Natural Killer Cells Generated From Human Induced Pluripotent Stem Cells Mature to CD56brightCD16+NKp80+/-In-Vitro and Express KIR2DL2/DL3 and KIR3DL1.

The differentiation of human induced pluripotent stem cells (hiPSCs) into T and natural killer (NK) lymphocytes opens novel possibilities for developmental studies of immune cells and in-vitro generation of cell therapy products. In particular, iPSC-derived NK cells gained interest in adoptive anti-cancer immunotherapies, since they enable generation of homogenous populations of NK cells with and without genetic engineering that can be grown at clinical scale. However, the phenotype of in-vitro generated NK cells is not well characterized. NK cells derive in the bone marrow and mature in secondary lymphoid tissues through distinct stages from CD56brightCD16- to CD56dimCD16+ NK cells that represents the most abandoned population in peripheral blood. In this study, we efficiently generated CD56+CD16+CD3- NK lymphocytes from hiPSC and characterized NK-cell development by surface expression of NK-lineage markers. Hematopoietic priming of hiPSC resulted in 31.9% to 57.4% CD34+CD45+ hematopoietic progenitor cells (HPC) that did not require enrichment for NK lymphocyte propagation. HPC were further differentiated into NK cells on OP9-DL1 feeder cells resulting in high purity of CD56brightCD16- and CD56brightCD16+ NK cells. The output of generated NK cells increased up to 40% when OP9-DL1 feeder cells were inactivated with mitomycine C. CD7 expression could be detected from the first week of differentiation indicating priming towards the lymphoid lineage. CD56brightCD16-/+ NK cells expressed high levels of DNAM-1, CD69, natural killer cell receptors NKG2A and NKG2D, and natural cytotoxicity receptors NKp46, NKp44, NKp30. Expression of NKp80 on 40% of NK cells, and a perforin+ and granzyme B+ phenotype confirmed differentiation up to stage 4b. Killer cell immunoglobulin-like receptor KIR2DL2/DL3 and KIR3DL1 were found on up to 3 and 10% of mature NK cells, respectively. NK cells were functional in terms of cytotoxicity, degranulation and antibody-dependent cell-mediated cytotoxicity.

Natural killer cell receptor variants and chronic hepatitis B virus infection in the Vietnamese population.

OBJECTIVES: Genes of host immunity play an important role in disease pathogenesis and are determinants of clinical courses of infections, including hepatitis B virus (HBV). Killer-cell immunoglobulin-like receptor (KIR), expressed on the surface of natural killer cells (NK), regulate NK cell cytotoxicity by interacting with human leukocyte antigen (HLA) class I molecules and are candidates for influencing the course of HBV. This study evaluated whether variations in KIR gene content and HLA-C ligands are associated with HBV and with the development of liver cirrhosis and hepatocellular carcinoma. METHODS: A Vietnamese study cohort (HBV n = 511; controls n = 140) was genotyped using multiplex sequence-specific polymerase chain reaction (PCR-SSP) followed by melting curve analysis. RESULTS: The presence of the functional allelic group of KIR2DS4 was associated with an increased risk of chronic HBV (OR = 1.86, pcorr = 0.02), while KIR2DL2+HLA-C1 (OR = 0.62, pcorr = 0.04) and KIR2DL3+HLA-C1 (OR = 0.48, pcorr = 0.04) were associated with a decreased risk. The pair KIR2DL3+HLA-C1 was associated with liver cirrhosis (OR = 0.40, pcorr = 0.01). The presence of five or more activating KIR variants was associated with hepatocellular carcinoma (OR = 0.53, pcorr = 0.04). CONCLUSIONS: KIR gene content variation and combinations KIR-HLA influence the outcome of HBV infection.

Imbalance of Genes Encoding Natural Killer Immunoglobulin-Like Receptors and Human Leukocyte Antigen in Patients With Biliary Cancer.

