NK Cell Surface Research Articles

Acquisition of activation receptor ligand by trogocytosis renders NK cells hyporesponsive.

Because NK cells secrete cytotoxic granules and cytokines that can destroy surrounding cells and help shape the subsequent immune response, they must be kept under tight control. Several mechanisms, at different levels, are in place to control NK cell function. In this study, we describe a novel mechanism regulating NK cell function in which NK cells acquire ligands for activating receptors from target cells by trogocytosis, rendering the NK cells hyporesponsive. In this model, murine NK cells acquire m157, the murine CMV-encoded ligand for the Ly49H-activating receptor, from target cells both in vitro and in vivo. Although acquisition of m157 requires cell-to-cell contact, it does not require the expression of the Ly49H receptor by the NK cell. Acquired m157 protein is expressed on the NK cell surface with a glycosylphosphatidylinisotol linkage and interacts with the Ly49H receptor expressed on the NK cell. This interaction results in blocking the Ly49H receptor that prevents the NK cells from recognizing m157-expressing targets and continuous engagement of the Ly49H-activating receptor, which results in the hyporesponsiveness of the Ly49H(+) NK cell to stimulation through other activating receptors. Thus, NK cell acquisition of a ligand for an activation receptor by trogocytosis renders them hyporesponsive. This mechanism, by which mature NK cell function can be altered, has important implications in regard to how NK cells respond to tumors in specific microenvironments as well as the use of expanded NK cells in treating various malignancies.

Effects of methylated CpG islands in promoter region on the expression of KIR3DL1 protein

Objective To investigate the effects of methylated CpG islands in the promoter region on the expression of killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1). Methods Three voluntary unpaid blood donors carrying high expression allele KIR3DL1*01502 and three donors carrying low expression allele KIR3DL1*005 were recruited in this study. The nucleotide sequences and the methylated CpG islands in the promoter regions of KIR3DL1*01502 allele and KIR3DL1*005 allele were analyzed. The NK cells expressing KIR3DL1*01502 and KIR3DL1*005 were respectively treated with 5-aza for the demethylation of CpG islands within the promoters. The expression of KIR3DL1 protein on the surface of NK cells was measured with flow cytometer. Results Two differences at nucleotide sites -65 and -269 were detected within the promoter regions of KIR3DL1*01502 and KIR3DL1*005, resulting in two distinct CpG islands. The CpG islands within the promoter of KIR3DL1*01502 allele were highly methylated. The expression of KIR3DL1 protein on NK cells which carried KIR3DL1*01502 allele was significantly increased after the demethylation of CpG islands. However, the treatment of demethylation had no significant effects on the expression KIR3DL1 protein on NK cells harboring KIR3DL1*005 allele. Conclusion The methylated CpG islands within the promoter of KIR3DL1*01502 allele affected the antigen expression on the surface of NK cells. Different KIR3DL1 alleles might show different mechanisms in regulating antigen expression. Key words: Killer cell immunoglobulin like receptor; Methylation; Promoter

Expression and purification of soluble and stable ectodomain of natural killer cell receptor LLT1 through high-density transfection of suspension adapted HEK293S GnTI− cells

