ T cells before CMV infection and its occurrence just afterwards (HR=1.39 for 1000 unit lower, P=0.04). However, this association did not remain significant after adjustment for CMV serological status. Conclusions This study suggests that immunological monitoring is not better than CNI whole blood levels for diagnosis of overimmunosuppression-induced CMV infection in KTR.

The ability of human γδ T cells to develop immunologic memory is still a matter of debate. We previously demonstrated the involvement of Vδ2− γδ T lymphocytes in the response of immunosuppressed organ recipients to cytomegalovirus (CMV). Here, we demonstrate their ability to mount an adaptive immune response to CMV in immunocompetent subjects. Vδ2− γδ T-cell peripheral blood numbers, repertoire restriction, and cytotoxicity against CMV-infected fibroblasts were markedly increased in CMV-seropositive, compared with CMV-seronegative, healthy persons. Whereas Vδ2− γδ T cells were found as naive cells in CMV− patients, they virtually all exhibited the cytotoxic effector/memory phenotype in CMV+ patients, which is also observed in transplanted patients challenged with CMV. This long-term complete remodeling of the Vδ2− γδ T-cell population by CMV predicts their ability to exhibit an adaptive anti-CMV immune response. Consistent with this, we observed that the secondary response to CMV was associated with a faster γδ T-cell expansion and a better resolution of infection than the primary response. In conclusion, the increased level of effector-memory Vδ2− γδ T cells in the peripheral blood is a specific signature of an adaptive immune response to CMV infection of both immunocompetent and immunosuppressed patients.

Background: Helicobacter pylori infection leads to different chronic diseases, suggesting that this bacterium can evade the host immune defense system. The ability to control lymphocyte proliferation may be a mechanism leading to the development of gastric pathologies. Our aim was to characterize the effects of mucosa-associated lymphoid tissue (MALT) associated H. pylori strains on lymphocyte proliferation. Materials and methods: We measured the in vitro proliferation of human lymphocytes originally from blood or tonsil samples in the presence or absence of viable bacteria or lysates. Results: We showed that MALT lymphoma-associated strains are not likely to be directly responsible for anarchical B-cell proliferation in vitro. On the other hand, proliferation of prestimulated T lymphocytes was abolished in vitro by the presence of all H. pylori strains, whether associated with MALT lymphoma or not. Conclusion: Inhibition of T-cell proliferation may be of major importance in the gastric colonization and in the persistence of the infection. Furthermore, this inhibition may favor anarchical B-cell proliferation in vivo and predispose the host to gastric MALT lymphoma, whereas MALT-associated H. pylori strains do not appear to possess a specific capability to directly stimulate B-lymphocyte proliferation.

Objective Recent studies have revealed a potential implication of CD8+ T lymphocytes in the pathogenesis of systemic lupus erythematosus (SLE) through their ability to induce tissue damage. The aim of the present study was to analyze the localization of CD8+ cells in the kidneys of patients with class III and class IV lupus nephritis and to establish correlations with histologic, biologic, and clinical features of SLE. Methods Twenty-five consecutive SLE patients with class III or class IV lupus nephritis were enrolled. Phenotype analyses of blood lymphocytes and renal immunohistochemistry studies were performed. Results CD8+ T cells were the predominant kidney-infiltrating subset of cells. The mean ± SD numbers of CD8+ T cells and CD4+ T cells were 66.2 ± 65.2/mm2 and 19.3 ± 29.4/mm2, respectively. There was a significant correlation between the percentage of blood CD3+,CD8+,DR+ cells and the total number of renal CD8+ T cells (r = 0.42, P = 0.039). Renal CD8+ T cell infiltration correlated well with the renal activity index (r = 0.63, P = 0.0007) and with high serum creatinine levels (r = 0.75, P = 0.0001). This CD8+ T cell infiltrate, which was predominantly in the periglomerular area, was correlated with cellular crescents and Bowman's capsule rupture and was associated with a poor response after conventional induction therapy. Conclusion CD8+ T lymphocytes infiltrate the periglomerular area in patients with severe (class III and class IV) lupus nephritis and are linked to a poor outcome after induction therapy. These results reveal a new potential effector pathway operant in lupus nephritis.

