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Introduction: Polymorphisms in the KIR and HLA genes contribute to the diversity of the NK cell repertoire. Extrinsic factors also play a role in modifying this repertoire. The best example is cytomegalovirus, which promotes the expansion of memory-like NK cells. However, the mechanisms governing this phenotypic structure are poorly understood. Furthermore, the influence of age and sex has been understudied., Methods: In this study, we examined these parameters in a cohort of 200 healthy volunteer blood donors, focusing on the major inhibitory KIR receptors and CD94/NKG2A, as well as the differentiation marker CD57 and the memory-like population marker NKG2C. Flow cytometry and two joint analyses, unsupervised and semi-supervised, helped define the impact of various intrinsic and extrinsic markers on the phenotypic structure of the NK cell repertoire., Results: In the KIR NK cell compartment, the KIR3DL1 gene is crucial, as unexpressed alleles lead to a repertoire dominated by KIR2D interacting only with HLA-C ligands, whereas an expressed KIR3DL1 gene allows for a greater diversity of NK cell subpopulations interacting with all HLA class I ligands. KIR2DL2 subsequently favors the KIR2D NK cell repertoire specific to C1/C2 ligands, whereas its absence promotes the expression of KIR2DL1 specific to the C2 ligand. The C2C2Bw4+ environment, marked by strong -21T motifs, favors the expansion of the NK cell population expressing only CD57, whereas the absence of HLA-A3/A11 ligands favors the population expressing only NKG2A, a population highly represented within the repertoire. The AA KIR genotype favors NK cell populations without KIR and NKG2A receptors, whereas the KIR B+ genotypes favor populations expressing KIR and NKG2A. Interestingly, we showed that women have a repertoire enriched in CD57- NK cell populations, while men have more CD57+ NK cell subpopulations., Discussion: Overall, our data demonstrate that the phenotypic structure of the NK cell repertoire follows well-defined genetic rules and that immunological history, sex, and age contribute to shaping this NK cell diversity. These elements can contribute to the better selection of hematopoietic stem cell donors and the definition of allogeneic NK cells for cell engineering in NK cell-based immunotherapy approaches.cters are displayed correctly., Competing Interests: The authors declare that this study was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2024 Ferron, David, Willem, Legrand, Salameh, Anquetil, Walencik, Gendzekhadze, Gagne and Retière.)

KIR3DL1 alleles are expressed at different levels on the natural killer (NK) cell surface. In particular, the non-expressed KIR3DL1*004 allele appears to be common in Caucasian populations. However, the overall distribution of non-expressed KIR3DL1 alleles and their clinical relevance after T-replete haploidentical hematopoietic stem cell transplantation (hHSCT) with post-transplant cyclophosphamide remain poorly documented in European populations. In a cohort of French blood donors (N = 278), we compared the distribution of expressed and non-expressed KIR3DL1 alleles using next-generation sequencing (NGS) technology combined with multi-color flow cytometry. We confirmed the predominance of the non-expressed KIR3DL1*004 allele. Using allele-specific constructs, the phenotype and function of the uncommon KIR3DL1*019 allotype were characterized using the Jurkat T cell line and NKL transfectants. Although poorly expressed on the NK cell surface, KIR3DL1*019 is retained within NK cells, where it induces missing self-recognition of the Bw4 epitope. Transposing our in vitro observations to a cohort of hHSCT patients (N = 186) led us to observe that non-expressed KIR3DL1 HSC grafts increased the incidence of relapse in patients with myeloid diseases. Non-expressed KIR3DL1 alleles could, therefore, influence the outcome of hHSCT.

Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym "NK-AML" (NCT03955848), and "MRD-NK". The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2 + donors and HLA-C1 + patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as potential conflict of interest., (Copyright © 2023 Meazza, Ruggeri, Guolo, Minetto, Canevali, Loiacono, Ciardelli, Bo, Luchetti, Serio, Zannoni, Retière, Colomar-Carando, Parisi, Curti, Lemoli and Pende.)

KIR 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., (© 2021. The Author(s).)

Since 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.

