Etude du polymorphisme allélique KIR pour optimiser la sélection des donneurs de greffes des cellules souches hématopoiétiques

Les cellules Natural Killer (NK) jouent un rôle important en greffes de CSH par leur rôle antileucémique. Les fonctions cytotoxiques des cellules NK sont gouvernées principalement par les récepteurs KIR spécifiques des molécules HLA de classe I. Ces récepteurs inhibiteurs ou activateurs sont codés par 15 gènes KIR connus pour être polymorphes au niveau allélique. Ce polymorphisme allélique peut affecter le phénotype et la fonction des cellules NK mais reste difficile à évaluer. Dans ce projet, nous avons développé une approche de séquençage-nouvelle-génération (NGS) pour déterminer les allèles de tous les gènes KIR de donneurs de sang. Les résultats ont montré la fiabilité de notre approche NGS.KIR pour le typage allélique KIR. Le polymorphisme des allèles KIR2DL1/2/3 a été mis en lien avec le phénotype et la fonction des cellules NK. Nos résultats montrent une diversité limitée d’allèles KIR2DL1, KIR2DL2/L3 qui impactent sur le phénotype et la fonction des souspopulations NK KIR2DL+. Le polymorphisme des allèles KIR2DL1/2/3 module également les interactions des récepteurs KIR correspondants avec les molécules HLA-Cw. Ce projet de Thèse contribue à une meilleure connaissance de l’impact du polymorphisme allélique KIR sur la structuration et fonction du répertoire des cellules NK KIR+. Ces données permettront d’affiner la sélection des donneurs de greffes de CSH haplo-identiques non T déplétées pour mieux évaluer le rôle de l’alloréactivité des cellules NK KIR+ sur l’effet antileucémique. La connaissance des allèles KIR peut aussi constituer un outil diagnostic dans certaines infections virales, associations KIR-maladies ou pathologies liées à la grossesse.Natural Killer (NK) cells are large granular lymphocytes able to kill leukemic cells after Hematopoietic Stem Cell Transplantation (HSCT). Their cytotoxic functions are governed by many receptors, especially by the KIR receptors that recognize HLA class I molecules. These inhibitory (KIR2DL, KIR3DL) and activating (KIR2DS, KIR3DS) receptors are encoded by a family of 15 genes known to be polymorphic at allelic level. This allelic polymorphism may impact on NK cell phenotype and function, but remains difficult to evaluate by lack of appropriate methods. In this thesis project, we developed a Next- Generation-Sequencing approach to investigate the KIR allele polymorphism in volunteer blood donors. The results showed the reliability of our NGS approach for KIR allele typing. Then, we correlated the KIR2DL1/2/3 allele polymorphism with the phenotype and function of KIR+ NK cells. We found a limited diversity of KIR2DL1 and KIR2DL2/3 alleles that impact the phenotype and function of KIR2DL+ NK cell subsets. This KIR2DL1/2/3 allele polymorphism modulates also the corresponding KIR-HLA-Cw interactions. At the fundamental level, this thesis project improves our knowledge on the impact of KIR allele polymorphism on the structuration and the function of KIR+ NK cell repertoire. Overall these data can help to refine the HSCT donor selection in the context of T replete haploidentical HSCT to unravel the role of alloreactive KIR+ NK cells mediating an antileukemic effect. More broadly, the knowledge of KIR alleles may also constitute a diagnostic tool in some viral infections, KIR associated diseases and pregnancy disorders

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

Killer Immunoglobulin-Like Receptor Allele Determination Using Next-Generation Sequencing Technology