Lectin-like transcript 1 (LLT1, gene clec2d) was identified to be a ligand for the single human NKR-P1 receptor present on NK and NK-T lymphocytes. Naturally, LLT1 is expressed on the surface of NK cells, stimulating IFN-γ production, and is up-regulated upon activation of other immune cells, e.g. TLR-stimulated dendritic cells and B cells or T cell receptor-activated T cells. While in normal tissues LLT1:NKR-P1 interaction (representing an alternative “missing-self” recognition system) play an immunomodulatory role in regulation of crosstalk between NK and antigen presenting cells, LLT1 is upregulated in glioblastoma cells, one of the most lethal tumors, where it acts as a mediator of immune escape of glioma cells.Here we report transient expression and characterization of soluble His176Cys mutant of LLT1 ectodomain in an eukaryotic expression system of human suspension-adapted HEK293S GnTI− cell line with uniform N-glycans. The His176Cys mutation is critical for C-type lectin-like domain stability, leading to the reconstruction of third canonical disulfide bridge in LLT1, as shown by mass spectrometry. Purified soluble LLT1 is homogeneous, deglycosylatable and forms a non-covalent homodimer whose dimerization is not dependent on presence of its N-glycans.As a part of production of soluble LLT1, we have adapted HEK293S GnTI− cell line to growth in suspension in media facilitating transient transfection and optimized novel high cell density transfection protocol, greatly enhancing protein yields. This transfection protocol is generally applicable for protein production within this cell line, especially for protein crystallography.

Establishment and Characterization of a CD20-Positive NK/T-Cell Lymphoma Cell Line.

CD20 positive NK/T-cell lymphoma is extremely rare and difficult for clinical treatment. Due to the lack of an established cell model for this disease, less is known about its biological characterization and potential therapeutic options. A cell line of NK/T-cell lymphoma, which was enriched by magnetic sorting with proper cell surface markers (CD56) from peripheral blood mononuclear cells (PBMCs) drawn from a 21-year-old male patient with nasal angiocentric NK/T-cell lymphoma, was designated as ZQNK-29. Immunophenotypic analysis of ZQNK-29 was performed by flow cytometric and immunohistochemical analysis. Comparative genomic hybridization (CGH) analysis was used for cytogenetic analysis of ZQNK-29. Potential rearrangements of the immunoglobulin gene and Epstein-Barr virus (EBV) infection were examined by PCR and RT-PCR, respectively. ZQNK-29 cells express the phenotypic T-cell marker (CD3), T cell activation markers (HLA-DR), markers for both NK and cytotoxic T lymphocytes (TIA-1), and B-lineage marker CD20; however, expression of CD56 was not detected in expanded ZQNK-29 cells although this NK cell surface marker was used as one of selective cell surface markers for the initial isolation of NK/T cells. RT-PCR analysis showed that the pattern of gene expressions for infected EBV was latency type III, with the expressions of LMP1, EBNA-1, and EBNA-2; no rearrangements were found in the heavy-chain of the immunoglobulin gene or in the y chain of the T cell receptors (TCRs) gene. CGH analysis demonstrated that ZQNK-29 possessed an abnormal karyotype, 46XY, 1p (dist)+, 4p (dist)+, 4q (mid)-, 5q (mid)-, 9q (dist)+, 16p (dist)+, 16q (dist)+, 17p+, 17q (dist)+, 19q (dist)+, 20p+, 20q+, 21q+, and 22q+. Of these, 1p (dist)+, which has been confirmed to be mitochondrial DNA amplification, is believed to be mainly caused by EBV infection. ZQNK-29 is a well characterized premature human NK/T-cell lymphoma cell line with expression of the B-cell marker CD20 and will provide a useful pre-clinic model for characterization and potential therapeutic studies of the aggressive NK/T-cell lymphoma.

Bacterial lipopolysaccharide activates CD57-negative human NK cells.

NK cells play an important regulatory role in sepsis by induction and augmentation of proinflammatory reactions in early stages of the septic process and by suppression of immune response in later stages of inflammation. The present work was aimed at the effect of bacterial lipopolysaccharide (LPS), the main pathogenic factor of sepsis development, on human NK cells ex vivo. We show that LPS activates immature CD57-negative NK cells, which typically constitute less than half of the normal NK cell population in human peripheral blood. Under conditions of NK cell stimulation with IL-2, addition of LPS provokes an increase in IFN-γ production. However, LPS both increased and inhibited NK cell cytotoxic activity. It is important to note that the activation of NK cells on LPS addition was observed in the absence of TLR4 on the NK cell surface. These results confirm our previous data arguing for a direct interaction of LPS with NK cells and evidence an atypical mechanism of LPS-induced NK cell activation without the involvement of surface TLR4.