Malaria induces potent activation and expansion of the Vγ9Vδ2 subpopulation of γδT cells, which inhibit the Plasmodium falciparum blood cycle through soluble cytotoxic mediators, abrogating merozoite invasion capacity. Intraerythrocytic stages efficiently trigger Vγ9Vδ2 T-cell activation and degranulation through poorly understood mechanisms. P. falciparum blood-stage extracts are known to contain phosphoantigens able to stimulate Vγ9Vδ2 T cells, but how these are presented by intact infected red blood cells (iRBCs) remains elusive. Here we show that, unlike activation by phosphoantigen-expressing cells, Vγ9Vδ2 T-cell activation by intact iRBCs is independent of butyrophilin expression by the iRBC, and contact with an intact iRBC is not required. Moreover, blood-stage culture supernatants proved to be as potent activators of Vγ9Vδ2 T cells as iRBCs. Bioactivity in the microenvironment is attributable to phosphoantigens, as it is dependent on the parasite DOXP pathway, on Vγ9Vδ2 TCR signaling, and on butyrophilin expression by Vγ9Vδ2 T cells. Kinetic studies showed that the phosphoantigens were released at the end of the intraerythrocytic cycle at the time of parasite egress. We document exquisite sensitivity of Vγ9Vδ2 T cells, which respond to a few thousand parasites. These data unravel a novel framework, whereby release of phosphoantigens into the extracellular milieu by sequestered parasites likely promotes activation of distant Vγ9Vδ2 T cells that in turn exert remote antiparasitic functions.

In type I cells, Fas-mediated cell death requires cytoplasmic membrane subdomains called microdomains or lipid rafts. On the contrary, Fas signaling is independent of these structures in type II cells. We report that in human T cells, CD28, CD59, and CD55 are all localized into lipid rafts and that CD28 is concentrated into microdomains enriched in ganglioside GM1, whereas CD59 and CD55 are not. Moreover, CD28 cross-linking leads to the formation of lipid raft clusters which exclude CD59 and CD55, and reciprocally. Coligation of Fas with CD55 or CD59 inhibits the apoptotic signal, whereas CD28 recruitment amplifies the Fas signaling pathway. Therefore, we conclude that 1) different types of microdomains exist on the cell surface, with distinct functional properties and 2) the recruitment of these distinct structures may differentially modulate the Fas pathway. Moreover, our results demonstrate that Fas-induced apoptosis can be controlled at the level of the cytoplasmic membrane.

Fas triggers apoptosis via the caspase cascade when bound to its ligand FasL. In type I cells, Fas is concentrated into the plasma membrane lipid rafts, and these domains are required for the apoptotic signal to occur. In contrast, Fas is excluded from the microdomains in type II cells. We report that the coligation with Fas of the membrane receptor CD28 strongly increases Fas-induced apoptosis in type II T lymphocytes, whereas it has no effect in a type I cell line. The effect of CD28 is independent of its intracellular region and requires the recruitment of the microdomains. Indeed, upon CD28 costimulation, Fas is redistributed in the lipid rafts, and their disruption with a cholesterol chelator abrogates the effect of CD28. The microdomain-mediated cell death amplification does not alter death-induced signaling complex formation and is mediated by the enhancement of the mitochondrial apoptotic pathway. These findings indicate that the sensitivity to Fas-induced apoptosis of type II cells can be amplified in vivo by the recruitment of lipid rafts following interactions between nonapoptotic ligand/receptor pairs during cell-to-cell contacts.

NK cell receptors for MHC class I molecules (MHC-NKR) can be expressed by T cell subsets. The restricted repertoire and phenotypic characteristics of MHC-NKR(+) T cells indicate that expression of MHC-NKR is acquired upon antigenic challenge and might promote expansion of T cells. Previous studies performed on in vitro generated alphabeta T cell clones concluded that MHC-NKR expression was not a clonal attribute. Here, we examined a massive monoclonal expansion of a non-leukemic gammadelta T cell population found in the peripheral blood of a lung-transplanted patient who suffered from a cytomegalovirus infection. Despite their monoclonality, these T cells displayed a heterogeneous and stable in vivo Ig- and lectin-like MHC-NKR phenotype. Twenty percent of the cells displayed a CD94(+)NKG2A(+) phenotype, and 10% were labeled with an anti-CD158b1/b2/j monoclonal antibody. A CD158b/j(+) gammadelta T cell clone derived in vitro from patient's peripheral blood lymphocytes was shown to express the activating form CD158j (KIR2DS2), which once cross-linked stimulated the clone cytolytic function and costimulated the TCR-induced production of cytokines, independently of the killer-activating receptor-associated protein (KARAP). In conclusion, heterogeneity of MHC-NKR expression confers a functional intraclonal diversity that may participate to induction of specific gammadelta T cell effector functions or proliferation upon pathogen challenge.

