On T lymphocyte alloreactivity

Alloreaktive ab-T-Zellen erkennen fremde MHC Moleküle, wobei die Erkennung durch Determinanten des MHC-Moleküls und/oder des Peptides vermittelt wird. Ein Teil der peptidabhängigen alloreaktiven T-Zellen, die allorestringierten T-Zellen, ist spezifisch für ein bestimmtes Peptid. Hier wurde Peptidbibliotheken dazu benutzt, den Einfluss engverwandter selbst-HLA-Moleküle auf das Repertoire an alloreaktiven T-Zellen zu untersuchen. Wir beobachteten keinen erhöhten Anteil an peptidspezifischen CTL im alloreaktiven Repertoire von HLA-A*0205-positiven Spendern verglichen mit HLA-A2-negativen Spendern. Ein Vergleich der alloreaktiven T-Zellantwort zweier Spender mit ähnlichem HLA-Haplotyp zeigte, daß das allorestringierte T-Zellrepertoire starke individuelle Unterschiede aufweist. In vitro eine Unterscheidung zwischen der Erkennung der MHC-Struktur und peptidspezifischer Erkennung ist schwierig. In dieser Promotionsarbeit wurde zum ersten Mal gezeigt, daß allorestringierte T-Zellen in der Lage sind, spezifisch HLA-Peptid-Tetramerkomplexe zu binden. Es wurde gezeigt, daß fluoreszierende HLA-Peptid-Tetramerkomplexe dazu geeignet sind, allorestringierte CTL gegen ein bestimmtes Peptid zu isolieren. Weiterhin wurden mit Hilfe von HLA-Peptid-Tetramerkomplexen alloreaktive T-Zellen nachgewiesen, die eine peptidselektive Konformation des allo-MHC-Moleküls erkennen. gd T-Zellen erkennen, im Gegensatz zu ab T-Zellen, ein breites Antigen Spektrum. Vg9/Vd2 T-Zellen werden durch Phosphoantigene aktiviert. In dieser Promotionsarbeit wird der erste alloreaktive Vg9/Vd2 T-Zellklon beschrieben, der spezifisch ein HLA Klasse I-Molekül erkennt. Obwohl dieser Klon den Vg9/Vd2 TCR exprimiert, wurde erstaunlicherweise kein Aktivierung durch bakterielle Phosphoantigene beobachtet. Für gd T-Zellrezeptoren, die keine Disulfidbrücken aufweisen, ist daher eine Konformation wahrscheinlich, die die Erkennung von Molekülen der MHC Superfamilie erlaubt.Alloreactive ab-T cells recognise framework or peptide-dependent determinants on foreign MHC molecules. Among peptide-dependent alloreactive T cells, a significant proportion is specific for one particular peptide presented by the allo-MHC molecule, the allorestricted T cells. We made use of peptide libraries to study the role of closely related self-HLA molecules on the shaping of the alloreactive T cell repertoire. We did not observe a bias towards peptide-specific CTLs in the alloreactive repertoire of HLA-A*0205 donors compared to HLA-A*02 negative donors. Comparison of the alloreactive T cell response between two donors having similar HLA haplotypes demonstrates that the allorestricted T cell repertoire is largely different between individuals. Allorestricted T cells specific for tumour associated antigens can be raised in vitro, however it is difficult to differentiate in vitro between framework and peptide-specific recognition. We provide the first evidence that allorestricted T cells can bind specifically HLA-peptide tetrameric complexes. We demonstrate that fluorescent HLA-peptide tetrameric complexes can be used for sorting and cloning of allorestricted CTLs specific against a peptide of interest. We also show by the mean of HLA-peptide tetramers the existence of peptide-selective alloreactive T cells that recognise a conformation on the allo-MHC. In contrast with ab T cells, gd T cells recognise a broad variety of antigens. Vg9/Vd2 T cell that represent the vast majority of gd T cells in the human periphery are activated by non-peptidic phosphoantigen. In this thesis, we describe the first alloreactive Vg9/Vd2 T cell clone specific for an HLA class I molecule. Although expressing the Vg9/Vd2 TCR, the B18 clone was not activated by bacterial phosphoantigen and does not express any known NK-receptors. Our results suggest that the particular conformation adapted by non-disulphide-linked gd TCRs is more prone to react with molecules of the MHC superfamily

Restriction Factors: From Intrinsic Viral Restriction to Shaping Cellular Immunity Against HIV-1