BACKGROUND & AIMS: Bile duct tumors are rare and have poor prognoses. Natural killer (NK) cells are frequent in human liver and infiltrate these tumors but do not control their progression. Responses of NK cells are regulated by NK immunoglobulin-like receptors (KIRs), which interact with HLA class I ligands. We aimed to characterize the features of the KIR gene loci and their ligands in patients with bile duct cancer (BDC). METHODS: We performed combined multidimensional characterization of genes that encode KIRs and their ligands in blood samples from patients with BDC from Sweden, followed for up to 8 years after diagnosis (n = 148), in 2 geographically matched cohorts of healthy individuals from Northern Europe (n = 204 and n = 900), and in healthy individuals from 6 geographically unrelated populations (n = 2917). We used real-time polymerase chain reaction, RNA sequencing, immunohistochemistry, and flow cytometry to evaluate NK-cell presence, as well as KIR and KIR-ligand expression in bile duct tumors and control tissues. RESULTS: Patients with bile duct tumors had multiple alterations at the KIR gene loci. KIR loci are grouped into genotypes that encode more inhibitory (group A) and more activating (group B) receptors, which can be subdivided into centromeric and telomeric fragments. Patients with BDC had a lower prevalence of KIR2DL3, which was linked to disequilibrium in centromeric A/B and B/B genotypes, compared with control individuals. The associations between KIRs and KIR ligands differed between patients with BDC and control individuals; patients had an altered balance between activating and inhibitory KIRs. KIR-positive NK cells infiltrated biliary tumors that expressed matched KIR ligands. CONCLUSIONS: In a multidimensional analysis of DNA from blood samples of patients with BDC in Europe, we found patients to have multiple alterations at the KIR and HLA gene loci compared with control individuals. These alterations might affect NK-cell tumor surveillance. NK cells from bile duct tumors expressed KIRs and were found in tumors that expressed cognate ligands. This should be considered in development of immune-based therapies for BDC.

A novel antibody combination to identify KIR2DS2high natural killer cells in KIR2DL3/L2/S2 heterozygous donors.

The killer cell immunoglobulin-like receptor (KIR) KIR2DS2 induces natural killer (NK) cell activation upon ligation and in genetic studies is associated with protection against certain cancers and viral infections. One of the difficulties in understanding KIR2DS2 has been that ligands have been hard to define. In part, this is because the high sequence homology between KIR2DS2 and KIR2DL3/KIR2DL2 has made it difficult to make antibodies that specifically detect NK cells expressing KIR2DS2. Using transfected NK cell line (NKL) cells and primary human samples, we report the identification of a novel antibody combination which allows identification of NK cells with relatively high expression of KIR2DS2. This separation is sufficient to examine primary human NK cell activation in response to KIR2DS2 specific ligands.

Siglec-7 Defines a Highly Functional Natural Killer Cell Subset and Inhibits Cell-Mediated Activities.

Sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) is an inhibitory receptor expressed on natural killer (NK) cells. In this study, we investigated the relationship between Siglec-7 expression and NK cell functions. Siglec-7 was highly expressed on NK cells and was preferentially expressed by mature NK cells from peripheral blood of healthy adults. Siglec-7(+) NK cells displayed higher levels of activating receptors CD38, CD16, DNAM1, NKp30 and NKp46, but lower levels of inhibitory receptors such as NKG2A and CD158b, compared with Siglec-7(-) NK cells. Functional tests showed that Siglec-7(+) NK cells displayed more CD107a degranulation and IFN-γ production than Siglec-7(-) NK cells. Siglec-7 inhibited NK cell functions when interacting with specific antibodies. These data suggest that Siglec-7 defines a highly functional NK cell subset and suppresses NK cell-mediated functions when cross-linked with specific antibodies.

KIR2DL2/2DL3-E(35) alleles are functionally stronger than -Q(35) alleles.

KIR2DL2 and KIR2DL3 segregate as alleles of a single locus in the centromeric motif of the killer cell immunoglobulin-like receptor (KIR) gene family. Although KIR2DL2/L3 polymorphism is known to be associated with many human diseases and is an important factor for donor selection in allogeneic hematopoietic stem cell transplantation, the molecular determinant of functional diversity among various alleles is unclear. In this study we found that KIR2DL2/L3 with glutamic acid at position 35 (E(35)) are functionally stronger than those with glutamine at the same position (Q(35)). Cytotoxicity assay showed that NK cells from HLA-C1 positive donors with KIR2DL2/L3-E(35) could kill more target cells lacking their ligands than NK cells with the weaker -Q(35) alleles, indicating better licensing of KIR2DL2/L3(+) NK cells with the stronger alleles. Molecular modeling analysis reveals that the glutamic acid, which is negatively charged, interacts with positively charged histidine located at position 55, thereby stabilizing KIR2DL2/L3 dimer and reducing entropy loss when KIR2DL2/3 binds to HLA-C ligand. The results of this study will be important for future studies of KIR2DL2/L3-associated diseases as well as for donor selection in allogeneic stem cell transplantation.

