Multifactorial determinants of NK cell repertoire organization: insights into age, sex, KIR genotype, HLA typing, and CMV influence.

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.

Non-Expressed Donor KIR3DL1 Alleles May Represent a Risk Factor for Relapse after T-Replete Haploidentical Hematopoietic Stem Cell Transplantation.

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.

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

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.

Low number of KIR ligands in lymphoma patients favors a good rituximab-dependent NK cell response.

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.

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

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.

Pseudomonas aeruginosa Infection Impairs NKG2D-Dependent NK Cell Cytotoxicity through Regulatory T-Cell Activation.

Natural killer (NK) cells play a key role in both antibacterial and antitumor immunity. Pseudomonas aeruginosa infection has already been reported to alter NK cell functions. We studied in vitro the effect of P. aeruginosa on NK cell cytotoxic response (CD107a membrane expression) to a lymphoma cell line. Through positive and negative cell sorting and adoptive transfer, we determined the influence of monocytes, lymphocytes, and regulatory T cells (Treg) on NK cell function during P. aeruginosa infection. We also studied the role of the activating receptor natural killer group 2D (NKG2D) in NK cell response to B221. We determined that P. aeruginosa significantly altered both cytotoxic response to B221 and NKG2D expression on NK cells in a Treg-dependent manner and that the NKG2D receptor was involved in NK cell cytotoxic response to B221. Our results also suggested that during P. aeruginosa infection, monocytes participated in Treg-mediated NK cell alteration. In conclusion, P. aeruginosa infection impairs NK cell cytotoxicity and alters antitumor immunity. These results highlight the strong interaction between bacterial infection and immunity against cancer.

Genetic and Molecular Basis of Heterogeneous NK Cell Responses against Acute Leukemia.

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.

Corrigendum: Broad Impairment of Natural Killer Cells From Operationally Tolerant Kidney Transplanted Patients.

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

Exoenzyme T Plays a Pivotal Role in the IFN-γ Production after Pseudomonas Challenge in IL-12 Primed Natural Killer Cells.

Pseudomonas aeruginosa (PA) expresses the type III secretion system (T3SS) and effector exoenzymes that interfere with intracellular pathways. Natural killer (NK) cells play a key role in antibacterial immunity and their activation is highly dependent on IL-12 produced by myeloid cells. We studied PA and NK cell interactions and the role of IL-12 using human peripheral blood mononuclear cells, sorted human NK cells, and a human NK cell line (NK92). We used a wild-type (WT) strain of PA (PAO1) or isogenic PA-deleted strains to delineate the role of T3SS and exoenzymes. Our hypotheses were tested in vivo in a PA-pneumonia mouse model. Human NK cells or NK92 cell line produced low levels of IFN-γ in response to PA without IL-12 stimulation, whereas PA significantly increased IFN-γ after IL-12 priming. The modulation of IFN-γ production by PA required bacteria-to-cell contact. Among T3SS effectors, exoenzyme T (ExoT) upregulates IFN-γ production and control ERK activation. In vivo, ExoT also increases IFN-γ levels and the percentage of IFN-γ+ NK cells in lungs during PA pneumonia, confirming in vitro data. In conclusion, our results suggest that T3SS could modulate the production of IFN-γ by NK cells after PA infection through ERK activation.

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

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.

Role of IL-12 in overcoming the low responsiveness of NK cells to missing self after traumatic brain injury.

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.

Broad Impairment of Natural Killer Cells from Operationally Tolerant Kidney Transplanted Patients.

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 CD56Dim 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 51Cr release assay compared to HVs. Conversely, despite impaired K562 cell lysis in the 51Cr 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.

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

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.

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.

The 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(-) 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(-) 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.

Amplified NKG2C+ NK cells in cytomegalovirus (CMV) infection preferentially express killer cell Ig-like receptor 2DL: functional impact in controlling CMV-infected dendritic cells.

CMV 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.

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

NK-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.

Discrimination between the main activating and inhibitory killer cell immunoglobulin-like receptor positive natural killer cell subsets using newly characterized monoclonal antibodies.

Natural killer (NK) cells are key components of the innate anti-viral and anti-tumour immune responses. NK cell function is regulated by the interaction of killer cell immunoglobulin-like receptors (KIR) with human leucocyte antigen (HLA) class I molecules. In this study, we report on the generation of KIR-specific antibodies allowing for discrimination between activating and inhibitory KIR. For this purpose, BALB/c mice were immunized with human KIR2DS2 recombinant protein. The precise specificity of KIR2DS2-specific clones was determined on KIR-transfected BW cells and KIR-genotyped NK cells. When used in combination with EB6 (KIR2DL1/2DS1) or GL183 (KIR2DL2/2DL3/2DS2), two KIR-specific monoclonal antibodies (mAbs), 8C11 (specific for KIR2DL1/2DL2/2DL3/2DS2) and 1F12 (specific for KIR2DL3/2DS2), discriminated activating KIR2DS1 (8C11(-) EB6(+)) from inhibitory KIR2DL1 (8C11(+) GL183(-)) and KIR2DL2 (1F12(-) GL183(+)), while excluding the main HLA-Cw-specific KIR. Using these mAbs, KIR2DS1 was shown to be expressed on the surface of NK cells from all individuals genotyped as KIR2DS1(+) (n = 23). Moreover, KIR2DS1 and KIR2DL1 were independently expressed on NK cells. We also determined the amino acid position recognized by the 8C11 and 1F12 mAbs, which revealed that some KIR2DL1 allele-encoded proteins are not recognized by 8C11. Because most available anti-KIR mAbs recognize both inhibitory and activating forms of KIR, these newly characterized antibodies should help assess the expression of activating and inhibitory KIR and their functional relevance to NK biology.