Phenotype and functions of natural killer cells in septic patients and its clinical significance

To investigate the possible mechanism of natural killer cells (NK cells) in immune dysfunction in sepsis by monitoring the phenotype and function of periphery NK cells in patients with sepsis. A retrospective study was conducted. The patients with systemic inflammatory response syndrome (SIRS, n=59) or sepsis (n=65) admitted to Department of Critical Care Medicine of Anhui Provincial Hospital from August 2011 to August 2013 were enrolled. Blood samples were collected within 48 hours after intensive care unit (ICU) admission, the phenotype and function of periphery NK cells were determined by flow cytometry. Twenty-eight healthy people served as controls. The proportion and number of peripheral blood CD3⁻ CD56⁺ NK cells in SIRS and sepsis groups were normal, and no statistical difference was found when compared with those of the healthy control group [cell proportion: 0.102 ± 0.019, 0.102 ± 0.108 vs. 0.106 ± 0.018, F = 0.018, P = 0.982; cell number (× 10⁶/L): 182.46 ± 65.98, 172.97 ± 63.51 vs. 179.25 ± 60.44, F=0.349, P=0.706]. It was shown by NK cell degranulation detection that there was no significant difference in the expression of CD107 and interferon-γ (IFN-γ) secretion [CD107: 0.135 ± 0.050, 0.140 ± 0.058, 0.128 ± 0.070, F = 0.583, P = 0.560; IFN-γ (kU/L): 14.36 ± 4.74, 12.49 ± 4.21, 13.45 ± 5.04, F=1.616, P=0.202] among healthy control group, SIRS group, and sepsis group. It was shown by antibody dependent cytotoxic effect (ADCC) test that there was no difference in the expression of CD107 among healthy control group, SIRS group, and sepsis group (0.574 ± 0.166, 0.643 ± 0.165, 0.581 ± 0.157, F = 0.808, P = 0.448). When compared with healthy controls, the secretion of IFN-γ was increased in SIRS patients (kU/L: 40.5 ± 13.2 vs. 28.4 ± 9.6, P = 0.001), while reduced in sepsis patients (kU/L: 19.8 ± 6.7 vs. 28.4 ± 9.6, P<0.01). Compared with SIRS group, only NK cell surface inhibitory receptors CD158e (KIR 3DL1) expression in sepsis group was significantly increased (0.203 ± 0.057 vs. 0.079 ± 0.021, t = 15.762, P<0.001), and there were no significant differences in the other phenotype between the two groups. Compared with SIRS group, the IFN-γ production of the sepsis group was significantly lowered (kU/L: 0.280 ± 0.040 vs. 0.310 ± 0.038, t = 3.390, P = 0.009), and the level of IL-12 was also significantly decreased (ng/L: 0.15 ± 0.03 vs. 0.30 ± 0.08, t = 32.832, P < 0.001). It was showed by NK cell phenotype and function assay that the function of NK cells in patients with sepsis was impaired and led to a poor production of IFN-γ. The IFN-γ mediated immune dysfunction may be a main reason for the disorder of NK cell function, which laid the foundation of the clinical immune intervention practice to improve to NK cell function.

T cell receptors of “mucosal-associated invariant T” cells in multiple sclerosis brain lesions

Mucosal-associated invariant T (MAIT) cells are innate T cells with limited receptor variability, characterised by the expression of an invariant T cell receptor (TCR) Valpha7.2-RD-Jalpha33 alpha-chain, the NK cell surface marker CD161 and restriction by the MHC-related protein 1 (MR1). The role of MAIT cells has not yet been fully understood; however, they seem to play a role in defence against microorganisms and have been associated with autoimmune disorders. Cells expressing the MAIT-typical TCR have been detected in brain homogenates from multiple sclerosis (MS) patients.

