ETUDE DES ALTERATIONS DE LA CHAINE V BETA DU RECEPTEUR DES LYMPHOCYTES T LORS DE LA RECONNAISSANCE DIRECTE ET INDIRECTE DES ALLO- OU XENOANTIGENES (DOCTORAT (IMMUNOLOGIE))

NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

KIR matching in hematopoietic stem cell transplantation

International audienceAlthough the key role of MHC-restricted T lymphocytes in hematopoietic stem cell transplantation (HSCT) has been known for a long time, recent data have focused on complementary or alternative effector cell populations, and in particular on NK cells. Spontaneously generated NK cell alloreactivity from stem cell grafts involves specific interactions between NK receptors, including killer immunoglobulin-like receptors (KIRs) and their MHC class I ligands. The combined effects of HLA and KIR polymorphic genes might explain discrepancies in the impact of donor-recipient matching observed in HSCT

Next-generation sequencing technology a new tool for killer cell immunoglobulin-like receptor allele typing in hematopoietic stem cell transplantation : Séquençage nouvelle génération, un nouvel outil pour typer les allèles killer cell immunoglobulin-like receptor en greffes de cellules souches hématopoïétiques

International audienceKiller cell Immunoglobulin-like Receptor (KIR) genes are a family of genes located together within the leukocyte receptor cluster on human chromosome 19q13.4. To date, 17 KIR genes have been identified including nine inhibitory genes (2DL1/L2/L3/L4/L5A/L5B, 3DL1/L2/L3), six activating genes (2DS1/S2/S3/S4/S5, 3DS1) and two pseudogenes (2DP1, 3DP1) classified into group A (KIR A) and group B (KIR B) haplotypes. The number and the nature of KIR genes vary between the individuals. In addition, these KIR genes are known to be polymorphic at allelic level (907 alleles described in July 2017). KIR genes encode for receptors which are predominantly expressed by Natural Killer (NK) cells. KIR receptors recognize HLA class I molecules and are able to kill residual recipient leukemia cells, and thus reduce the likelihood of relapse. KIR alleles of Hematopoietic Stem Cell (HSC) donor would require to be known (Alicata et al. Eur J Immunol 2016) because the KIR allele polymorphism may affect both the KIR+ NK cell phenotype and function (Gagne et al. Eur J Immunol 2013; Bari R, et al. Sci Rep 2016) as well as HSCT outcome (Boudreau et al. JCO 2017). The introduction of the Next Generation Sequencing (NGS) has overcome current conventional DNA sequencing method limitations, known to be time consuming. Recently, a novel NGS KIR allele typing approach of all KIR genes was developed by our team in Nantes from 30 reference DNAs (Maniangou et al. Front in Immunol 2017). This NGS KIR allele typing approach is simple, fast, reliable, specific and showed a concordance rate of 95% for centromeric and telomeric KIR genes in comparison with high-resolution KIR typing obtained to those published data using exome capture (Norman PJ et al. Am J Hum Genet 2016). This NGS KIR allele typing approach may also be used in reproduction and to better study KIR+ NK cell implication in the control of viral infections.Les gènes Killer cell Immunoglobulin-like Receptor (KIR) sont une famille de 15 gènes, localisés chez l’homme sur le bras long du chromosome 19. Ces gènes KIR peuvent être inhibiteurs (2DL1/L2/L3/L4/L5A/L5B, 3DL1/L2/L3) ou activateurs (2DS1/S2/S3/S4/S5, 3DS1) et sont organisés en deux groupes d’haplotypes : haplotype A ou B. Le nombre et la nature des gènes KIR présents varient selon les individus. De plus, ces gènes KIR sont connus pour être polymorphes au niveau allélique (907 allèles décrits en juillet 2017). Les gènes KIR codent pour des récepteurs KIR inhibiteurs ou activateurs, exprimés principalement sur les cellules tueuses naturelles (NK). Les récepteurs KIR ont pour ligands les molécules HLA de classe I et sont capables de lyser les cellules leucémiques résiduelles des patients après greffe de cellules souches hématopoïétiques (CSH). Le contenu en allèles KIR de chaque donneur de CSH nécessiterait d’être connu (Alicata et al. Eur J Immunol 2016) car ce polymorphisme allélique KIR peut affecter le phénotype et la fonction des cellules NK KIR+(Gagne et al. Eur J Immunol 2013; Bari R, et al. Sci Rep 2016) ainsi que le devenir des greffes de CSH (Boudreau et al. JCO 2017). L’arrivée de nouvelles technologies de séquençage à haut débit (NGS) a permis d’aller au-delà des limites des techniques de séquençages conventionnelles, connues pour prendre plus de temps car spécifique d’un seul locus KIR. Récemment, une nouvelle approche NGS de typage allélique de tous les gènes KIR en entier a été développée par notre équipe nantaise à partir de 30 ADNs de référence (Maniangou et al. Front in Immunol 2017). Cette approche NGS.KIR est simple, rapide, fiable, spécifique et a montré une concordance des résultats alléliques KIR proche de 95 % avec ceux effectués sur les mêmes ADN dans une étude de l’exome aux États-Unis (Norman PJ et al. Am J Hum Genet 2016). Cette approche NGS de typage des allèles KIR peut aussi être utilisée en reproduction et pour étudier plus finement l’implication des cellules NK KIR+ dans le contrôle des infections virales