Phenotype of NK Cells Determined on the Basis of Selected Immunological Parameters in Children Treated due to Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy. The chemotherapy for ALL is associated with a profound secondary immune deficiency.We evaluated the number and phenotype of natural killer (NK) cells at diagnosis, after the intensive chemotherapy and following the completion of the entire treatment for patients with ALL. The fraction, absolute number, and percentage of NK cells expressing interferon-γ were determined in full blood samples. The fraction of NK cells expressing CD158a, CD158b, perforin, A, B, and K granzymes was examined in isolated NK cells.We have shown that patients assessed at ALL diagnosis showed significantly lower values of the fraction of NK cells and percentage of NK cells with the granzyme A expression. Additionally, the absolute number of NK cells, the expression of CD158a, CD158b, perforin, and granzyme A were significantly lower in patients who completed intensive chemotherapy. Also, there was a significantly higher fraction of NK cells expressing granzyme K in patients who completed the therapy.Abnormalities of NK cells were found at all stages of the treatment; however, the most pronounced changes were found at the end of intensive chemotherapy.

NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors.

IL-15 bound to the IL-15Rα-chain (IL-15Rα) is presented in trans to cells bearing the IL-2Rß-chain and common γ-chain. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor-ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR-HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to downregulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A(+) cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Coengagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across, inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15-dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.

Polymorphic HLA-C Receptors Balance the Functional Characteristics of KIR Haplotypes.

The human killer cell Ig-like receptor (KIR) locus comprises two groups of KIR haplotypes, termed A and B. These are present in all human populations but with different relative frequencies, suggesting they have different functional properties that underlie their balancing selection. We studied the genomic organization and functional properties of the alleles of the inhibitory and activating HLA-C receptors encoded by KIR haplotypes. Because every HLA-C allotype functions as a ligand for KIR, the interactions between KIR and HLA-C dominate the HLA class I-mediated regulation of human NK cells. The C2 epitope is recognized by inhibitory KIR2DL1 and activating KIR2DS1, whereas the C1 epitope is recognized by inhibitory KIR2DL2 and KIR2DL3. This study shows that the KIR2DL1, KIR2DS1, and KIR2DL2/3 alleles form distinctive phylogenetic clades that associate with specific KIR haplotypes. KIR A haplotypes are characterized by KIR2DL1 alleles that encode strong inhibitory C2 receptors and KIR2DL3 alleles encoding weak inhibitory C1 receptors. In striking contrast, KIR B haplotypes are characterized by KIR2DL1 alleles that encode weak inhibitory C2 receptors and KIR2DL2 alleles encoding strong inhibitory C1 receptors. The wide-ranging properties of KIR allotypes arise from substitutions throughout the KIR molecule. Such substitutions can influence cell surface expression, as well as the avidity and specificity for HLA-C ligands. Consistent with the crucial role of inhibitory HLA-C receptors in self-recognition, as well as NK cell education and response, most KIR haplotypes have both a functional C1 and C2 receptor, despite the considerable variation that occurs in ligand recognition and surface expression.

ERAP1 regulates natural killer cell function by controlling the engagement of inhibitory receptors.

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by MHC class I (MHC-I) molecules. Herein, we demonstrate that genetic or pharmacological inhibition of ERAP1 on human tumor cell lines perturbs their ability to engage several classes of inhibitory receptors by their specific ligands, including killer cell Ig-like receptors (KIR) by classical MHC-I-peptide (pMHC-I) complexes and the lectin-like receptor CD94-NKG2A by nonclassical pMHC-I complexes, in each case leading to natural killer (NK) cell killing. The protective effect of pMHC-I complexes could be restored in ERAP1-deficient settings by the addition of known high-affinity peptides, suggesting that ERAP1 was needed to positively modify the affinity of natural ligands. Notably, ERAP1 inhibition enhanced the ability of NK cells to kill freshly established human lymphoblastoid cell lines from autologous or allogeneic sources, thereby promoting NK cytotoxic activity against target cells that would not be expected because of KIR-KIR ligand matching. Overall, our results identify ERAP1 as a modifier to leverage immune functions that may improve the efficacy of NK cell-based approaches for cancer immunotherapy.