The antibody-dependent cellular cytotoxicity (ADCC) effector function of natural killer (NK) cells is one of the known mechanisms of action for rituximab-based anti-cancer immunotherapy. Inhibition of the ADCC function of NK cells through interactions between inhibitory killer cell immunoglobulin-like receptors (KIRs) and HLA class I ligands is associated with resistance of cancers to rituximab. In this study, we deeply investigated the impact of KIR, HLA class I, and CD16 genotypes on rituximab-dependent NK cell responses in both an in vitro cellular model from healthy blood donors and ex vivo rituximab-treated non-Hodgkin lymphoma (NHL) patients. We highlight that an HLA environment with limited KIR ligands is beneficial to promoting a higher frequency of KIR + NK cells including both educated and uneducated NK cells, two NK cell compartments that demonstrate higher rituximab-dependent degranulation than KIR - NK cells. In contrast, a substantial KIR ligand environment favors a higher frequency of poorly effective KIR - NK cells and numerous functional KIR/HLA inhibitions of educated KIR + NK cells. These phenomena explain why NHL patients with limited KIR ligands respond better to rituximab. In this HLA environment, CD16 polymorphism appears to have a collateral effect. Furthermore, we show the synergic effect of KIR2DS1, which strongly potentiates NK cell ADCC from C2 - blood donors against C2 + target cells. Taken together, these results pave the way for stronger prediction of rituximab responses for NHL patients. HLA class I typing and peripheral blood KIR + NK cell frequency could be simple and useful markers for predicting rituximab response., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The closely related inhibitory killer-cell immunoglobulin-like receptors (KIR), KIR2DL2 and KIR2DL3, regulate the activation of natural killer cells (NK) by interacting with the human leukocyte antigen-C1 (HLA-C1) group of molecules. KIR2DL2, KIR2DL3 and HLA-C1 are highly polymorphic, with this variation being associated with differences in the onset and progression of some human diseases. However, the molecular bases underlying these associations remain unresolved. Here, we determined the crystal structures of KIR2DL2 and KIR2DL3 in complex with HLA-C*07:02 presenting a self-epitope. KIR2DL2 differed from KIR2DL3 in docking modality over HLA-C*07:02 that correlates with variabilty of recognition of HLA-C1 allotypes. Mutagenesis assays indicated differences in the mechanism of HLA-C1 allotype recognition by KIR2DL2 and KIR2DL3. Similarly, HLA-C1 allotypes differed markedly in their capacity to inhibit activation of primary NK cells. These functional differences derive, in part, from KIR2DS2 suggesting KIR2DL2 and KIR2DL3 binding geometries combine with other factors to distinguish HLA-C1 functional recognition.

We 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.

A higher incidence of graft-versus-host disease (GVHD) has been observed after haploidentical hematopoietic stem cell transplantation (h-HSCT) with posttransplant cyclophosphamide (PTCY) using peripheral blood stem cells (PBSC) as a source of graft. Moreover, combining PTCY with antithymocyte globulin (ATG) may help to reduce GVHD incidence. In this study, early immune reconstitution, especially of T and NK cell compartments, was compared after both types of transplant (PTCY versus PTCY + ATG) investigate their influence on patient outcomes. This retrospective study included 58 adults who received a reduced intensity conditioning to PBSC h-HSCT with cyclosporine and mycophenolate mofetyl + PTCY ( n = 32) or PTCY + ATG ( n = 26) as GVHD prophylaxis. Both groups shared similar characteristics except for the median number of CD3 + T cells infused, significantly higher for PTCY + ATG patients. Blood samples from all patients were collected three times a week from day 0 until day 30 then at day 60 and day 90/100 to evaluate T and NK cells reconstitution by flow cytometry. The results show that PTCY + ATG versus PTCY alone significantly limits the occurrence of acute grade 2-4 GVHD after reduced intensity conditioning PBSC h-HSCT, perhaps because of the combined effect of T and NK cell reconstitution. Indeed, although a slower T cell reconstitution with PTCY + ATG may limit GVHD occurrence, the quicker reconstitution of some NK cell subtypes may help with avoiding relapse. Larger prospective studies are needed to better determine which NK cell subsets may influence the incidence of relapse after h-HSCT and optimize donor selection., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Natural killer (NK) cells are key cytotoxic effectors against malignant cells. Polygenic and polymorphic Killer cell Immunoglobulin-like Receptor (KIR) and HLA genes participate in the structural and functional formation of the NK cell repertoire. In this study, we extensively investigated the anti-leukemic potential of NK cell subsets, taking into account these genetic parameters and cytomegalovirus (CMV) status. Hierarchical clustering analysis of NK cell subsets based on NKG2A, KIR, CD57 and NKG2C markers from 68 blood donors identified donor clusters characterized by a specific phenotypic NK cell repertoire linked to a particular immunogenetic KIR and HLA profile and CMV status. On the functional side, acute lymphoblastic leukemia (ALL) was better recognized by NK cells than acute myeloid leukemia (AML). However, a broad inter-individual disparity of NK cell responses exists against the same leukemic target, highlighting bad and good NK responders. The most effective NK cell subsets against different ALLs expressed NKG2A and represented the most frequent subset in the NK cell repertoire. In contrast, minority CD57 + or/and KIR + NK cell subsets were more efficient against AML. Overall, our data may help to optimize the selection of hematopoietic stem cell donors on the basis of immunogenetic KIR/HLA for ALL patients and identify the best NK cell candidates in immunotherapy for AML.