Carbohydrate-mediated modulation of NK cell receptor function: structural and functional influences of heparan sulfate moieties expressed on NK cell surface.

OPINION article Front. Oncol., 16 July 2014Sec. Molecular and Cellular Oncology Volume 4 - 2014 | https://doi.org/10.3389/fonc.2014.00185

KIR Haplotype and Genotype Distribution in Hematopoietic Stem Cell Transplant Donors in Panama.

AIM Published reports have described KIR gene frequency distributions in more than 120 populations. Besides the classical HLA matching, KIR genes form an independent diverse immunogenetic system that implicated in influencing the clinical outcomes of allogeneic HSCT. KIRs are NK cell surface protein, and are encoded by the Leukocyte Receptor Complex (LCR) on chromosome 19 (19q13.4) and expressed on NK cell and subset of T cells. They play important role in the prevention of autoreactivity and elimination of viral-infected cells, tumor cells and anti-leukemia effect. The Aim of this study is to study the distribution of KIR gene polymorphisms in the Panamanian population for future consideration of incorporation in our Laboratory evaluation of donors as an additional selection criterion. Methods Stored genomic DNA (86 oC) of a total of 116 hematopoietic stem cell transplant (HSCT) donors evaluated between 2000-2014, from Instituto Oncologico, Hospital del Niño, Complejo Metropolitano of Panamá, were tested for KIR genotyping using LIFECODES KIR TYPING KIT ( Gen-Probe) and PCR-SSO. Results 30 patients (26%) were homozygous for haplotype A whereas 86 patients had at least one haplotype B (Bx, 74%). The most frequently seen KIR genotypes in Bx haplotypes in this cohort are: 3DL3, 2DL3,2DL5, 2DS5, 2DP1, 2DL1, 3DP1, 2DL4, 3DL1, 3DS1, 2DS1, 2DS4, 3DL2 (18 patients, 16%); 3DL3, 2DS2; 2DL2, 2DL3, 2DL5, 2DS5, 2DP1, 2DL1, 3DP1, 2DL4, 3DL1, 3DS1, 2DS1, 2DS4, 3DL2 (9 patients, 8%); 3DL3,2DS2,2DL2,2DL3,2DL5,2DS3,2DP1,2DL1,3DP1,2DL4,3D L1,2DS4,3DL2(8 patients, 7%); 3DL3,2DS2,2DL2,2DL3,2DP1,2DL1,3DP1,2DL 4,3DL1,2DS4,3DL2 (8 patients, 7%); 3DL3,2DS2,2DL2,2DL5,2DS3,2DP1,2DL 1,3DP1,2DL4,3DL1,2DS4,3DL2( 7 patients, 6%). CONCLUSION This is the second report to describe KIR haplotypes and genotypes in the Panamanian population.

A phase II trial of pan-KIR2D blockade with IPH2101 in smoldering multiple myeloma

Natural killer (NK) cells are involved in immune surveillance of various malignancies, including multiple myeloma (MM).[1][1] IPH2101 is a fully human monoclonal antibody that blocks HLA-C binding KIR2D receptors (KIR2DL/DS-1, -2 -3) expressed on the surface of NK-cells, enhancing their cytotoxicity

The DNA Damage Response: A Common Pathway in the Regulation of NKG2D and DNAM-1 Ligand Expression in Normal, Infected, and Cancer Cells

NKG2D and DNAM-1 are two activating receptors, present on the surface of NK cells and other cells of the immune system. Their ligands – MICA, MICB, ULBP1-6 for NKG2D, PVR/CD155 and Nectin-2/CD112 for DNAM-1 – can be constitutively expressed at low levels in some normal cells, but they are more often defined as “stress-induced,” since different stimuli can positively regulate their expression. In this review, we describe the molecular mechanisms involved in the up-regulation of NKG2D and DNAM-1 ligands under different physiological and pathological “stress” conditions, including mitosis, viral infections, and cancer. We will focus on the DNA damage response, as recent advances in the field have uncovered its important role as a common signaling pathway in the regulation of both NKG2D and DNAM-1 ligand expression in response to very diverse conditions and stimuli.