Objective To compare changes in CD4+, CD8+ and total lymphocyte counts after initiation of highly active anti-retroviral therapy (HAART) between HIV-infected patients with and without a recent history of Mycobacterium avium complex (MAC) infection. Method Matched exposed-non-exposed retrospective cohort study. Results Fifty-one patients with a recent history of MAC infection (MAC+) started a combination of at least three antiretroviral drugs. They were individually matched to 145 patients without any history of MAC infection (MAC-) according to CD4+ T-cell count (±30 cells/mm3), previous experience of antiretroviral treatment, AIDS clinical stage at the time of HAART initiation (baseline), age (±10 years) and gender. MAC+ and MAC- patients presented comparable median levels of total lymphocytes (488 vs 688/mm3, P=0.09), CD4+ (11 vs 16/mm3, P=0.15), CD8+ count (359 vs 386/mm3, P=0.39) and plasma HIV RNA (5.3 vs 5.1 log10 copies/ml, P=0.22) at baseline. After 6 months on HAART, the median increase of CD4+ T-cell count was 28 cells/mm3 (interquartile range [IQR]: 1–63) in MAC+ and 72 cells/mm3 (IQR: 34–120) in MAC- patients ( P+ T cells was not significantly different between the two groups ( P=0.13). Comparable differences were observed for total lymphocytes and CD8+ T cells ( P10 copies/ml in MAC- patients, P=0.65). Results were confirmed after adjustment for other characteristics than the matching variables and after taking into account potential informative bias due to unbalanced number of deaths between the two groups. Conclusion MAC infection at the time of HAART initiation is an important deleterious factor for immune reconstitution. A better understanding of the underlying mechanism and an evaluation of additional treatment strategies are necessary to help immune restoration in such circumstances.

Long-lasting expansion of Vdelta2(neg) gammadelta T cells is a hallmark of cytomegalovirus (CMV) infection in kidney transplant recipients. The ligands of these cells and their role remain elusive. To better understand their immune function, we generated gammadelta T cell clones from several transplanted patients. Numerous patient Vdelta1(+), Vdelta3(+), and Vdelta5(+) gammadelta T cell clones expressing diverse Vgamma chains, but not control Vgamma9Vdelta2(+) T clones, displayed strong reactivity against CMV-infected cells, as shown by their production of tumor necrosis factor-alpha. Vdelta2(neg) gammadelta T lymphocytes could also kill CMV-infected targets and limit CMV propagation in vitro. Their anti-CMV reactivity was specific for this virus among herpesviridae and required T cell receptor engagement, but did not involve major histocompatibility complex class I molecules or NKG2D. Vdelta2(neg) gammadelta T lymphocytes expressed receptors essential for intestinal homing and were strongly activated by intestinal tumor, but not normal, epithelial cell lines. High frequencies of CMV- and tumor-specific Vdelta2(neg) gammadelta T lymphocytes were found among patients' gammadelta T cells. In conclusion, Vdelta2(neg) gammadelta T cells may play a role in protecting against CMV and tumors, probably through mucosal surveillance of cellular stress, and represent a population that is largely functionally distinct from Vgamma9Vdelta2(+) T cells.

The leukemia inhibitory factor (LIF) receptor comprises the low affinity binding chain gp190 and the high affinity converter gp130. The ectodomain of gp190 is among the most complex in the hematopoietin receptor family, because it contains two typical cytokine receptor homology domains separated by an immunoglobulin-like (Ig-like) domain. Human and murine gp190 proteins share 76% homology, but murine gp190 binds human LIF with a much higher affinity, a property attributed to the Ig-like domain. Using alanine-scanning mutagenesis of the Ig-like domain, we mapped a LIF binding site at its carboxyl terminus, mainly involving residue Phe-328. Mutation of selected residues into their orthologs in the murine receptor (Q251E and N321D) significantly increased the affinity for human LIF. Interestingly, these residues, although localized at both the amino and carboxyl terminus, make a spatially unique LIF binding site in a structural model of the Ig-like module. These results demonstrate definitively the role of the Ig-like domain in LIF binding and the potential to modulate receptor affinity in this family with very limited amino acid changes.