Peptide selectivity discriminates NK cells from KIR2DL2- and KIR2DL3-positive individuals.

Natural killer cells are controlled by peptide selective inhibitory receptors for MHC class I, including the killer cell immunoglobulin-like receptors (KIRs). Despite having similar ligands, KIR2DL2 and KIR2DL3 confer different levels of protection to infectious disease. To investigate how changes in peptide repertoire may differentially affect NK cell reactivity, NK cells from KIR2DL2 and KIR2DL3 homozygous donors were tested for activity against different combinations of strong inhibitory (VAPWNSFAL), weak inhibitory (VAPWNSRAL), and antagonist peptide (VAPWNSDAL). KIR2DL3-positive NK cells were more sensitive to changes in the peptide content of MHC class I than KIR2DL2-positive NK cells. These differences were observed for the weakly inhibitory peptide VAPWNSRAL in single peptide and double peptide experiments (p < 0.01 and p < 0.03, respectively). More significant differences were observed in experiments using all three peptides (p < 0.0001). Mathematical modeling of the experimental data demonstrated that VAPWNSRAL was dominant over VAPWNSFAL in distinguishing KIR2DL3- from KIR2DL2-positive donors. Donors with different KIR genotypes have different responses to changes in the peptide bound by MHC class I. Differences in the response to the peptide content of MHC class I may be one mechanism underlying the protective effects of different KIR genes against infectious disease.

Human NK cells licensed by killer Ig receptor genes have an altered cytokine program that modifies CD4+ T cell function.

NK cells are innate immune cells known for their cytolytic activities toward tumors and infections. They are capable of expressing diverse killer Ig-like receptors (KIRs), and KIRs are implicated in susceptibility to Crohn's disease (CD), a chronic intestinal inflammatory disease. However, the cellular mechanism of this genetic contribution is unknown. In this study, we show that the "licensing" of NK cells, determined by the presence of KIR2DL3 and homozygous HLA-C1 in host genome, results in their cytokine reprogramming, which permits them to promote CD4(+) T cell activation and Th17 differentiation ex vivo. Microfluidic analysis of thousands of NK single cells and bulk secretions established that licensed NK cells are more polarized to proinflammatory cytokine production than unlicensed NK cells, including production of IFN-γ, TNF-α, CCL-5, and MIP-1ß. Cytokines produced by licensed NK augmented CD4(+) T cell proliferation and IL-17A/IL-22 production. Ab blocking indicated a primary role for IFN-γ, TNF-α, and IL-6 in the augmented T cell-proliferative response. In conclusion, NK licensing mediated by KIR2DL2/3 and HLA-C1 elicits a novel NK cytokine program that activates and induces proinflammatory CD4(+) T cells, thereby providing a potential biologic mechanism for KIR-associated susceptibility to CD and other chronic inflammatory diseases.

Associations between genes for killer immunoglobulin-like receptors and their ligands in patients with epithelial ovarian cancer.

Killer immunoglobulin-like receptors (KIRs) regulate function of NK cells and subsets of T cells. HLA class I molecules are ligands for inhibitory KIRs while specificity of activating KIRs is mainly unknown. Both KIR and HLA genotypes are highly polymorphic. In this study we analyzed associations of KIR and KIR ligand genes with the incidence and clinical course of epithelial ovarian cancer. DNA of 142 patients was analyzed for KIR genes and 103 samples were typed for HLA class I. Control group consisted of 200 healthy individuals, including 83 women, analyzed separately. The frequency of KIR genes in patients and controls were comparable. HLA-C group 1 (ligand for KIR2DL2/3) was more frequent in patients than in controls (86.4% vs. 67.5%, p=0.002). The frequency of KIR2DS4fl was higher in patients with endometrioid cancer (72.3%) compared with other histological subtypes (36.5%, p=0.004) and controls (29.5%, p=0.0001). KIR and KIR ligand genotype did not influence significantly the clinical course of the disease. We conclude that the genotype of KIR ligands is strongly associated with the incidence of epithelial ovarian cancer while KIR2DS4fl confers susceptibility to endometrioid subtype of the disease.

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.

Modulation of the natural killer cell KIR repertoire by cytomegalovirus infection.