HLA-G1 and HLA-G5 isoforms have an additive effect on NK cytolysis

The immunotolerant HLA-G could generate seven isoforms including HLA-G1–-G7. The suppressive function of either HLA-G1 or HLA-G5 isoform to NK cell cytolysis has been well established. Whether HLA-G1 and HLA-G5 isoform have an additive effect on the cytolysis of NK cells remain to be explored. In this study, effects of expression of HLA-G1 and HLA-G5 isoforms and their combination on NK cytolysis was investigated. NK cell cytolysis was analyzed by detecting the NK cell surface CD107a expression. In this study, data showed that the inhibition capacity is dependent on the level of both HLA-G1 and HLA-G5 expression, but the HLA-G5 isoform has a more potent inhibition effect on the NK cytolysis (p<0.01). Furthermore, HLA-G1 and HLA-G5 have an additive effect on the suppression of NK cell cytolysis. Our study provided further understanding for the roles of HLA-G1 and HLA-G5 isoform expression in target cells immune escaping from NK cells.

KIR3DL1 and HLA-Bw4 Allotypes Predict The Extent Of NK Cell Licensing

Members of the killer immunoglobulin family (KIR) and their HLA class I ligands contribute to establishing natural killer cell reactive potential. NK cells bearing inhibitory KIR that bind self-HLA are termed “licensed” and are highly responsive to targets lacking self HLA, but tolerant to autologous, healthy cells. Among KIR:HLA partnerships, KIR3DL1 and HLA-Bw4 alleles demonstrate the greatest polymorphism. Allelic subgroups of KIR3DL1 are distinguished by their density on the surface of NK cells and demonstrate different sensitivity to inhibition by HLA-Bw4 allotypes. Specifically, KIR3DL1 alleles expressed with high surface density (3DL1high) are more potently inhibited by HLA-Bw4 epitopes possessing isoleucine (80I) compared with threonine (80T) at position 80. It is not currently known whether the same KIR-HLA interactions that mediate higher inhibitory response also endow higher effector capacity. Retrospective analyses of patients with HIV have demonstrated that the same allelic combinations of HLA-B and KIR3DL1 predictive of high inhibition are associated with delayed progression to AIDS. Indeed, a potential target for NK cells is created by the HIV nef protein, which mediates downregulation of HLA-B expression. Taken together, these finding have led us to hypothesize that the sensitivity of NK cells for inhibition by HLA predicts the extent to which they may be licensed for functional responsiveness.We undertook functional analyses of NK cells exclusively expressing KIR3DL1 from healthy HLA-Bw4+ or Bw4-/- donors, using HLA-negative 721.221 target cells to examine licensing function. First, we developed and validated a multiplex PCR array allowing identification of allelic groups of KIR3DL1 that correspond to expression densities. This genomic analysis informed the division of 59 subjects into groups stratified by the inhibitory potential conveyed by self HLA-B and KIR3DL1+ alleles. As expected and consistent with NK education or “licensing” by self-specific inhibitory KIR with cognate HLA ligand, KIR3DL1+Bw4+ NK cells demonstrated greater responsiveness 721.221 cells compared with KIR3DL1+ cells obtained from Bw4-/- donors, as assessed by CD107α externalization. The proportion of KIR3DL1+ NK cells degranulating in response to HLA class I-negative target cells was highly variable and not reflective of either HLA-Bw4 nor KIR3DL1 allele groups alone; however, when both KIR3DL1 and Bw4 allele groups were considered, highly inhibitory allotype pairs were indeed associated with higher NK effector function (p=0.0065). This finding was particularly pronounced among partnerships involving high-density KIR3DL1 alleles: compared with 80T, 80I conditioned 3DL1high NK cells for superior 721-221-stimulated degranulation (p=0.0035). We further investigated whether NK cell licensing could be mediated and/or maintained by HLA intrinsic to NK cells. We found that diminution of HLA expression in licensed KIR3DL1+ by shRNA-mediated knockdown reduced responsiveness to HLA class I-negative target cells, demonstrating that HLA is required on NK cells to maintain their licensed potential.Collectively, these findings reveal that an NK cell's capacity for effector response is not only determined by the presence of a self-specific receptor, but that functional hierarchies exist among NK cells bearing different allotypes of one KIR for its ligand in a manner correlated with inhibitory capacity. Finally, cis-interactions between KIR and HLA contribute to NK licensing, indicating that human NK education is at least partly determined by molecules intrinsic to the cell itself. These findings of differential NK licensing among KIR3DL1-Bw4 allotype combinations now provide the biological basis for the clinical findings of variable HIV control among patients with different KIR3DL1-Bw4 allotype combinations. Disclosures:No relevant conflicts of interest to declare.