Objective The effects on T-lymphocyte populations of two interferon-alfa-2a (IFN) regimens associated with ribavirin were evaluated in 36 HCV-HIV co-infected patients with chronic hepatitis C, T-CD4 cell count > 250 cells/µL and a plasma viral load of

International audience; Objectives: Innate immune system alterations, including dendritic cell loss, have been reproducibly observed in patients with septic shock and correlated to adverse outcomes or nosocomial infections. The goal of this study is to better understand the mechanisms behind this observation in order to better assess septic shock pathogenesis.Design: Prospective, controlled experimental study. Setting: Research laboratory at an academic medical center. Subjects: The study enrolled 71 patients, 49 with septic shock and 22 with cardiogenic shock. Seventeen healthy controls served as reference. In vitro monocyte-derived dendritic cells were generated from healthy volunteers. Interventions: Sera were assessed for their ability to promote in vitro dendritic cell death through flow cytometry detection in each group of patients. The percentage of apoptotic or necrotic dendritic cells was evaluated by annexin-V and propidium iodide staining. Measurements and Main Results: We observed that only patients with septic shock and not patients with pure cardiogenic shock were characterized by a rapid and profound loss of circulating dendritic cells. In vitro analysis revealed that sera from patients with septic shock induced higher dendritic cell death compared to normal sera or cardiogenic shock (p < 0.005). Sera from surviving patients induced dendritic cell death through a caspase-dependent apoptotic pathway, whereas sera from nonsurviving patients induced dendritic cell-regulated necrosis. Dendritic cell necrosis was not due to necroptosis but was dependent of the presence of circulating histone. The toxicity of histones toward dendritic cell could be prevented by recombinant human activated protein C. Finally, we observed a direct correlation between the levels of circulating histones in patients and the ability of the sera to promote dendritic cell-regulated necrosis. Conclusions: The study demonstrates a differential mechanism of dendritic cell death in patients with septic shock that is dependent on the severity of the disease.

Common variable immunodeficiency (CVID) is a heterogeneous group of immunodeficiency syndromes that involves defective production of specific antibodies and decreased serum concentrations of > or =1 immunoglobulin isotype. We describe a patient with an atypical case of CVID who had extensive granulomatous lesions that were partially attributable to mycobacterial infection. In the peripheral blood, there was a massive increase in the number of double-negative CD3+ T cells that expressed the gammadelta T cell receptor.

CD4 T lymphocyte proliferative responses to hepatitis C virus (HCV) antigens were evaluated before and during an anti-HCV regimen (interferon-alpha2a and ribavirin) in 36 patients coinfected with HCV and human immunodeficiency virus (HIV), to determine whether immune responses against HCV antigens are present in such patients, whether these responses are modified by anti-HCV treatment, and whether they are correlated with treatment efficacy. The CD4 responses against HCV antigens (primarily core antigens) detected at study entry in one-half of the patients did not correlate with anti-HCV treatment efficacy. Of 36 patients, 8 had patterns of persistent immune response to infection by genotypes 3 or 4 that were significantly correlated with sustained virologic response. Persistent immunologic reactivity and sustained virologic response coexisted only in patients infected with genotype 3. These findings suggest that HCV genotype may influence specific immune response, which, in turn, is implicated in virologic control.

FasL and TRAIL are apoptotic ligands of the TNF-like cytokines family, acting via activation of the transmembrane death domain containing receptors Fas for FasL, and DR4 or DR5 for TRAIL. A glycosylphosphatidylinositol-linked TRAIL receptor called DcR1 behaves as a decoy receptor inhibiting TRAIL-mediated cell death in several cellular systems. We engineered and stably expressed a chimeric GPI-linked Fas receptor (Fas-GPI) in T-lymphocyte cell lines constitutively expressing functional transmembrane Fas. Surprisingly, despite lacking the death domain region of functional Fas, Fas-GPI was able to significantly increase Fas-mediated cell death triggered by membrane bound or soluble FasL, whereas engagement of Fas-GPI alone did not trigger apoptosis. This potentiating effect, but not transmembrane Fas activation, was selectively inhibited by protein kinase C activation with phorbol esters, demonstrating that Fas-GPI activated a specific synergistic signal transduction pathway. Fas-GPI and transmembrane Fas were localized in distinct membrane compartments, since Fas-GPI, but not transmembrane Fas, was found in the glycolipid-rich membrane microdomains. These results suggest that apoptosis induced by members of this ligand/receptors family may be differentially modulated through other and parallel signalling pathways.

In addition to antibodies targeting native class I human leukocyte antigens (HLA), the single antigen flow beads assay (SAFB) detects antibodies recognizing denatured forms (anti-dHLA). Acid treated SAFB and the modified SAFB reagent named iBeads are expected to distinguish anti-native (anti-nHLA) from anti-dHLA. Sera from 280 class I HLA-sensitized SAFB-positive kidney transplant candidates were retested with acid-treated SAFB and iBeads. Concordance between SAFB and iBeads, taking into account acid-treatment results, was described at global and locus levels. T-lymphocyte flow cytometry crossmatches (FCXM) were performed to identify an accurate iBeads MFI threshold allowing predicting FCXM results. Concordance between acid-treatment and iBeads assays was observed for 86.9% of alleles. The iBeads MFI were lower than for classical SAFB, especially for HLA-B and C alleles. Anti-dHLA identified with acid-treated SAFB were more frequently negative with iBeads for HLA-B and -C alleles. An iBeads MFI threshold of 1000 allowed predicting positive FCXM with 95.6% sensitivity, 91.6% negative predictive value and 0.08 negative likelihood ratio. The iBeads assay still has limitations, but might represent an invaluable alternative to SAFB for virtual crossmatch strategies in organ transplant allocation programs.