Relevance of Polymorphic KIR and HLA Class I Genes in NK-Cell-Based Immunotherapies for Adult Leukemic Patients

International audienceSince the mid-1990s, the biology and functions of natural killer (NK) cells have been deeply investigated in healthy individuals and in people with diseases. These effector cells play a particularly crucial role after allogeneic hematopoietic stem-cell transplantation (HSCT) through their graft-versus-leukemia (GvL) effect, which is mainly mediated through polymorphic killer-cell immunoglobulin-like receptors (KIRs) and their cognates, HLA class I ligands. In this review, we present how KIRs and HLA class I ligands modulate the structural formation and the functional education of NK cells. In particular, we decipher the current knowledge about the extent of KIR and HLA class I gene polymorphisms, as well as their expression, interaction, and functional impact on the KIR+ NK cell repertoire in a physiological context and in a leukemic context. In addition, we present the impact of NK cell alloreactivity on the outcomes of HSCT in adult patients with acute leukemia, as well as a description of genetic models of KIRs and NK cell reconstitution, with a focus on emergent T-cell-repleted haplo-identical HSCT using cyclosphosphamide post-grafting (haplo-PTCy). Then, we document how the immunogenetics of KIR/HLA and the immunobiology of NK cells could improve the relapse incidence after haplo-PTCy. Ultimately, we review the emerging NK-cell-based immunotherapies for leukemic patients in addition to HSCT

T Cell Response in Xenorecognition and Xenografts: A Review

International audienceXenotransplantation has recently become a subject of interest for the transplantation community due to the current organ shortage, which could be partially or even totally solved by the development of this strategy. The humoral response, which arises as a result of species disparities, is the major obstacle to the success of xenotransplantation. However, if the use of different strategies such as plasmapheresis, immunoadsorption, the utilization of organs from transgenic pigs for complement regulatory molecules and new immunosuppressive drugs, may allow to overcome or reduce the early antibody mediated rejections (hyperacute or acute vascular rejection), delayed responses based on cellular activations will still occur. In this review, despite the fact that different cell populations have been shown to be implicated in these phenomena (NK, granulocytes, macrophages), we will focus on recent published information concerning T cell response only, in xenorecognition

Natural Killer Cells and Killer-cell Immunoglobulin-like Receptor Polymorphisms Their role in haematopoietic stem cell transplantation

International audienceNatural Killer (NK) cells are important effector cells in the early control of infected, malignant and 'non-self' cells. Various receptor families are involved in enabling NK cells to detect and efficiently eliminate these target cells. The Killer-cell Immunoglobulin-like Receptor (KIR) family are a set of receptors that are very polymorphic with regards to gene content, expression level and expression pattern. KIRs are responsible for the induction of a NK cell alloreactive response through their interaction with HLA class I molecules. The role of NK cells in Haematopoietic Stem Cell Transplantation (HSCT) has been studied for many years and induction of antileukaemic responses by donor NK cells have been reported. Conflicting data still exist on the exact circumstances in which the KIR repertoire affects and influences clinical outcome after HSCT. More large-scale studies are needed on well-defined cohorts to unravel the mechanism of action of the NK cell-mediated alloresponse in an HSCT setting

Improved KIR gene and HLA-C KIR ligand sequence-specific primer polymerase chain reaction genotyping using whole genome amplification