Purpose: Cellular immunotherapies are currently being explored to eliminate highly invasive and chemoradioresistant glioblastoma (GBM) cells involved in rapid relapse. We recently showed that concomitant stereotactic injections of nonalloreactive allogeneic Vγ9Vδ2 T lymphocytes eradicate zoledronate-primed human GBM cells. In the present study, we investigated the spontaneous reactivity of allogeneic human Vγ9Vδ2 T lymphocytes toward primary human GBM cells, in vitro and in vivo , in the absence of any prior sensitization., Experimental Design: Through functional and transcriptomic analyses, we extensively characterized the immunoreactivity of human Vγ9Vδ2 T lymphocytes against various primary GBM cultures directly derived from patient tumors., Results: We evidenced that GBM cells displaying a mesenchymal signature are spontaneously eliminated by allogeneic human Vγ9Vδ2 T lymphocytes, a reactivity process being mediated by γδ T-cell receptor (TCR) and tightly regulated by cellular stress-associated NKG2D pathway. This led to the identification of highly reactive Vγ9Vδ2 T lymphocyte populations, independently of a specific TCR repertoire signature. Moreover, we finally provide evidence of immunotherapeutic efficacy in vivo , in the absence of any prior tumor cell sensitization., Conclusions: By identifying pathways implicated in the selective natural recognition of mesenchymal GBM cell subtypes, accounting for 30% of primary diagnosed and 60% of recurrent GBM, our results pave the way for novel targeted cellular immunotherapies., (©2019 American Association for Cancer Research.)

Several 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 allotypes 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 interferon (IFN-γ), 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 + 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 + NK cells of CCL4, IFN-γ, and/or CD107a. Both NK cell education and HIV-mediated changes in HLA expression influenced NK cell responses to iCD4 cells. IMPORTANCE Epidemiological studies show that natural killer (NK) cells have anti-HIV activity: they are able to reduce the risk of HIV infection and/or slow HIV disease progression. How NK cells contribute to these outcomes is not fully characterized. We used primary NK cells and autologous HIV-infected cells to examine the role of education through four inhibitory killer immunoglobulin-like receptors (iKIRs) from persons with HLA types that are able to educate NK cells bearing one of these iKIRs. HIV-infected cells activated NK cells through missing-self mechanisms due to the downmodulation of cell surface HLA expression mediated by HIV Nef and Vpu. A higher frequency of educated than uneducated NK cells expressing each of these iKIRs responded to autologous HIV-infected cells by producing CCL4, IFN-γ, and CD107a. Since NK cells were from non-HIV-infected individuals, they model the consequences of healthy NK cell-HIV-infected cell interactions occurring in the HIV eclipse phase, when new infections are susceptible to extinction., (Copyright © 2019 American Society for Microbiology.)