Phenotypic and functional analyses of KIR3DL1+ and KIR3DS1+ NK cell subsets demonstrate differential regulation by Bw4 molecules and induced KIR3DS1 expression on stimulated NK cells.

Recently, the Z27 mAb was shown to recognize the NK cell-activating receptor KIR3DS1, and several genetic studies suggest that the most probable ligands of KIR3DS1 are HLA class I molecules with the Bw4 motif. Despite these findings, the attempts to establish a functional interaction between KIR3DS1 and its potential ligand have been unsuccessful. Here, we study the proliferation and cytotoxicity of KIR3DS1(+) NK cells, compared with KIR3DL1(+) NK cells, according to the Bw4(+) or Bw4(-) allogeneic environment. Our results show for the first time that KIR3DS1 expression on NK cells can be induced after exposure to stimulator cells (221, K562, EBV-B cell lines, and B cells), polyinosinic-polycytidylic acid, IL-15, or IL-2. Furthermore, whereas KIR3DL1(+) NK cell proliferation and cytotoxicity were inhibited in a Bw4(+) but not a Bw4(-) context, KIR3DS1(+) NK cell functions were not influenced by the presence of Bw4 on target cells. Nevertheless, despite the absence of demonstrated regulation of KIR3DS1(+) NK cell functions by HLA-Bw4 molecules, we found a higher KIR3DS1(+) NK cell frequency and higher levels of KIR3DS1 expression in Bw4(+) compared with Bw4(-) individuals. Altogether, these results suggest that KIR3DS1 does not recognize HLA-Bw4 molecules in a physiological context, and they highlight the induced expression of KIR3DS1 observed on stimulated NK cells and the higher frequency of KIR3DS1(+) NK cells in Bw4(+) individuals. Because a protective KIR3DS1-Bw4 association has been reported in viral infections, our results further the understanding of the role of KIR3DS1(+) NK cells in controlling viral infections.

Autologous and allogeneic HLA KIR ligand environments and activating KIR control KIR NK-cell functions.

NK-cell function is regulated by a balance between inhibitory and activating killer cell immunoglobulin-like receptors (KIR) that specifically recognize HLA class I molecules. Using KIR-specific mAb to discriminate between KIR2DS1 and KIR2DL1 receptors, we show that KIR2DS1(+) NK cells are C2-alloreactive only from C2(-) individuals. Moreover, using an in vitro model of NK-cell expansion, we show here that the frequency of KIR2DL1(+) NK cells is significantly higher in the absence of C2 ligand on stimulator EBV-B cells than in its presence. This observation was made regardless of the presence or absence of the autologous C2 ligand, suggesting that the C2(-) EBV-B stimulator cells used in this in vitro model could activate unlicensed KIR2DL1(+) NK cells. In the case of KIR2DL1(+)/S1(+) genotyped individuals, KIR2DS1(+) NK-cell frequency was increased after stimulation with C2(+) compared with C2(-) stimulator B cells, but only from C2(-) individuals. Altogether, these data highlight the C2 alloreactivity of KIR2DS1(+) NK cells that is only observed in C2(-) individuals. These results provide new insights into the way in which NK KIR cell expansion might be regulated in an allogeneic environment.

Flow cytometric evaluation of red blood cell chimerism after bone marrow transplantation in Iranian patients: a preliminary study.

The aim of this study was to evaluate mixed red cells population and red blood cell chimerism after hematopoietic stem cell transplantation. Red blood cell chimerism after hematopoietic stem cell transplantation was analyzed using a series of fluorescein isothiocyanate-conjugated monoclonal antibodies (BioAtlantic, France) directed against ABH, Rh (D, C, E, c, e), Kell, Duffy, Kidd, and Ss antigens on blood samples of 14 patients with hematologic disorders undergoing hematopoietic stem cell transplantation, by flow cytometric method on days 15, 30, and 60 after transplantation. All patients showed expression of donor red cell antigens within days 15 - 30 after hematopoietic stem cell transplantation. Graft versus host disease and ABO incompatibility did not affect the expression of chimerism. Flow cytometric analysis is a simple, accurate, and valuable test which is of significant help in monitoring chimerism in allogeneic hematopoietic stem cell transplantation.