International audienceMolecular analysis of genetic polymorphism for clinical or research purposes may be compromised by genomic DNA of limited quality and quantity. In this study, we have successfully tested the feasibility of using whole genome amplification (WGA) to allow genotyping for killer cell immunoglobulin-like receptor (KIR) genes and human leucocyte antigen (HLA)-C KIR ligand dimorphism on HLA-C. WGA was achieved by multiple displacement amplification (MDA) using bacteriophage phi29 polymerase. For KIR genotyping, a revised sequence-specific primer polymerase chain reaction protocol consisting of 23 primer pairs was used avoiding hitherto undetected cross-priming involving KIR2DL1, KIR2DS1, KIR3DL1 and KIR3DS1 alleles. Similarly, MDA-amplified genomic DNA was analyzed for the detection of the HLA-C KIR ligand groups C1 and C2, based on the amino acid K/N dimorphism in position 80

Large Spectrum of HLA-C Recognition by Killer Ig–like Receptor (KIR)2DL2 and KIR2DL3 and Restricted C1 Specificity of KIR2DS2: Dominant Impact of KIR2DL2/KIR2DS2 on KIR2D NK Cell Repertoire Formation

International audienceThe interactions of killer Ig-like receptor 2D (KIR2D) with HLA-C ligands contribute to functional NK cell education and regulate NK cell functions. Although simple alloreactive rules have been established for inhibitory KIR2DL, those governing activating KIR2DS function are still undefined, and those governing the formation of the KIR2D repertoire are still debated. In this study, we investigated the specificity of KIR2DL1/2/3 and KIR2DS1/2, dissected each KIR2D function, and assessed the impact of revisited specificities on the KIR2D NK cell repertoire formation from a large cohort of 159 KIR and HLA genotyped individuals. We report that KIR2DL2 + and KIR2DL3 + NK cells reacted similarly against HLA-C + target cells, irrespective of C1 or C2 allele expression. In contrast, KIR2DL1 + NK cells specifically reacted against C2 alleles, suggesting a larger spectrum of HLA-C recognition by KIR2DL2 and KIR2DL3 than KIR2DL1. KIR2DS2 + KIR2DL2 2 NK cell clones were C1-reactive irrespective of their HLA-C environment. However, when KIR2DS2 and KIR2DL2 were coexpressed, NK cell inhibition via KIR2DL2 overrode NK cell activation via KIR2DS2. In contrast, KIR2DL1 and KIR2DS2 had an additive enhancing effect on NK cell responses against C1C1 target cells. KIR2DL2/3/S2 NK cells predominated within the KIR repertoire in KIR2DL2/S2 + individuals. In contrast, the KIR2DL1/S1 NK cell compartment is dominant in C2C2 KIR2DL2/S2 2 individuals. Moreover, our results suggest that together with KIR2DL2, activating KIR2DS1 and KIR2DS2 expression limits KIR2DL1 acquisition on NK cells. Altogether, our results suggest that the NK cell repertoire is remolded by the activating and inhibitory KIR2D and their cognate ligands

T cell repertoire alterations of vascularized xenografts

International audienceThe role of T cells in the rejection of vascularized xenografts has been little explored. Because of the high potential diversity of xenoantigens, it has been suggested that xenotransplantation could induce a strong cellular response that could contribute to delayed rejection. Alternatively, alterations in molecular interactions could impair the T cell response. Because the analysis of TCR repertoire in vivo indirectly reflects the nature and the magnitude of T cell xenorecognition, we took advantage of the possibility of obtaining long term survival of hamster heart xenografts in rat recipients treated with a combination of cobra venom factor and cyclosporin A (CsA), to analyze T cell infiltration and, for the first time, V beta TCR usage, at the complementarity-determining region 3 level, in accommodated and rejected xenografts, compared with allografts. After withdrawal of CsA (on day 40), the analysis of V beta family expression and corresponding complementarity-determining region 3 lengths in rejected xenografts revealed a Gaussian pattern, in contrast to a much more restricted pattern in rejected allografts (p = 0.002), suggesting that, after withdrawal of CsA, all the underrepresented T cell clones are rapidly expanded in xenografts. These results correlate with the rapid kinetics of rejection (4 +/- 1 days), the high number of T cells, the rapid expression of markers of activation (IL-2 receptor alpha-chain and class II receptor), and the strong deposit of IgG Abs in rejected xenografts. Taken together, these results suggest that the intensity and diversity of the T cell response to xenografts could be stronger than the response to allografts in vivo