Variants in genes encoding ribosomal proteins have thus far been associated with Diamond-Blackfan anemia, a rare inherited bone marrow failure, and isolated congenital asplenia. Here, we report one de novo missense variant and three de novo splice variants in RPL13, which encodes ribosomal protein RPL13 (also called eL13), in four unrelated individuals with a rare bone dysplasia causing severe short stature. The three splice variants (c.477+1G>T, c.477+1G>A, and c.477+2 T>C) result in partial intron retention, which leads to an 18-amino acid insertion. In contrast to observations from Diamond-Blackfan anemia, we detected no evidence of significant pre-rRNA processing disturbance in cells derived from two affected individuals. Consistently, we showed that the insertion-containing protein is stably expressed and incorporated into 60S subunits similar to the wild-type protein. Erythroid proliferation in culture and ribosome profile on sucrose gradient are modified, suggesting a change in translation dynamics. We also provide evidence that RPL13 is present at high levels in chondrocytes and osteoblasts in mouse growth plates. Taken together, we show that the identified RPL13 variants cause a human ribosomopathy defined by a rare skeletal dysplasia, and we highlight the role of this ribosomal protein in bone development., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Little is known regarding the effect of KIR/HLA incompatibilities (inc.) in the setting of T-replete haploidentical allogeneic hematopoietic 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 posttransplant. 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., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Killer-cell Ig-like receptor (KIR) genes are inherited as haplotypes. They are expressed by NK cells and linked to outcomes of infectious diseases and pregnancy in humans. Understanding how genotype relates to phenotype is difficult because of the extensive diversity of the KIR family. Indeed, high-resolution KIR genotyping and phenotyping in single NK cells in the context of disease association is lacking. In this article, we describe a new method to separate NK cells expressing allotypes of the KIR2DL1 gene carried by the KIR A haplotype (KIR2DL1A) from those expressing KIR2DL1 alleles carried by the KIR B haplotype (KIR2DL1B). We find that in KIR AB heterozygous individuals, different KIR2DL1 allotypes can be detected in both peripheral blood and uterine NK cells. Using this new method, we demonstrate that both blood and uterine NK cells codominantly express KIR2DL1A and KIR2DL1B allotypes but with a predominance of KIR2DL1A variants, which associate with enhanced NK cell function. In a case-control study of pre-eclampsia, we show that KIR2DL1A , not KIR2DL1B , associates with increased disease risk. This method will facilitate our understanding of how individual KIR2DL1 allelic variants affect NK cell function and contribute to disease risk., (Copyright © 2018 by The American Association of Immunologists, Inc.)

[This corrects the article on p. 1721 in vol. 8, PMID: 29312288.].

We have compared prospectively the outcome and immune reconstitution of patients receiving either post-transplant cyclophosphamide (PTCY) ( n = 30) or anti-thymocyte globulin ATG ( n = 15) as Graft-versus-host disease (GVHD) prophylaxis after reduced-intensity conditioning (RIC) allogeneic peripheral blood stem cell (PBSC) transplantation (allo-SCT). The outcome and immune reconstitution of patients receiving either of these two regimens were compared prospectively. This study allowed also to investigate the impact of PTCY between haplo-identical vs matched donors and of clofarabine as part of the RIC regimen. The γ/δ T-cells, α/β T-cells (CD8 + and CD4 + ), NK T-cells, NK cells, B-cells, Tregs and monocytes were analyzed by flow cytometry from a total of 583 samples. In the PTCY group significant delayed platelets recovery, higher CD3+ donor chimerism, higher HHV-6 and lower EBV reactivations were observed. Early survival advantage for CD4+ T-cells, Tregs and α/β T-cells was documented in the PTCY group while it was the case for α/β T-cells, NK cells and monocytes in the ATG group. Higher counts of NK and monocytes were observed at days +30 and/or day+60 in the ATG group. Both results were retained even in the case of mismatched donors. However, higher percentages of CD4+ T-cells, α/β T-cells and Tregs were observed with haplo-identical donors in the PTCY group. Finally, clofarabine was responsible for early survival advantage of NK T-cells in the PTCY group while it abrogated the early survival advantage of γ/δ T-cells in the ATG group. In conclusion, there are marked differences in the immunological effects of ATG vs PTCY as GVHD prophylaxis for RIC PBSC allo-SCT., Competing Interests: CONFLICTS OF INTEREST The authors declared no conflicts of interest.