International audience; Cytomegalovirus (CMV) infection in solid-organ transplantation is associated with increased morbidity and mortality, particularly if a CMV mutant strain with antiviral resistance emerges. Monitoring CMV-specific T cell response could provide relevant information for patient care. We and others have shown the involvement of Vδ2(neg) γδ T cells in controlling CMV infection. Here, we assessed if Vδ2(neg) γδ T cell kinetics in peripheral blood predict CMV infection resolution and emergence of a mutant strain in high-risk recipients of kidney transplants, including 168 seronegative recipients receiving organs from seropositive donors (D+R-) and 104 seropositive recipients receiving antithymocyte globulins (R+/ATG). Vδ2(neg) γδ T cell percentages were serially determined in patients grafted between 2003 and 2011. The growing phase of Vδ2(neg) γδ T cells was monitored in each infected patient, and the expansion rate during this phase was estimated individually by a linear mixed model. A Vδ2(neg) γδ T cell expansion rate of ˃0.06% per day predicted the growing phase. The time after infection at which an expansion rate of 0.06% per day occurred was correlated with the resolution of CMV DNAemia (r=0.91; P

Human cytomegalovirus infection in transplant recipients has been associated with adverse renal allograft outcome and with a large γδ T-cell response, but whether both mechanisms are connected is unknown. We previously showed that most expanded circulating cytomegalovirus-responsive γδ T cells express the Fcγ-receptor CD16, suggesting that γδ T cells may participate in allograft lesions mediated by donor-specific antibodies through antibody-dependent cellular cytotoxicity. Here, we show that cytomegalovirus-specific CD16(pos) γδ T cells can perform antibody-dependent cellular cytotoxicity against stromal cells coated with donor-specific antibodies in vitro. In vivo, graft-infiltrating γδ T cells localized in close contact with endothelial cells only in patients who experienced cytomegalovirus infection and were more frequent within peritubular capillaries and glomeruli from antibody-mediated acute rejections than within those from T cell-mediated acute rejections. Finally, a persistently increased percentage of circulating cytomegalovirus-induced γδ T cells correlated inversely with the 1-year eGFR only in kidney recipients with donor-specific antibodies. Collectively, these data support the conclusion that cytomegalovirus-induced γδ T cells are involved in, and may serve as a clinical biomarker of, antibody-mediated lesions of kidney transplants. Moreover, these findings offer a new physiopathologic link between cytomegalovirus infection and allograft dysfunction in recipients with donor-specific antibodies.

The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp190 and the high affinity converter gp130, both of which are members of the family of hematopoietic receptors characterized by the cytokine receptor homology (CRH) domain. The gp190 is among the very few members of this large family to contain two CRH domains. The membrane-distal one (herein called D1) is followed by an Ig-like domain, a membrane-proximal CRH domain called D2, and three type III fibronectin repeats. We raised a series of monoclonal antibodies specific for the human gp190. Among them was the blocking antibody 1C7, which was directed against the D1Ig region and which impaired the binding of LIF to gp190. Another blocking antibody, called 12D3, was directed against domain D2 and interfered with the reconstitution of the high affinity receptor complex, independently of the interaction between LIF and gp190. The blocking effect of these two antibodies concerned four cytokines known to use gp190, i.e. LIF, oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. Among 23 antibodies tested alone or in combination (two anti-D2 and 21 anti-D1Ig), only the mixture of the two anti-D2 antibodies displayed agonistic activity in the absence of the cytokine. Taken together, these results demonstrate that the two CRH domains of gp190 play different functions in ligand binding and receptor activation.

