KIR Ligands Research Articles

Improved KIR gene and HLA-C KIR ligand sequence-specific primer polymerase chain reaction genotyping using whole genome amplification

Molecular analysis of genetic polymorphism for clinical or research purposes may be compromised by genomic DNA of limited quality and quantity. In this study, we have successfully tested the feasibility of using whole genome amplification (WGA) to allow genotyping for killer cell immunoglobulin-like receptor (KIR) genes and human leucocyte antigen (HLA)-C KIR ligand dimorphism on HLA-C. WGA was achieved by multiple displacement amplification (MDA) using bacteriophage phi29 polymerase. For KIR genotyping, a revised sequence-specific primer polymerase chain reaction protocol consisting of 23 primer pairs was used avoiding hitherto undetected cross-priming involving KIR2DL1, KIR2DS1, KIR3DL1 and KIR3DS1 alleles. Similarly, MDA-amplified genomic DNA was analyzed for the detection of the HLA-C KIR ligand groups C1 and C2, based on the amino acid K/N dimorphism in position 80.

Reconstitution of natural killer cell receptors influences natural killer activity and relapse rate after haploidentical transplantation of T- and B-cell depleted grafts in children

Natural killer cells have been demonstrated to exert remarkable graft-versus-leukemia effects after haploidentical transplantation. Acquisition of both, inhibiting and activating, receptors on developing natural killer cells is an important step in their functional maturation. Here, we report on the reconstitution of natural killer receptors after haploidentical transplantation of T-and B-cell (CD3/CD19) depleted grafts with co-transfusion of natural killer cells in children and its influence on natural killer cell activity and clinical outcome. We analyzed reconstitution patterns of natural killer receptors at different time intervals after haploidentical transplantation by multi-color flow cytometry. Natural killer cell activity and antibody-dependent cellular cytotoxicity was tested against cell lines and leukemic blasts in vitro. Survival was analyzed using Kaplan-Meier estimates. Recovery of CD56(+)/CD16(+) cells was fast with high cytolytic activity against K562 and strong antibody-dependent cellular cytotoxicity activity against neuroblastoma and leukemic blasts as early as day 14 posttransplant. KIR reconstitution showed a predominance of KIR negative natural killer cells early after transplantation and an early reconstitution of CD158b compared to CD158a and CD158e. These differences were independent of presence or absence of the corresponding KIR ligands in donors or recipients. This reconstitution pattern was associated with a higher relapse probability of patients homozygous for HLA-C1-alleles compared to patients homozygous or even heterozygous for HLA-C2-alleles. Our results indicate a fast recovery of functional and alloreactive natural killer cells with a constant KIR pattern after haploidentical transplantation with T- and B-cell depleted grafts. Moreover, these natural killer cells can mediate antibody-dependent cellular cytotoxicity and therefore may allow for an early use of antibodies against residual malignant cells.

Natural Killer Cell Killing of Acute Myelogenous Leukemia and Acute Lymphoblastic Leukemia Blasts by Killer Cell Immunoglobulin-Like Receptor–Negative Natural Killer Cells after NKG2A and LIR-1 Blockade

Although the study of natural killer (NK) cell alloreactivity has been dominated by studies of killer cell immunoglobulin-like receptors (KIRs), we hypothesized that NKG2A and LIR-1, present on 53% +/- 13% and 36% +/- 18% of normal NK cells, respectively, play roles in the NK cell killing of primary leukemia targets. KIR(-) cells, which compose nearly half of the circulating NK cell population, exhibit tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both acute myelogenous leukemia and acute lymphoblastic leukemia targets were rendered susceptible to lysis by fresh resting KIR(-) NK cells when inhibitory receptor-major histocompatibility class I interactions were blocked by pan-HLA antibodies, demonstrating that these cells are functionally competent. Blockade of a single inhibitory receptor resulted in slightly increased killing, whereas combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR(-) NK cells, demonstrating that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR(-) NK cells is mediated by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemias.