The role of natural killer (NK) cells in organ transplantation is controversial. This study aims to decipher their role in kidney transplant tolerance in humans. Previous studies highlighted several modulated genes involved in NK cell biology in blood from spontaneously operationally tolerant patients (TOLs; drug-free kidney-transplanted recipients with stable graft function). We performed a phenotypic, functional, and genetic characterization of NK cells from these patients compared to kidney-transplanted patients with stable graft function under immunosuppression and healthy volunteers (HVs). Both operationally TOLs and stable patients harbored defective expression of the NKp46 activator receptor and lytic molecules perforin and granzyme compared to HVs. Surprisingly, NK cells from operationally TOLs also displayed decreased expression of the CD16 activating marker (in the CD56 Dim NK cell subset). This decrease was associated with impairment of their functional capacities upon stimulation, as shown by lower interferon gamma (IFNγ) production and CD107a membranous expression in a reverse antibody-dependent cellular cytotoxicity (ADCC) assay, spontaneous lysis assays, and lower target cell lysis in the 51 Cr release assay compared to HVs. Conversely, despite impaired K562 cell lysis in the 51 Cr release assay, patients with stable graft function harbored a normal reverse ADCC and even increased amounts of IFNγ + NK cells in the spontaneous lysis assay. Altogether, the strong impairment of the phenotype and functional cytotoxic capacities of NK cells in operationally TOLs may accord with the establishment of a pro-tolerogenic environment, despite remaining highly activated after transplantation in patients with stable graft function.

Background: 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.

The 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.

Blood samples from 32 patients with severe Traumatic brain injury (TBI) were studied and compared with 11 cardiac surgery patients, and 29 healthy controls. A dramatic decreased expression of HLA class I molecules on monocytes was associated with increased KIR+ NK cell frequency in TBI patients. Overall, the phenotype of TBI NK cells marked by KIR and CD57 expression and lower level of NKp46 and DNAM-1 reflected a differentiated state. The NK-cell response to missing self was marked by lower degranulation and lower IFN-γ production after stimulation with HLA class I deficient cell line. In contrast, the NK-cell ADCC was not altered. IL-12 was able to restore both IFN-γ production and the cytotoxicity capacities of NK cells. This study provides the first extensive description of the phenotype and functions of NK cells in TBI patients. Further evaluation of IL-12 treatment to overcome immunosuppression-induced nosocomial infections is warranted., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Primary human cytomegalovirus (HCMV) infection usually goes unnoticed, causing mild or no symptoms in immunocompetent individuals. However, some rare severe clinical cases have been reported without investigation of host immune responses or viral virulence. In the present study, we investigate for the first time phenotypic and functional features, together with gene expression profiles in immunocompetent adults experiencing a severe primary HCMV infection. Twenty primary HCMV-infected patients (PHIP) were enrolled, as well as 26 HCMV-seronegative and 39 HCMV-seropositive healthy controls. PHIP had extensive lymphocytosis marked by massive expansion of natural killer (NK) and T cell compartments. Interestingly, PHIP mounted efficient innate and adaptive immune responses with a deep HCMV imprint, revealed mainly by the expansion of NKG2C + NK cells, CD16 + Vδ2(-) γδ T cells, and conventional HCMV-specific CD8 + T cells. The main effector lymphocytes were activated and displayed an early immune phenotype that developed toward a more mature differentiated status. We suggest that both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage observed in PHIP. Taken together, these findings bring new insights into the comprehensive understanding of immune mechanisms involved during primary HCMV infection in immunocompetent individuals. IMPORTANCE HCMV-specific immune responses have been extensively documented in immunocompromised patients and during in utero acquisition. While it usually goes unnoticed, some rare severe clinical cases of primary HCMV infection have been reported in immunocompetent patients. However, host immune responses or HCMV virulence in these patients has not so far been investigated. In the present study, we show massive expansion of NK and T cell compartments during the symptomatic stage of acute HCMV infection. The patients mounted efficient innate and adaptive immune responses with a deep HCMV imprint. The massive lymphocytosis could be the result of nonadapted or uncontrolled immune responses limiting the effectiveness of the specific responses mounted. Both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage. Furthermore, we cannot exclude a delayed immune response caused by immune escape established by HCMV strains., (Copyright © 2017 American Society for Microbiology.)

By affecting immunological presentation, the presence of cytomegalovirus in some glioblastomas may impact progression. In this study, we examined a hypothesized role for natural killer (NK) cells in impacting disease progression in this setting. We characterized 108 glioblastoma patients and 454 healthy controls for HLA-A,-B,-C, NK-cell KIR receptors, and CMV-specific antibodies and correlated these metrics with clinical parameters. Exome sequences from a large validation set of glioblastoma patients and control individuals were examined from in silico databases. We demonstrated that the KIR allele KIR2DS4*00101 was independently prognostic of prolonged survival. KIR2DS4*00101 displayed 100% concordance with cognate HLA-C1 ligands in glioblastoma patients, but not controls. In the context of both HLA-C1/C2 ligands for the KIR2DS4 receptor, patient survival was further extended. Notably, all patients carrying KIR2DS4*00101 alleles were CMV seropositive, but not control individuals, and exhibited increased NK-cell subpopulations, which expressed the cytotoxicity receptors CD16, NKG2D, and CD94/NKG2C. Finally, healthy controls exhibited a reduced risk for developing glioblastoma if they carried two KIR2DS4*00101 alleles, where protection was greatest among Caucasian individuals. Our findings suggest that KIR2DS4*00101 may offer a molecular biomarker to identify intrinsically milder forms of glioblastoma. Cancer Res; 76(18); 5326-36. ©2016 AACR., (©2016 American Association for Cancer Research.)

