RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

International audienceEndothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiationinduced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton

The Th1 immune response against HIV-1 Gag p24-derived peptides in mice expressing HLA-A02.01 and HLA-DR1

Using HLA-DR1-transgenic H-2 class II knockout mice, we identified two new HLA-DR1-restricted HIV-1 Gag p24-derived epitopes (Gag321–340 and Gag331–350) and confirmed the immunogenicity of seven that have been previously described. The human relevance was confirmed for the two new ones (Gag321–340 and Gag331–350) assaying peripheral blood mononuclear cells from HLA-DR1+ HIV-1-infected long-term asymptomatic subjects and showing that Gag331–350 could prime CD4+ T cells from two HLA-DR1+ HIV-1 seronegative donors in vitro. Seven of these epitopes, structurally conserved among HIV-1 clade B isolates, were selected for a comparative evaluation of their Th1 helper potential by immunizing HLA-A02.01/HLA-DR1-transgenic, H-2 class I/class II knockout mice with recombinant mouse invariant chain constructs in which each helper epitope was inserted in association with two reporter HIV-1-derived HLA-A02.01-restricted CD8+ T cell epitopes. A T helper effect was demonstrated in all cases, and was particularly strong with epitopes Gag301–320, Gag321–340 and Gag271–290, which should, therefore, be considered in the design of new vaccines

Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective Innate and Adaptive Immune Responses

International audiencePrimary human cytomegalovirus (HCMV) infection usually goes unnoticed, causing mild or no symptoms in immunocompetent individuals. However, some rare severe clinical cases have been reported without investigation of host immune responses or viral virulence. In the present study, we investigate for the first time phenotypic and functional features, together with gene expression profiles in immunocompetent adults experiencing a severe primary HCMV infection. Twenty primary HCMV-infected patients (PHIP) were enrolled, as well as 26 HCMV-seronegative and 39 HCMV-seropositive healthy controls. PHIP had extensive lymphocytosis marked by massive expansion of natural killer (NK) and T cell compartments. Interestingly, PHIP mounted efficient innate and adaptive immune responses with a deep HCMV imprint, revealed mainly by the expansion of NKG2C+ NK cells, CD16+ Vδ2(-) γδ T cells, and conventional HCMV-specific CD8+ T cells. The main effector lymphocytes were activated and displayed an early immune phenotype that developed toward a more mature differentiated status. We suggest that both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage observed in PHIP. Taken together, these findings bring new insights into the comprehensive understanding of immune mechanisms involved during primary HCMV infection in immunocompetent individuals.IMPORTANCE HCMV-specific immune responses have been extensively documented in immunocompromised patients and during in utero acquisition. While it usually goes unnoticed, some rare severe clinical cases of primary HCMV infection have been reported in immunocompetent patients. However, host immune responses or HCMV virulence in these patients has not so far been investigated. In the present study, we show massive expansion of NK and T cell compartments during the symptomatic stage of acute HCMV infection. The patients mounted efficient innate and adaptive immune responses with a deep HCMV imprint. The massive lymphocytosis could be the result of nonadapted or uncontrolled immune responses limiting the effectiveness of the specific responses mounted. Both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage. Furthermore, we cannot exclude a delayed immune response caused by immune escape established by HCMV strains

Levels of intracellular HIV-DNA in patients with suppressive antiretroviral therapy

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European HIV-HCV co-infected individuals Short title: HLA-C and spontaneous clearance in HIV-HCV coinfection

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Deciphering anti-HCMV HLA-E-restricted unconventional CD8 T-cell responses in seropositive HCMV+ hosts

International audienceHuman cytomegalovirus (HCMV) causes severe illness and poor outcome in immunocompromised hosts such as transplant recipients and HIV-infected patients. Cytotoxic CD8 T cells against HCMV antigens (pp65, IE1) presented by classical HLA class-I molecules are major cellular components of the protective anti-HCMV immunity. HLA-E-restricted CD8 T cells targeting HCMV UL40 leader peptides (Lp) have been recently reported as unconventional T-cell responses also observed in some hosts but they still need clinical and functional characterization. Our study aimed to provide a qualitative and quantitative ex vivo analysis of HLA-EUL40 CD8 Tcell responses, in a large cohort (n=144) of kidney transplant recipients and healthy volunteers, and to elucidate determining factors. HLA-EUL40 CD8 T-cells were detected in >30% of seropositive HCMV+ hosts and may represent >30% of total circulating CD8 αβT cells at a time point. We identified host-related genetic factors (HLA-A*02 and HLA-E genotype) and HCMV strain, determining the sequence of UL40Lp, as critical parameters for this response. HLA-EUL40 CD8 are effector memory T cells that appear early post-infection as monoclonal/oligoclonal populations and persist for life. Although specifically induced in response to HCMV infection, a key feature of these cells is their potential ability to be also responsive against self and allogeneic HLA resulting from sequence homology between HLA-ILp and UL40Lp. Thus we established that unconventional HLA-EUL40 CD8 T cells belong to the common immune arsenal against HCMV. Their functions remain to be defined toward infection as well as their potential side effect in contexts such as autoimmunity and transplantation