Patients carrying activating killer cell immunoglobulin-like receptor (KIR) genes are significantly protected from CMV-associated complications after solid organ or hematopoietic stem cell transplantation. Whether previous infection with CMV affects NK-cell function in healthy donors is unknown. We studied the KIR repertoire and alterations of KIR expression after in vitro exposure to CMV in 54 healthy donors. The expression of neither activating nor inhibitory KIRs was different at baseline between 23 seropositive and 31 seronegative donors. However, after co-culture of NK cells with CMV-infected fibroblast cells, expression of the inhibitory receptors KIR2DL1 and KIR2DL3 and the activating receptor KIR3DS1 significantly increased in CMV-seropositive donors. In CMV-seronegative donors, changes were subtle and restricted to the subset of NK cells expressing NK-cell group antigen 2C (NKG2C). Expansion of inhibitory KIRs occurred exclusively in donors carrying the cognate HLA class I ligands, whereas the presence of the putative ligand HLA-Bw4 was not necessary for the expansion of KIR3DS1-expressing NK cells. Our data show that previous infection with CMV does not alter the resting NK-cell receptor repertoire, but appears to modify how NK cells respond to re-exposure to CMV in vitro.

[Effects of blocking inhibitory KIR receptors on cytotoxic activity of human NK cells in vitro].

OBJECTIVE: To investigate the effect of blocking the inhibitory receptors KIR2DL1 and KIR2DL2/2DL3 with monoclonal antibody on cytotoxic activity of human NK cells. METHODS: Human peripheral blood NK cells were isolated by Rosettesep NK sorting kit. The cytotoxic activity of NK cells against human leukemia NB4, K-562, Raji cells and allogeneic mature or dendritic cells (DCs) was detected before or after KIR2DL1 and KIR2DL2/2DL3 were blocked. The effect of NK cells on T lymphocyte proliferation was detected by mixed lymphocyte reaction and TGF-ß1 concentration in culture supernatant was measured. RESULTS: The cytotoxicity of NK cells to NB4 cells was augmented with increasing concentration of the antibody. Combination of both antibodies enhanced killing activity of NK cells. NK cells had strong cytotoxicity to K-562 cells, but were not enhanced by the blockade of inhibitory receptors. The cytotoxicity to Raji cells was not evidently augmented. The cytotoxicity of NK cells to mature DC was enhanced remarkably with the increase of concentration of the antibodies (2.20% ±1.10% compared with 37.59% ±5.06%, P<0.05). In mixed lymphocyte reaction, the blockade of two antibodies enhanced the inhibition effect of NK cells on T cell proliferation (77.85% ± 8.31% compared with 43.05% ± 5.95%, P<0.05) and the content of TGF-ß1 in the supernatant was increased. CONCLUSION: The cytotoxic effects of human NK cells against target cells were significantly enhanced with the blockade of inhibitory KIR receptor; and the cytokine TGF-ß1 secreted by NK cells further inhibits T cells proliferation.

NK-cell education is shaped by donor HLA genotype after unrelated allogeneic hematopoietic stem cell transplantation.

The rules governing natural killer (NK)-cell education in the allogeneic environment created by unrelated hematopoietic stem-cell transplantation (HSCT) are still largely elusive, especially in an unrelated donor setting. NK-cell inhibitory receptors for self-human leukocyte antigen (HLA) play a central role in the acquisition or maintenance of NK-cell functional competence. Therefore, the responsiveness of different NK-cell subsets was assessed as a function of their expression or absence of expression of self-HLA-specific inhibitory receptors, in a large cohort (n = 60) of unrelated HSCT recipients. A fully effective NK-cell education process was achieved within the first year after allogeneic HSCT and lasted for at least 3 years thereafter. In addition, HLA-mismatched HSCT led to a stable education pattern that was determined by the donor's HLA ligands. These data suggest that the NK cell's education partner could be of hematopoietic rather than extrahematopoietic origin. This donor-ligand-driven NK-cell education model would suggest a sustained graft-versus-leukemia effect after HLA-mismatched HSCT.

Associations between genes for killer immunoglobulin-like receptors and their ligands in patients with solid tumors.