Antiviral restriction factors are host cellular proteins that constitute a first line of defense blocking viral replication and propagation. In addition to interfering at critical steps of the viral replication cycle, some restriction factors also act as innate sensors triggering innate responses against infections. Accumulating evidence suggests an additional role for restriction factors in promoting antiviral cellular immunity to combat viruses. Here, we review the recent progress in our understanding on how restriction factors, particularly APOBEC3G, SAMHD1, Tetherin, and TRIM5α have the cell-autonomous potential to induce cellular resistance against HIV-1 while promoting antiviral innate and adaptive immune responses. Also, we provide an overview of how these restriction factors may connect with protein degradation pathways to modulate anti-HIV-1 cellular immune responses, and we summarize the potential of restriction factors-based therapeutics. This review brings a global perspective on the influence of restrictions factors in intrinsic, innate, and also adaptive antiviral immunity opening up novel research avenues for therapeutic strategies in the fields of drug discovery, gene therapy, and vaccines to control viral infections

Identification of Cryptic MHC I–restricted Epitopes Encoded by HIV-1 Alternative Reading Frames

Human immunodeficiency virus (HIV) 1 major histocompatibility complex (MHC) I–restricted epitopes are widely believed to be derived from viral proteins encoded by primary open reading frames. However, the HIV-1 genome contains alternative reading frames (ARFs) potentially encoding small polypeptides. We have identified a panel of epitopes encoded by ARFs within the gag, pol, and env genes. The corresponding epitopic peptides were immunogenic in mice humanized for MHC-I molecules. In addition, cytotoxic T lymphocytes recognizing these epitopes were found in HIV-infected patients. These results reveal the existence of atypical mechanisms of HIV-1 epitope generation. They indicate that the repertoire of epitopes recognized by the cellular anti–HIV-1 immune response is broader than initially thought. This should be taken into account when designing vaccine strategies aimed at activating these responses

The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells

The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti–human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1–specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system

Dendritic Cells from HIV Controllers Have Low Susceptibility to HIV-1 Infection In Vitro but High Capacity to Capture HIV-1 Particles

ANRS CO21 CODEX cohortInternational audienceHIV controllers (HICs), rare HIV-1 infected individuals able to control viral replication without antiretroviral therapy, are characterized by an efficient polyfunctional and cytolytic HIV-specific CD8+ T cell response. The mechanisms underlying the induction and maintenance of such response in many HICs despite controlled viremia are not clear. Dendritic cells play a crucial role in the generation and reactivation of T cell responses but scarce information is available on those cells in HICs. We found that monocyte derived dendritic cells (MDDCs) from HICs are less permissive to HIV-1 infection than cells from healthy donors. In contrast MDDCs from HICs are particularly efficient at capturing HIV-1 particles when compared to cells from healthy donors or HIV-1 patients with suppressed viral load on antiretroviral treatment. MDDCs from HICs expressed on their surface high levels of syndecan-3, DC-SIGN and MMR, which could cooperate to facilitate HIV-1 capture. The combination of low susceptibility to HIV-1 infection but enhanced capacity to capture particles might allow MDDCs from HICs to preserve their function from the deleterious effect of infection while facilitating induction of HIV-specific CD8+ T cells by cross-presentation in a context of low viremia

CTL Escape Mediated by Proteasomal Destruction of an HIV-1 Cryptic Epitope

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes

Pediatric Measles Vaccine Expressing a Dengue Antigen Induces Durable Serotype-specific Neutralizing Antibodies to Dengue Virus

Dengue disease is an increasing global health problem that threatens one-third of the world's population. Despite decades of efforts, no licensed vaccine against dengue is available. With the aim to develop an affordable vaccine that could be used in young populations living in tropical areas, we evaluated a new strategy based on the expression of a minimal dengue antigen by a vector derived from pediatric live-attenuated Schwarz measles vaccine (MV). As a proof-of-concept, we inserted into the MV vector a sequence encoding a minimal combined dengue antigen composed of the envelope domain III (EDIII) fused to the ectodomain of the membrane protein (ectoM) from DV serotype-1. Immunization of mice susceptible to MV resulted in a long-term production of DV1 serotype-specific neutralizing antibodies. The presence of ectoM was critical to the immunogenicity of inserted EDIII. The adjuvant capacity of ectoM correlated with its ability to promote the maturation of dendritic cells and the secretion of proinflammatory and antiviral cytokines and chemokines involved in adaptive immunity. The protective efficacy of this vaccine should be studied in non-human primates. A combined measles–dengue vaccine might provide a one-shot approach to immunize children against both diseases where they co-exist

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

TAX1BP1 a novel player in antigen presentation

International audienceCD4$^+$ T lymphocytes play a major role in establishing and maintaining antiviral immunity. They are activated by anti- genic peptides derived from extracellular or newly synthesized (endogenous) proteins presented by the MHC-II molecules. Little is known about the mechanisms leading to endogenous MHC-II-restricted antigen presentation. We recently revealed a novel function of the macroautophagy/autophagy receptor (AR), TAX1BP1, in the presentation of endogenous viral antigens to CD4$^+$ T cells. In this punctum, we will focus on MHC-II-restricted endogenous antigen presentation and the involvement of TAX1BP1 in the biology of MHC-II molecules