TLR8 agonist VTX-2337 enhances NKG2D-mediated cytotoxicity of NK cells

Meeting abstracts NK cells express an array of activating and inhibitory receptors, which facilitate the recognition and lysis of virally infected and transformed cells, but safeguard healthy cells from attack. NKG2D is an activating receptor expressed on the surface of NK cells that recognizes the

KIR3DL1 genetic diversity and phenotypic variation in the Chinese Han population

Allelic polymorphism and expression variation of killer cell immunoglobulin-like receptor (KIR) 3DL1 on natural killer (NK) cells differ among populations. To determine whether the phenotypic variants are due to KIR polymorphism, transcription or copy number, the allelic polymorphism, mRNA levels and antigen expression of KIR3DL1 were assessed in 162 individuals. We characterized 13 KIR3DL1 alleles, five of which were novel. In addition, 21 genotypes were identified. The correlation between the binding patterns of NK cells to anti-KIR3DL1 and KIR3DL1 alleles was also examined. NK cells with different 3DL1 alleles showed distinct binding levels to anti-KIR3DL1. The binding frequencies of NK cells to anti-KIR3DL1 were not accordant with their binding levels, but both associated with the allele copy numbers. The mRNA expression amounts of individuals with two copy alleles were higher than those of individuals with one copy allele. Our data indicate that both the allele copy number and polymorphism of KIR3DL1 influence the antigen expression on the NK-cell surface, but only the copy number was associated with mRNA expression.

97-P: KIR HAPLOTYPE AND GENOTYPE DISTRIBUTION IN HEMATOPOIETIC STEM CELL TRANSPLANT DONORS IN PANAMA

Aim Published reports have described KIR gene frequency distributions in more than 120 populations. Besides the classical HLA matching, KIR genes form an independent diverse immunogenetic system that implicated in influencing the clinical outcomes of allogeneic HSCT. KIRs are NK cell surface protein, and are encoded by the Leukocyte Receptor Complex (LCR) on chromosome 19 (19q13.4) and expressed on NK cell and subset of T cells. They play important role in the prevention of autoreactivity and elimination of viral-infected cells, tumor cells and anti-leukemia effect. The Aim of this study is to study the distribution of KIR gene polymorphisms in the Panamanian population for future consideration of incorporation in our Laboratory evaluation of donors as an additional selection criterion. Methods Stored genomic DNA (86 oC) of a total of 84 hematopoietic stem cell transplant (HSCT) donors evaluated between 2000-2012, from Instituto Oncologico, Hospital del Nino, Complejo Metropolitano of Panama, were tested for KIR genotyping using LIFECODES KIR TYPING KIT (Gen-Probe) and PCR-SSO. Results 22 patients (26%) were homozygous for haplotype A whereas 62 patients had at least one haplotype B (Bx, 74%). The most frequently seen KIR genotypes in Bx haplotypes in this cohort are: 3DL3, 2DL3, DL5, 2DS5, 2DP1, 2DL1, 3DP1, 2DL4, 3DL1, 3DS1, 2DS1, 2DS4, 3DL2 (13 patients, 15%); 3DL3, 2DS2; 2DL2, 2DL3, 2DL5, 2DS5, 2DP1, 2DL1, 3DP1, 2DL4, 3DL1, 3DS1, 2DS1, 2DS4, 3DL2 (6 patients, 7%); 3DL3, 2DS2, 2DL2, 2DL3, 2DL5, 2DS3, 2DP1, 2DL1, 3DP1, 2DL4, 3DL1 (6 patients, 7%). Conclusions This is the first report to describe KIR haplotypes and genotypes in the Panamanian population.

