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

Use of killer cell immunoglobulin-like receptor genes as early markers of hematopoietic chimerism after double-umbilical cord blood transplantation

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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

Deciphering the biology of KIR2DL3+ T lymphocytes that are associated to relapse in haploidentical HSCT

International audienceKIR are mainly expressed on NK cells and to a lesser extent on T lymphocytes. Although the KIR NK cell repertoire was well explored in haploidentical Hematopoietic Stem Cell Transplantation (HSCT), KIR T cell compartment remains to be investigated in this context. In this study, the investigation of NK receptors on T lymphocytes during immune reconstitution after T-cell-replete haploidentical HSCT with Post-Transplant Cyclophosphamide (PTCy) has shown a significant increase of KIR2DL2/3 + T cell frequency at day 25. This was especially observed at day 30 in recipients who relapsed. IL-15 but not IL-12 increased in vitro KIR + T cell expansion suggesting that the raised IL-15 serum concentration observed after PTCy in haploidentical HSCT might increase KIR + T cell frequency. Moreover, investigations from healthy blood donors showed a higher inhibiting effect of KIR2DL3 on CMV specific T cell response against allogeneic than autologous C1 + target cells. The association of KIR + T cell subset with relapse may suggest that inhibitory KIR2DL2/3 limit anti-leukemic effect of specific T lymphocytes at this early step of immune reconstitution. Further phenotypic and mechanistic investigations on this cell subset from a broader cohort of HSCT recipients should clarify its potential implication in relapse occurrence. Our results demonstrate that KIR-HLA interactions known to modulate NK cell functions also modulate T cell immune responses in the context of allogeneic HSCT

Amplified NKG2C+NK Cells in Cytomegalovirus (CMV) Infection Preferentially Express Killer Cell Ig-like Receptor 2DL: Functional Impact in Controlling CMV-Infected Dendritic Cells

International audienceCMV infection represents a major complication in hematopoietic stem cell transplantation, which compromises graft outcome. Downregulation of HLA class I expression is one mechanism by which CMV evades T cell-mediated immune detection, rendering infected cells vulnerable to killer cell Ig-like receptor (KIR)(+) NK cells. In this study, we observed that the amplified NKG2C(+) NK cell population observed specifically in CMV seropositive individuals mainly expressed KIR2DL receptors. We have shown that HLA class I expression was downregulated on CMV-infected immature dendritic cells (iDCs), which escape to HLA-A2-pp65-specific T lymphocytes but strongly trigger the degranulation of KIR2D(+) NK cells. CMV infection conferred a vulnerability of C2C2(+) iDCs to educated KIR2DL1(+) and KIR2DL3(+) NK cell subsets. Alloreactivity of KIR2DL1(+) NK cell subsets against C1C1(+) iDCs was maintained independently of CMV infection. Unexpectedly, CMV-infected C1C1(+) iDCs did not activate KIR2DL3(+) NK cell reactivity, suggesting a potential CMV evasion to KIR2DL3 NK cell recognition. Altogether, the coexpression of KIR and NKG2C on expanded NK cell subsets could be related to a functional contribution of KIR in CMV infection and should be investigated in hematopoietic stem cell transplantation, in which the beneficial impact of CMV infection has been reported on the graft-versus-leukemia effect

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

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Both the nature of KIR3DL1 alleles and the KIR3DL1/S1 allele combination affect the KIR3DL1 NK-cell repertoire in the French population

International audienceNK-cell functions are regulated by many activating and inhibitory receptors including KIR3DL1. Extensive allelic polymorphism and variability in expression can directly alter NK-cell phenotype and functions. Here we investigated the KIR3DL1 + NK-cell repertoire, taking into account the allelic KIR3DL1/S1 polymorphism, KIR3DL1 phenotype, and function. All 109 studied individuals possessed at least one KIR3DL1 allele, with weak KIR3DL1*054, or null alleles being frequently present. In KIR3DL1 high/null individuals, we observed a bimodal distribution of KIR3DL1 + NK cells identified by a different KIR3DL1 expression level and cell frequency regardless of a similar amount of both KIR3DL1 transcripts, HLA background, or KIR2D expression. However, this bimodal distribution can be explained by a functional selection following a hierarchy of KIR3DL1 receptors. The higher expression of KIR3DL1 observed on cord blood NK cells suggests the expression of the functional KIR3DL1*004 receptors. Thus, the low amplification of KIR3DL1 high , KIR3DL1*004 NK-cell subsets during development may be due to extensive signaling via these two receptors. Albeit in a nonexclusive manner, individual immunological experience may contribute to shaping the KIR3DL1 NK-cell repertoire. Together, this study provides new insight into the mechanisms regulating the KIR3DL1 NK-cell repertoire

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