Persistent BK Polyomavirus Viruria Is Associated with Accumulation of VP1 Mutations and Neutralization Escape

International audienceTo investigate the relationship between neutralization escape and persistent high-level BK polyomavirus replication after kidney transplant (KTx), VP1 sequences were determined by Sanger and next-generation sequencing in longitudinal samples from KTx recipients with persistent high-level viruria (non-controllers) compared to patients who suppressed viruria (controllers). The infectivity and neutralization resistance of representative VP1 mutants were investigated using pseudotype viruses. In all patients, the virus population was initially dominated by wild-type VP1 sequences, then non-synonymous VP1 mutations accumulated over time in non-controllers. BC-loop mutations resulted in reduced infectivity in 293TT cells and conferred neutralization escape from cognate serum in five out of six non-controller patients studied. When taken as a group, non-controller sera were not more susceptible to neutralization escape than controller sera, so serological profiling cannot predict subsequent control of virus replication. However, at an individual level, in three non-controller patients the VP1 variants that emerged exploited specific “holes” in the patient’s humoral response. Persistent high-level BK polyomavirus replication in KTx recipients is therefore associated with the accumulation of VP1 mutations that can confer resistance to neutralization, implying that future BKPyV therapies involving IVIG or monoclonal antibodies may be more effective when used as preventive or pre-emptive, rather than curative, strategies

HCMV triggers frequent and persistent UL40- specific unconventional HLA-E-restricted CD8 T-cell responses with potential autologous and allogeneic peptide recognition

International audienceImmune response against human cytomegalovirus (HCMV) includes a set of persistent cytotoxic NK and CD8 T cells devoted to eliminate infected cells and to prevent reactivation. CD8 T cells against HCMV antigens (pp65, IE1) presented by HLA class-I molecules are well characterized and they associate with efficient virus control. HLA-E-restricted CD8 T cells targeting HCMV UL40 signal peptides (HLA-E[UL40]) have recently emerged as a non-conventional T-cell response also observed in some hosts. The occurrence, specificity and features of HLA-E[UL40] CD8 T-cell responses remain mostly unknown. Here, we detected and quantified these responses in blood samples from healthy blood donors (n = 25) and kidney transplant recipients (n = 121) and we investigated the biological determinants involved in their occurrence. Longitudinal and phenotype ex vivo analyses were performed in comparison to HLA-A*02/pp65-specific CD8 T cells. Using a set of 11 HLA-E/UL40 peptide tetramers we demonstrated the presence of HLA-E[UL40] CD8 αβT cells in up to 32% of seropositive HCMV+ hosts that may represent up to 38% of total circulating CD8 T-cells at a time point suggesting a strong expansion post-infection. Host's HLA-A*02 allele, HLA-E *01:01/*01:03 genotype and sequence of the UL40 peptide from the infecting strain are major factors affecting the incidence of HLA-E[UL40] CD8 T cells. These cells are effector memory CD8 (CD45RA[high]RO[low], CCR7-, CD27-, CD28-) characterized by a low level of PD-1 expression. HLA-E[UL40] responses appear early post-infection and display a broad, unbiased, Vβ repertoire. Although induced in HCMV strain-dependent, UL40[15-23]-specific manner, HLA-E[UL40] CD8 T cells are reactive toward a broader set of nonapeptides varying in 1-3 residues including most HLA-I signal peptides. Thus, HCMV induces strong and lifelong lasting HLA-E[UL40] CD8 T cells with potential allogeneic or/and autologous reactivity that take place selectively in at least a third of infections according to virus strain and host HLA concordance

An NS5A single optimized method to determine genotype, subtype and resistance profiles of Hepatitis C strains.

The objective was to develop a method of HCV genome sequencing that allowed simultaneous genotyping and NS5A inhibitor resistance profiling. In order to validate the use of a unique RT-PCR for genotypes 1-5, 142 plasma samples from patients infected with HCV were analysed. The NS4B-NS5A partial region was successfully amplified and sequenced in all samples. In parallel, partial NS3 sequences were analyzed obtained for genotyping. Phylogenetic analysis showed concordance of genotypes and subtypes with a bootstrap >95% for each type cluster. NS5A resistance mutations were analyzed using the Geno2pheno [hcv] v0.92 tool and compared to the list of known Resistant Associated Substitutions recently published. In conclusion, this tool allows determination of HCV genotypes, subtypes and identification of NS5A resistance mutations. This single method can be used to detect pre-existing resistance mutations in NS5A before treatment and to check the emergence of resistant viruses while undergoing treatment in major HCV genotypes (G1-5) in the EU and the US