KIR3DS1/L1 and HLA-Bw4-80I are associated with HIV disease progression among HIV typical progressors and long-term nonprogressors

BackgroundNatural killer (NK) cells have emerged as pivotal players in innate immunity, especially in the defense against viral infections and tumors. Killer immunoglobulin-like receptors (KIRs) – an important recognition receptor expressed on the surface of NK cells – regulate the inhibition and/or activation of NK cells after interacting with human leukocyte antigen (HLA) class I ligands. Various KIR genes might impact the prognosis of many different diseases. The implications of KIR-HLA interaction in HIV disease progression remains poorly understood.MethodsHere, we studied KIR genotypes, mRNA levels, HLA genotypes, CD4+ T cell counts and viral loads in our cohort of Human Immunodeficiency Virus (HIV)-infected individuals, a group that includes HIV long-term nonprogressors (LTNPs) and typical progressors (TPs).ResultsWe found that the frequency of KIR3DS1/L1 heterozygotes with HLA-Bw4-80I gene was much higher in LTNPs than in TPs (P = 0.001) and that the KIR3DL1 homozygotes without HLA-Bw4-80I gene had higher viral loads and lower CD4+ T cell counts (P = 0.014 and P = 0.021, respectively). Our study also confirmed that homozygosity for the HLA-Bw6 allele was associated with rapid disease progression. In addition to the aforementioned results on the DNA level, we observed that higher level expression of KIR3DS1 mRNA was in LTNP group, and that higher level expression of KIR3DL1 mRNA was in TP group.ConclusionsOur data suggest that different KIR-HLA genotypes and different levels of transcripts associate with HIV disease progression.

Abstract 493: Antitumoral efficacy of therapeutic human anti-KIR antibody (BMS-986015/IPH2102) in a preclinical xenograft tumor model.