Summary A major problem in the management of SLE patients is to predict a flare or to distinguish between active and quiescent disease. Serological markers are widely used to assess disease activity, but many patients have close to or normal values for these parameters while exhibiting obvious disease-related signs and symptoms. This study aimed to determine which serological parameters, among ESR, ANA and anti-dsDNA antibody titres, CH50 and the HLA-DR expression on circulating T-lymphocyte subsets, best reflected the development of SLE flares. Sixty SLE patients were included, 34 with quiescent disease throughout the entire follow-up period and 26 who experienced an SLE flare defined as having active disease. According to univariate analysis, all parameters were significantly higher for patients with active disease, with the percentage of CD8+DR+ cells being the most significant parameter (P = 10−7). Multivariate logistic regression analysis identified three independent variables enabling the identification of a lupus flare: CH50, the CD8+DR+ and CD4+DR+ cell percentages among total lymphocytes. The CD8+DR+ cell percentage is the biological parameter most significantly associated with a flare (P

CD40 ligand (CD40L)/CD40 interactions play a central role in T-cell–dependent B-cell activation as previously shown by in vitro studies, the phenotype of CD40L knockout mice and the defective expression of CD40L in patients who have X-linked immunodeficiency with hyper-IgM. The distribution of CD40 in cells other than of myeloid and lymphoid lineages has suggested additional functions for this receptor/ligand couple. Here we show that CD40L stimulates myelopoiesis with a noticeable effect on megakaryocytopoiesis in cocultures of hematopoietic progenitor cells and bone marrow stromal cells. These results suggest a mechanism by which T-cell or platelet-associated or soluble CD40L may regulate myelopoiesis.

The aim of the present study was to better characterize the expansion of double-negative (DN) T cells in vivo in AIDS patients and to ascertain the discrepant response of an immunodepressed immune system towards two distinct mycobacterial infections. In a large cohort of HIV-1 seropositive patients with low CD4(+) T cell counts (

In a retrospective study, an increase in double-negative (CD3+ CD4- CD8-) (DN) T lymphocytes has been shown to be an independent predictor of disseminated Mycobacterium avium complex (D.MAC) infection in patients with less than 100 CD4+ T cells per mm3. To better characterize this cell expansion, a prospective study was designed. From July 1995 to April 1997, 206 HIV-infected patients with less than 100 CD4+ T cells per mm3 were prospectively followed up and immunophenotyped. The median followup was 1.1 year (+/-0.5 year), and 14 new D.MAC infections were diagnosed among 84 first AIDS-defining events. In univariate and multivariate analyses, D.MAC infections were the only opportunistic infection with a significant increase in DN T-cell percentage (median = 6.6; range = 1.7 to 24.5, P = 0.004) compared with patients without any opportunistic infection. This alteration in T-lymphocyte count could constitute a predictor for D.MAC infection in clinical practice.

In normal individuals, gammadelta T cells account for less than 6% of total peripheral T lymphocytes and mainly express T-cell receptor (TCR) Vdelta2-Vgamma9 chains. We have previously observed a dramatic expansion of gammadelta T cells in the peripheral blood of renal allograft recipients only when they developed cytomegalovirus (CMV) infection. This increase was long lasting (more than 1 year), was associated with an activation of gammadelta T cells, and concerned only Vdelta1 or Vdelta3 T-cell subpopulations. Analysis of gammadelta TCR junctional diversity revealed that CMV infection in these patients was accompanied by (a) a marked restriction of CDR3 size distribution in Vdelta3 and, to a lesser extent, in Vdelta1 chains; and (b) a selective expansion of Vdelta1 cells bearing recurrent junctional amino acid motifs. These features are highly suggestive of an in vivo antigen-driven selection of gammadelta T-cell subsets during the course of CMV infection. Furthermore, Vdelta1 and Vdelta3 T cells from CMV-infected kidney recipients were able to proliferate in vitro in the presence of free CMV or CMV-infected fibroblast lysates but not uninfected or other herpes virus-infected fibroblast lysates. This in vitro expansion was inhibited by anti-gammadelta TCR mAb's. These findings suggest that a population of gammadelta T cells might play an important role in the immune response of immunosuppressed patients to CMV infection.

In normal persons, circulating gammadelta T cells comprise a minor cell subset (0.5%-6% of total lymphocytes). gammadelta T cells were studied in the context of therapeutic immunosuppression in transplanted patients. Flow cytometry detected an expansion of gammadelta T cells in 31 of 205 renal allograft recipients and in 2 of 41 uremic patients but in none of 45 healthy subjects. Univariate statistical analysis identified cytomegalovirus (CMV) infection (P. 001), second graft (P.001), and antithymocyte globulin treatment (P=.01) as three variables associated with high levels (=6%) of circulating gammadelta T cells in allograft recipients. Multivariate analysis further indicated that CMV infection was the only independent parameter associated with6% gammadelta T cells. gammadelta T cell expansion directly followed CMV infection and was never observed in persons who did not develop CMV infection. Thus gammadelta T cells may represent a first-line defense mechanism against CMV infection in a person whose alphabeta T cell response has been weakened by immunosuppression.

