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

The Education of NK Cells Determines Their Responsiveness to Autologous HIV-Infected CD4 T Cells

International audienceSeveral studies support a role for specific killer immunoglobulin-like receptor (KIR)-HLA combinations in protection from HIV infection and slower progression to AIDS. Natural killer (NK) cells acquire effector functions through education, a process that requires the interaction of inhibitory NK cell receptors with their major histocompatibility complex (MHC) class I (or HLA class I [HLA-I]) ligands. HLA-C allo-types are ligands for the inhibitory KIRs (iKIRs) KIR2DL1, KIR2DL2, and KIR2DL3, whereas the ligand for KIR3DL1 is HLA-Bw4. HIV infection reduces the expression of HLA-A,-B, and-C on the surfaces of infected CD4 (iCD4) T cells. Here we investigated whether education through iKIR-HLA interactions influenced NK cell responses to autologous iCD4 cells. Enriched NK cells were stimulated with autologous iCD4 cells or with uninfected CD4 cells as controls. The capacities of single-positive (sp) KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1 NK cells to produce CCL4, gamma inter-feron (IFN-␥), and/or CD107a were assessed by flow cytometry. Overall, we observed that the potency of NK cell education was directly related to the frequency of each spiKIR ϩ NK cell's ability to respond to the reduction of its cognate HLA ligand on autologous iCD4 cells, as measured by the frequency of production by spiKIR ϩ NK cells of CCL4, IFN-␥, and/or CD107a. Both NK cell education and HIV-mediated changes in HLA expression influenced NK cell responses to iCD4 cells

Impact of Graft-Versus-Graft Natural Killer Cell Alloreactivity on Single Unit Dominance After Double Umbilical Cord Blood Transplantation

International audienceBACKGROUND:Natural killer (NK) cell alloreactivity is favored after double umbilical cord blood transplantation (dUCBT) in which cord blood (UCB) units and patients are often HLA class I mismatched. Generally, only 1 UCB unit persists after dUCBT. We hypothesize, that NK cell alloreactivity mediated by killer cell immunoglobulin-like receptor (KIR)-HLA interactions may explain the dominance of 1UCB unit over the other after dUCBT.METHODS:We investigated the impact of KIR NK cell alloreactivities on the dominance of 1 full UCB unit in 50 dUCBT. We analyzed the effects of the KIR/HLA genetic incompatibilities and studied cord blood cells at both the phenotypic and functional levels.RESULTS:The genetic combination of KIR3DL1 loser UCB unit/Bw4 winner UCB unit determined both the dominance of 1 UCB unit (hazards ratio, 2.88 [1.32-6.27], P = 0.0077) and correlated with an increased incidence of relapse (hazards ratio, 4.91 [1.39-17.3], P = 0.0134). It is interesting to note that cord blood cells exhibited extremely low HLA class I expression. Moreover, resting cord blood KIR3DL1 NK cells exhibited a basal alloreactivity against Bw4 target cells that increased upon activation, thus triggering death by apoptosis.CONCLUSIONS:Our unicentric study suggests, for the first time, the significant impact of KIR NK cell alloreactivity in the determination of which UCB unit will dominate in dUCBT

New insights on the natural killer cell repertoire from a thorough analysis of cord blood cells

International audienceAlthough CB NK cells are characterized as immature lymphocytes, their impressive expansion and efficient graft-versus-leukemia response have been highlighted early after UCBT. To better evaluate their potential as source of effective NK cells, we revisited the study of NK cell repertoire from a large cohort of CB samples. Our study showed that the CB NK cell repertoire appears to be constructed early, depending on KIR gene content, but not on the autologous HLA environment. NKG2A was expressed on a large proportion of CB NK cells that inversely correlated with KIR + NK cell frequency. Self-HLA class I molecule-educated CB KIR + NK cells present a lower spontaneous lysis than do their adult counterparts, which is probably related to the low expression of activating NK receptors. We describe for the first time a proliferative and cytotoxic NKG2C + NK cell subset representing more than 10% of CB NK cells. NKG2A strongly inhibited CB NK cell degranulation, and its coexpression on NKG2C + NK cells may contribute to limiting their activation. Overall, the CB NK cell repertoire is constructed early and harbors numerous functional abilities shared by adult NK cells. In addition, their naïve viral status and fast expansion confer numerous advantages in immunotherapy on CB NK cells

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

HLA Reduces Killer Cell Ig-like Receptor Expression Level and Frequency in a Humanized Mouse Model

Transplantation and autoimmunit