Although 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., (© Society for Leukocyte Biology.)

Background: Occurring frequently after solid organ and hematopoietic stem cell transplantation, cytomegalovirus (CMV) replication remains a relevant cause of mortality and morbidity in affected patients. Despite these adverse effects, an increased alloreactivity of natural killer (NK) cells after CMV infection has been assumed, but the underlying physiopathological mechanisms have remained elusive., Methods: We used serial analyses of NK cells before and after CMV infection in kidney transplant recipients as an in vivo model for CMV primary infection to explore the imprint of CMV infection using every patient as their own control: We analyzed NK cell phenotype and function in 47 CMV seronegative recipients of CMV seropositive kidney grafts, who developed CMV primary infection posttransplant. Seronegative recipients of seronegative kidney grafts served as controls., Results: We observed a significant increase of NKG2C expressing NK cells after CMV infection (mean increase, 17.5%; 95% confidence interval [95% CI], 10.2-24.9, P < 0.001), whereas cluster of differentiation (CD)57 expressing cells decreased (mean decrease, 14.1%; 95% CI, 8.0-20.2; P < 0.001). Analysis of killer immunoglobulin-like receptor (KIR) expression showed an increase of cells expressing KIR2DL1 as their only inhibitory KIR in patients carrying the cognate ligand HLA-C2 (mean increase, 10.0%; 95% CI, 1.7-18.3; P = 0.018). In C2-negative individuals, KIR2DL1 expression decreased (mean decrease, 3.9%; 95% CI, 1.6-6.2; P = 0.001). As for activating KIR, there was no conclusive change pattern. Most importantly, we observed a significantly higher NK cell degranulation and IFNγ production in response to different target cells (target K562, CD107a: mean increase, 9.9%; 95% CI, 4.8-15.0; P < 0.001; IFNγ: mean increase, 6.6%; 95% CI, 1.6-11.1; P < 0.001; target MRC-5, CD107a: mean increase, 6.9%; 95% CI, 0.7-13.1; P = 0.03; IFNγ: mean increase, 4.8%; 95% CI, 1.7-7.8; P = 0.002)., Conclusions: We report evidence for an increased function of NK cells induced by CMV infection. This increased in vitro functionality was seen in NKG2C-positive and NKG2C-negative subsets, arguing for an NKG2C independent mechanism of action., Competing Interests: The authors declare no conflicts of interest.

Among innate cells, natural killer (NK) cells play a crucial role in the defense against cytomegalovirus (CMV). In some individuals, CMV infection induces the expansion of NKG2C+ NK cells that persist after control of the infection. We have previously shown that KIR2DL+ NK cells, in contrast to NKG2C+ NK cells, contribute to controlling CMV infection using a CMV-infected monocyte-derived dendritic cell (MDDC) model. However, the nature of CMV-infected cells contributing to the expansion of the NKG2C+ NK cell subset remains unclear. To gain more insight into this question, we investigated the contribution of NKG2C+ NK cell activation by CMV-infected primary human aortic endothelial cells (EC) isolated from kidney transplant donors, which constitutively express the human leukocyte antigen (HLA)-E molecule. Here, we show that, although classic HLA class I expression was drastically downregulated, nonclassic HLA-E expression was maintained in CMV-infected EC. By comparing HLA expression patterns in CMV-infected EC, fibroblasts and MDDC, we demonstrate a cell-dependent modulation of HLA-E expression by CMV infection. NKG2C+ NK cell degranulation was significantly triggered by CMV-infected EC regardless of the nature of the HLA-E allele product. EC, predominantly present in vessels, may constitute a privileged site for CMV infection that drives a 'memory' NKG2C+ NK cell subset., (© 2016 S. Karger AG, Basel.)