SUMMARYWe investigated the production of IL-2, IFN-γ, IL-10 and IL-4 by PBMC from 24 patients with SLE and 10 healthy individuals. Basal and mitogen-stimulated (lipopolysaccharide and phytohaemagglutinin (LPS + PHA)) cytokine production was determined in a whole blood assay (WBA). Supernatants were collected and assayed with specific ELISAs. Although the IL-2 and IFN-γ contents did not differ significantly between patients and controls under both conditions, statistically significant correlations were found between each cytokine and disease activity (SLAM index) after stimulation (respectively, r= 0.501, P = 0.01 and r = 0.631, P = 0.001). PBMC IL-10 production was significantly higher for patients than controls (P = 0.05), but no correlation between IL-10 levels and the SLAM index was obtained. IL-4 production was not statistically different between SLE patients and controls. For stimulated WBAs, the IL-10/IL-2 and IL-10/IFN-γ ratios were significantly correlated with disease severity (P = 0.02; P = 0.001, respectively). Overall, our data suggest that SLE is characterized by an elevated production of IL-10, reflecting the basal state of activation of the immune system. During exacerbation of SLE, IL-2 and IFN-γ are synthesized in larger amounts and may cause the tissue damage observed.

Background Symptomatic Primary Humoral Immunodeficiency Diseases (PHID) constitute a highly heterogeneous group of diseases characterized by a shared hypogammaglobulinemia, resulting in increased risk of recurrent or severe infections. Associations have been described with a variety of immunological abnormalities involving B and T-cell differentiation, T-cell activation and innate immunity. However, PHID discrimination remains based on B-lymphocyte abnormalities and other components of the immune system have not been sufficiently taken into account. We carried out unsupervised and supervised methods for classification in a cohort of 81 symptomatic PHID patients to evaluate the relative importance of 23 immunological parameters and to select relevant markers that may be useful for diagnosis and prognosis. Results We identified five groups of patients, among which the percentage of PHID complications varied substantially. Combining the set of markers involved in PHID supported the existence of two distinct mechanisms associated with complications. Switched memory B-cell attrition and CD8+ HLA-DR + activated T-cell increase were the prominent abnormalities observed in PHID complications. Furthermore, in a subgroup of 57 patients with common variable immunodeficiency, the classification that added CD8+ HLA-DR + to the consensual EUROclass classification was better than the EUROclass model in predicting complications. Conclusion These results highlight the importance of T-cell activation that may improve discrimination of PHID patients in specific subgroups and help to identify patients with different clinical outcomes.

The single antigen flow bead (SAFB) assay greatly improves the identification of antigenic specificity of anti-HLA alloantibodies. However, it may underestimate or miss high titer antibodies due to the prozone phenomenon caused by a competition between the fluorescent anti-IgG conjugate and serum complement, for the alloantibody. We explored this effect in our cohort of transplant candidates and transplanted recipients. Among a total of 292 and 269 patients with at least three different sera tested with class I and/or II SAFB assays respectively, we identified 9 patients (6 in class I and 3 in class II) who displayed a profound drop (≥ 75%) followed by a subsequent rise (≥ 100%), in strong (mean fluorescence intensity >8000) antibody levels, across an 18-month period. We postulated that such abrupt fluctuations were not explainable by naturally occurring transient desensitization. Sera were analysed with the SAFB assay using EDTA-treated serum and direct complement C1q staining, and with complement-dependent cytotoxicity and flow cytometry crossmatches (CDCXM and FCXM respectively). The prozone phenomenon was involved in all cases. Because it relies on complement activation, the CDCXM was not sensitive to this phenomenon, but the FCMXM was not either, although it resembles in its principle to the SAFB assay. Four additional anti-human conjugates targeting the IgG Fc fragment or the light chains did not circumvent the SAFB drawback. Therefore, a quick decrease in antibody strength must alert against a potential risk for recipients at the time of the transplant, using virtual crossmatch strategies. A prospective pre-transplant crossmatch still remains an ultimate safeguard.

Human immunodeficiency virus type 1 (HIV-1)-infected persons frequently have increased numbers of T cells bearing the gamma delta T cell receptor for antigen (gamma delta TCR). HIV-1-seropositive patients with < 100 CD4+ cells/mm3 were selected and divided into 9 AIDS-defining illness groups. The percentages of CD4+, CD8+, or double-negative CD4-CD8- (DN) T cells (most of the latter expressing the gamma delta TCR) for 8 symptomatic groups were compared with those for a reference group of asymptomatic HIV-1-infected patients. DN T cells were increased only in patients with disseminated Mycobacterium avium-intracellulare complex (MAC) infection, toxoplasmosis, or Kaposi's sarcoma. Multivariate logistic regression analysis revealed that the percentage of DN T cells was a better predictor of MAC infection than was the percentage of CD4+T cells. The increased percentage of DN T cells might have important implications for the understanding of gamma delta T cell physiology and for the early diagnosis and management of MAC infections in AIDS patients.