Abstract Natural Killer cells (NK cells) are lymphocytes able to recognize and kill tumors for which the expression of Major Histocompatibility Complex (MHC) class I molecules is altered. This “missing self” recognition is mediated in humans by the lack of engagement of MHC class I i.e. Human Leucocyte Antigens (HLA) molecules with NK cell inhibitory receptors that include Killer Immunoglobulin-like Receptors (KIR). Some tumors escape NK cell immune surveillance by increasing the expression of HLA molecules on their surface. Consequently, blocking interactions between KIR and HLA molecules constitutes an interesting therapeutic strategy. The anti-KIR2DL1/2/3-specific monoclonal antibody, BMS-986015/IPH2102, is a human IgG4 that is being developed for treating both hematologic malignancies and solid tumors. In rodents, the MHC class I inhibitory system regulating NK cell activation is based on lectin-like family Ly49 but the KIR molecules are not expressed. The objective of this study was to develop a preclinical model to assess the efficacy of the drug candidate used in clinical trials, BMS-986015/IPH2102. Mice expressing the human NK inhibitory KIR2DL3, on the surface of NK cells were generated on a RAG-1deficient background (KIRtgRAG mice). The human B cell lymphoma cell line, 721.221, transduced with HLA-Cw3 molecule, a ligand of KIR2DL3, was intra-venously engrafted in these mice. The expression of HLA-C by tumor cells was sufficient to allow them to escape control of NK cells, leading to mice death in around 30 days. IPH2102 treatment increased mice survival in a dose dependent manner when injected at the same time as the tumor challenge. This protective effect was NK cell mediated and directly correlated with the duration of KIR saturation. Interestingly, BMS-986015/IPH2102 treatment also improved survival in therapeutic conditions i.e. when the antibody was injected 5 days after the tumor, also in a NK cell dependent manner. In conclusion, this study showed efficacy of BMS-986015/IPH2102 as single agent in a HLA-Cw3-expressing tumor model and this xenogenic pre-clinical model will be an excellent tool to investigate the therapeutic benefits of combination treatments. Citation Format: Caroline Sola, Fabien Chanuc, Ariane Thielens, Nicolas Fuseri, Isabelle Palacios, Mathieu Blery, Pascale André, Eric Vivier, Sophie Ugolini, Robert Graziano, François Romagne, Cécile Bonnafous. Antitumoral efficacy of therapeutic human anti-KIR antibody (BMS-986015/IPH2102) in a preclinical xenograft tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 493. doi:10.1158/1538-7445.AM2013-493

Effects of micro environmental factors on natural killer activity (NK) of Beta Thalassemia major patients

The physiological mechanisms of decreased NK activity of β-Thalassemia major (BTM) patients are unknown. To assess in vitro effects of mononuclear cells and their cytokine secretion on NK activity, we compared activator receptor levels and cytotoxic activity of purified NK cells and NK cells in mononuclear cells (MNC) pools. We collected cell supernatant from unincubated and incubated MNC with K562 cells and measured their secreted cytokines levels. CD16 was lower on the surface of NK cells in MNC pools from BTM patients compared to healthy volunteers. This inhibition does not appear when NK cells were purified. NKp30 levels in NK cells decreased both as purified cells and as part of a pool of MNC in BTM patients. After incubation of MNC pools with K562 target cells, we found that supernatant levels of IL10, TGFβ1 and IL15 cytokines were also significantly higher in BTM patients compared to healthy volunteers.

Analysis of NKp30/NCR3 isoforms in untreated HIV-1-infected patients from the ANRS SEROCO cohort

Natural killer (NK) cells play a prominent role at the intersection between innate and cognate immunity, thus influencing the development of multiple pathological conditions including HIV-1-induced AIDS. Not only NK cells directly kill HIV-1-infected cells, but also control the maturation and/or elimination of dendritic cells (DCs). These functions are regulated by the delicate balance between activating and inhibiting receptors expressed at the NK-cell surface. Among the former, NKp30 has raised significant interest since the alternative splicing of its intracellular domain leads to differential effector functions, dictating the prognosis of patients bearing gastrointestinal sarcoma, and B7-H6 has recently been identified as its main ligand. Since NKp30 is downregulated in CD56-/CD16+ NK cells expanded in viremic, chronically infected HIV-1+ patients, we decided to investigate the predictive value of NKp30 splice variants for spontaneous disease progression in 89 therapy-naïve HIV-1-infected individuals enrolled in an historical cohort of patients followed since diagnosis (ANRS SEROCO cohort). We found no difference in the representation of NK-cell subsets (CD56bright, CD56dim, CD56neg) in HIV-1-infected patients as compared with healthy subjects. NKp30 downregulation was detected in CD56dim and CD56neg NK-cell subsets, yet this did not convey any prognostic value. None of the NKp30 isoforms did affect disease progression, as measured in terms of time-to-loss of circulating CD4+ T cells, time-to-AIDS-defining events and overall survival. NKp30 isoforms do not seem to play a major role in the outcome of HIV-1 infection, but the heterogeneity of the immuno-virological status of patients at enrollment could have to be taken into account.