Killer immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) genotypes were analyzed from panels of lung (non-small-cell lung cancer [NSCLC] and small-cell lung cancer [SCLC]), colon, and kidney cancer patients and compared with normal control subjects. No significant differences were noted between KIR gene frequencies in patients compared with normal subjects. When combinations of KIR genes and their HLA ligands were considered, there were significant decreases in frequencies of both KIR2DL2 and KIR2DL3 in homozygotes for their ligand HLA-C1, and an increase in the frequency of KIR3DL1 and its ligand HLA-Bw4 in kidney cancer patients compared with controls. Both associations were partly attributable to changes in ligand frequencies alone. NSCLC patients showed a significant increase in the frequency of KIR2DL1 and its ligand HLA-C2 and a corresponding decrease in frequency of KIR2DL3 and its ligand HLA-C1 in homozygotes. In NSCLC, the Ile80 form of HLA-Bw4 was decreased in KIR3DL1+ HLA-Bw4+ patients, whereas in SCLC the Ile80 form was increased and the Thr80 form decreased in KIR3DS1+ HLA-Bw4+ patients. These findings are consistent with increased co-expression of high-affinity inhibitory KIRs and their ligands, potentially resulting in decreased natural killer cell function, and hence with natural killer cells having a protective role in lung and kidney cancer but not colon cancer.

Combined genotypic and phenotypic killer cell Ig-like receptor analyses reveal KIR2DL3 alleles displaying unexpected monoclonal antibody reactivity: identification of the amino acid residues critical for staining.

In humans, recent clinical and experimental data from hematopoietic stem cell transplantation revealed that donor-derived alloreactive NK cells exert a beneficial graft versus leukemia effect. The existence of donor-derived alloreactive NK cells can be predicted on the basis of donor killer cell Ig-like receptor (KIR) gene profile and HLA class I typing of both donor and recipient. Moreover, the size of the alloreactive NK cell population can be directly assessed by the combined use of anti-KIR-specific mAb. In this study, in an attempt to improve the definition of alloreactive NK cell subsets, we assessed the KIR genotype and phenotype in a cohort of 44 donors. This approach allowed the identification of two different KIR2DL3 alleles (KIR2DL3*005 and the novel allele KIR2DL3*015) that did not react with the anti-KIR2DL3-specific ECM41 mAb. In contrast, both alleles were recognized at the cell surface by several mAb reacting with KIR2DL2/L3/S2. Notably, KIR2DL3*005 was also stained by the anti-KIR2DL1/S1-specific EB6B and 11PB6 mAb. Functional analysis revealed that, despite its particular mAb reactivity, the specificity of KIR2DL3*005 for HLA-C molecules did not differ from that of other KIR2DL2/L3 alleles. Finally, site-directed mutagenesis demonstrated that glutamine at position 35 is required for ECM41 staining, whereas glutamic acid 35 and arginine 50 are relevant for staining with EB6B or 11PB6 mAb. Our present data represent a substantial progress in the characterization of the NK cell repertoire and an improved phenotypic/functional definition of given KIR(+) subsets.

B-lymphoma cells escape rituximab-triggered elimination by NK cells through increased HLA class I expression.

OBJECTIVE: Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is a major effector mechanism of the monoclonal anti-CD20 antibody rituximab in eliminating B-cell lymphomas. Resistance to this treatment occurs, although CD20 antigen is expressed on the tumor cells. MATERIALS AND METHODS: A model of ADCC was established by stimulating human bulk NK cells and inhibitory killer immunoglobulin receptor (KIR)-defined NK cells from human leukocyte antigen (HLA)-typed donors. NK-cell activation was triggered via stimulation of the Fc receptor with immunoglobulin G aggregates, rituximab-labeled HLA-defined CD20-positive B-lymphoblast cell lines or CD20-positive B-lymphoma cell lines. The effect of KIR ligation by anti-KIR antibodies and HLA, the HLA expression density and rituximab concentrations on the efficacy of ADCC were analyzed in granzyme B ELISPOT measuring NK-cell activation and fluorescein-activated cell sorting cytotoxicity assay. RESULTS: HLA, but not CD20 expression density correlated with NK-cell activity against rituximab-labeled targets. ADCC was increased or decreased following HLA shielding or KIR activation by anti-KIR antibodies, respectively. Herein we show that rituximab-induced ADCC is attenuated upon ligation of KIR by HLA molecules expressed on human B-lymphoma target cells. Moreover, anti-KIR antibodies do not only block KIR/HLA interactions, but display agonistic effects at the KIR, which has to be considered for therapeutical applications. CONCLUSION: KIR activation and HLA expression density are critical determinants for the efficacy of rituximab treatment. An explanation for the failure of rituximab treatment may be the protection of the tumor cells from ADCC by inhibiting NK-cell function with their surface HLA.