International audienceThe impact of natural killer (NK) cell alloreactivity on hematopoietic stem cell transplantation (HSCT) outcome is still debated due to the complexity of graft parameters, HLA class I environment, the nature of killer cell immunoglobulin-like receptor (KIR)/KIR ligand genetic combinations studied, and KIR+ NK cell repertoire size. KIR genes are known to be polymorphic in terms of gene content, copy number variation, and number of alleles. These allelic polymorphisms may impact both the phenotype and function of KIR+ NK cells. We, therefore, speculate that polymorphisms may alter donor KIR+ NK cell phenotype/function thus modulating post-HSCT KIR+ NK cell alloreactivity. To investigate KIR allele polymorphisms of all KIR genes, we developed a next-generation sequencing (NGS) technology on a MiSeq platform. To ensure the reliability and specificity of our method, genomic DNA from well-characterized cell lines were used; high-resolution KIR typing results obtained were then compared to those previously reported. Two different bioinformatic pipelines were used allowing the attribution of sequencing reads to specific KIR genes and the assignment of KIR alleles for each KIR gene. Our results demonstrated successful long-range KIR gene amplifications of all reference samples using intergenic KIR primers. The alignment of reads to the human genome reference (hg19) using BiRD pipeline or visualization of data using Profiler software demonstrated that all KIR genes were completely sequenced with a sufficient read depth (mean 317× for all loci) and a high percentage of mapping (mean 93% for all loci). Comparison of high-resolution KIR typing obtained to those published data using exome capture resulted in a reported concordance rate of 95% for centromeric and telomeric KIR genes. Overall, our results suggest that NGS can be used to investigate the broad KIR allelic polymorphism. Hence, these data improve our knowledge, not only on KIR+ NK cell alloreactivity in HSCT but also on the role of KIR+ NK cell populations in control of viral infections and diseases

Centromeric KIR AA Individuals Harbor Particular KIR Alleles Conferring Beneficial NK Cell Features with Implications in Haplo-Identical Hematopoietic Stem Cell Transplantation

International audienceWe have recently shown a broad disparity of Natural Killer (NK) cell responses against leukemia highlighting good and bad responders resting on the Killer cell Immunoglobulin-like Receptors (KIR) and HLA genetics. In this study, we deeply studied KIR2D allele expression, HLA-C recognition and functional effect on NK cells in 108 blood donors in combining high-resolution KIR allele typing and multicolor flow cytometry. The KIR2DL1*003 allotype is associated with centromeric (cen) AA motif and confers the highest NK cell frequency, expression level and strength of KIR/HLA-C interactions compared to the KIR2DL1*002 and KIR2DL1*004 allotypes respectively associated with cenAB and BB motifs. KIR2DL2*001 and *003 allotypes negatively affect the frequency of KIR2DL1+ and KIR2DL3+ NK cells. Altogether, our data suggest that cenAA individuals display more efficient KIR2DL alleles (L1*003 and L3*001) to mount a consistent frequency of KIR2DL+ NK cells and to confer an effective NK cell responsiveness. The transposition of our in vitro observations in the T-replete haplo-identical HSCT context led us to observe that cenAA HSC grafts limit significantly the incidence of relapse in patients with myeloid diseases after T-replete haplo-identical HSCT. As NK cells are crucial in HSCT reconstitution, one could expect that the consideration of KIR2DL1/2/3 allelic polymorphism could help to refine scores used for HSC donor selection

Impact of KIR/HLA Incompatibilities on NK Cell Reconstitution and Clinical Outcome after T Cell–Replete Haploidentical Hematopoietic Stem Cell Transplantation with Posttransplant Cyclophosphamide

P.C. and C.R. have equally contributed to this workInternational audienceLittle is known regarding the effect of KIR/HLA incompatibilities (inc.) in the setting of T-replete haploidentical allogeneic he-matopoietic stem cell transplantation using posttransplant cyclophosphamide (PTCy). In this retrospective study, the impact of KIR/HLA inc. on clinical outcomes and NK cell reconstitution was studied in a cohort of 51 consecutive patients receiving a T cell-replete haploidentical allogeneic hematopoietic stem cell transplantation after a reduced-intensity conditioning using peripheral blood stem cells as the source of the graft and PTCy as graft-versus-host disease (GvHD) prophylaxis. The NK cell repertoire reconstitution was examined by multiparameter flow cytometry in 34 of these 51 patients from day 0 to day 100 post-transplant. Genetic KIR2DL/HLA inc. were found to be significantly associated with more GvHD (81.2 versus 45.7%, p = 0.01) and less relapse (6.2 versus 42.8%, p = 0.008) in this context. GvHD is associated with increased levels of differentiated and activated NK cells. A significant loss of KIR2DL2/3 + NK cells was observed at day 30 in patients with inhibitory KIR/HLA inc., suggesting that responsive KIR NK cells are particularly targeted by the immunosuppressive PTCy treatment. Further investigations are needed from a larger cohort with an identical clinical approach to consolidate these results and to identify the NK cell subsets that may be beneficial for the graft-versus-leukemia effect observed. Because many haploidentical donors can be identified in a family, the prediction of KIR NK cell alloreactivity could be of crucial importance for donor selection and patient outcome