Allogeneic bone marrow transplantation is still limited by the morbidity and mortality caused by graft-versus-host disease (GVHD), resulting from host recognition by donor T lymphocytes. It is possible to drastically reduce the T-cell content of the graft. However, transplanted T cells can also have a beneficial effect by graft enhancement and the graft-versus-leukemia effect. How can we keep the beneficial GVL effect while protecting the patient from possible GVHD? A recent report proposed the ex vivo transfer of the herpes simplex thymidine kinase (HSv-tk) gene into donor T cells before their infusion with hematopoietic stem cells. This procedure is expected to allow selective donor T-cell depletion with ganciclovir should GVHD occur, but it has two major drawbacks: reinjection of a fraction of untransfected T cells cannot be avoided and heterogeneity of the transfected population results in increased risks such as HSv-tk gene instability or dysfunction of some of the transfected T cell. Alternative approaches must be considered. We demonstrate here the feasibility of generating HSv-tk transfected HLA-specific CD4+ cytotoxic T-cell clonal populations, in which 100% of the cells have the HSv-tk gene inserted at a single site within their genome. These clones retained their specificity, their function, and their sensitivity to ganciclovir treatment. Our approach is not limited to bone marrow transplantation. Indeed, this procedure represents a useful alternative to retroviral gene transduction and is applicable to every circumstance where clinical use of gene modified T-cell clones is to be considered.

Human cytomegalovirus (CMV) infection is often associated with myelosuppression and acute inflammatory reaction in immunocompromised patients. We have previously documented that CMV exposure of bone marrow (BM) stromal cells reduces the capacity of these cells to support hematopoiesis because of a decreased production of colony- stimulating factors. This study examines the potential role of CMV on constitutive and lipopolysaccharide (LPS)-stimulated production of cytokines involved in inflammatory reaction, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) by BM stromal cells. The release of IL- 6 was already detectable 2 hours post CMV-infection (2.5-fold increase in production) and the cumulative production of IL-6 after 5 days of infection was 23 +/- 1.2 ng/mL (ninefold increase in production). CMV was also able to induce a time-dependent production of LIF that was maximal 8 hours after CMV infection (2.5-fold increase in production). Concomitantly, there was no detectable release of granulocyte colony- stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) by CMV-infected stromal cells. The similar IL-6 and LIF production in the presence of polymyxin B ruled out the possibility that this increase could be caused by contamination of the viral stock by endotoxin. In addition, ultraviolet-inactivated virus behaved similarly to live virus and caused the release of IL-6 and LIF. However, heat-inactivated CMV was unable to induce IL-6 and LIF secretion by BM stromal cells. The production of IL-6 and LIF was also evaluated after stimulation by LPS. After 5 days of CMV exposure, the LPS-stimulated production of IL-6 and LIF was significantly lower than uninfected controls. This LPS-induced release of cytokine production was found to be dependent of viral replication. The experiments have shown that CMV is a potent inducer of IL-6 and LIF with differential effect on constitutive and LPS- stimulated cytokine production by stromal cells; we suggest that CMV induction of IL-6 and LIF during the first hours of infection could play a role in CMV-induced inflammatory reaction. Moreover, our results show that human CMV can disturb the balanced cytokine network involved in the regulation of hematopoiesis.

International audience; We evaluated the impact of cytomegalovirus (CMV)-induced immune responses, autoimmune-induced immune responses, and microbial translocation on immune activation in 191 human immunodeficiency virus type 1-infected patients from the ANRS CO3 Aquitaine Cohort. All enrolled subjects had achieved long-term virological suppression during receipt of combination antiretroviral therapy (cART). HLA-DR(+)/CD38(+) expression was 16.8% among CD8(+) T cells. Independent of age, CD4(+) T-cell count, 16S ribosomal DNA load, and regulatory T-cell count, positive results of Quantiferon CMV analysis (P = .02), positive results of CMV-pp65 enzyme-linked immunosorbent spot analysis (P = .01), positive results of CMV-pp65-specific CD8(+) T-cell analysis (P = .05), and CMV seropositivity (P = .01) were associated with a higher percentage of CD8+ T cells that expressed HLA-DR+/CD38+. Autoimmune response and microbial translocation were not associated with immune activation. Therefore, the CMV-induced immune response seems to be associated with chronic immune activation in cART recipients with sustained virological suppression.