Study of killer immunoglobulin-like receptor genes and human leukocyte antigens class I ligands in a Caucasian Brazilian population with Crohn's disease and ulcerative colitis

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the bowel, of unknown origin. Exposure to specific environmental factors by genetically susceptible individuals, leading to an inadequate response of the immune system, is one of the potential explanations for the occurrence of these diseases. Natural killer cells are part of the innate immune system recognizing class I HLA (human leukocyte antigen) molecules on target cells through their membrane receptors. The main receptors of the natural killer cells are the killer immunoglobulinlike receptors (KIRs). Our study aimed to evaluate the association between the KIR genes in patients with inflammatory bowel diseases and healthy controls. We typed 15 KIR genes and HLA class I ligands in 248 unrelated Brazilian Caucasians, of which 111 had UC and 137 had CD, and 250 healthy controls by polymerase chain reaction using sequence-specific oligonucleotides and sequence-specific primers. We found an increase in KIR2DL2 in controls (inflammatory bowel disease [IBD]: p < 0.001; UC: p = 0.01; CD: p = not significant [NS]). The genotype 2DL2+/HLA-C lys(80)+ was also more common in controls (IBD: p = 0.005; UC: p = 0.01; CD: p = NS); as well as 2DL1+/HLA-C Asn(80)+ (IBD: p = 0.026; UC: p = NS;CD: p = NS). The imbalance between activating and inhibitory KIR and HLA ligands may explain, at least in part, the pathogenesis of these inflammatory bowel diseases.

KIRandHLAGenotypes Are Associated with Disease Progression and Survival following Autologous Hematopoietic Stem Cell Transplantation for High-Risk Neuroblastoma

NK cells exhibit cytotoxicity against neuroblastoma. Gene polymorphisms governing NK cell function, therefore, may influence prognosis. Two highly polymorphic genetic loci instrumental in determining NK cell responses encode the NK cell killer immunoglobulin-like receptors (KIR) and their class I human leukocyte antigen (HLA) ligands. We hypothesized that patients with a "missing ligand" KIR-HLA compound genotype may uniquely benefit from autologous hematopoietic stem cell transplantation (HSCT). One hundred sixty-nine patients treated with autologous HSCT for stage IV neuroblastoma underwent KIR and HLA genotyping. Patients were segregated according to the presence or absence of HLA ligands for autologous inhibitory KIR. Univariate and multivariate analyses were done for overall and progression-free survival. Sixty-four percent of patients lacked one or more HLA ligands for inhibitory KIR. Patients lacking a HLA ligand had a 46% lower risk of death [hazard ratio, 0.54; 95% confidence interval (95% CI), 0.35-0.85; P = 0.007] and a 34% lower risk of progression (hazard ratio, 0.66; 95% CI, 0.44-1.0; P = 0.047) at 3 years compared with patients who possessed all ligands for his/her inhibitory KIR. Among all KIR-HLA combinations, 16 patients lacking the HLA-C1 ligand for KIR2DL2/KIR2DL3 experienced the highest 3-year survival rate of 81% (95% CI, 64-100). Survival was more strongly associated with "missing ligand" than with tumor MYCN gene amplification. KIR-HLA immunogenetics represents a novel prognostic marker for patients undergoing autologous HSCT for high-risk neuroblastoma.

Artificial Antigen Presenting Cells (aAPC) Expanded NK-Mediated Enhanced Lysis of Allogeneic Tumor Cells Regardless of HLA Class I/KIR Mismatch and Its Implication of Use in Eradicating Acute Lymphoblastic Leukemia (ALL).

Abstract 3023Poster Board II-999NK Killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA) ligands play critical roles in maintaining natural killer (NK) cell tolerance, while providing surveillance against pathogens and malignant transformation. Natural killer (NK) cells have been explored as tools for adoptive anti-tumor or leukemia immunotherapy and current models hold that a mismatch or absence of KIR ligands on target cells is essential for efficient NK cell mediated cytolysis. However, new approaches are now available to activate NK cells and the role for KIR mediated signaling in regulating cytotoxicity of activated NK cells has not been well studied. In this study, aAPCs comprising IL15Ra+K562 cells engineered to express 4-1BBL activated and expanded peripheral NK cells in the presence of exogenous IL15 up to 1000-fold in 3 weeks. Compared to resting NK cells, 4-1BBL/IL15-activated NK cells upregulated TRAIL and NKp30, 44, 46 expression, and showed significantly enhanced cytotoxicity against a multitude of tumor targets including K562, Daudi, Ewing's tumors, osteosarcoma, as well as autologous tumors (50%-90% killing vs. 0%-8% with non-activated NK cells). Meanwhile we could detect little to no influence of KIR signaling in regulating cytotoxicity by aAPC activated NK cells, since sorted CD158a+ and CD158b+ activated NK cells showed similar killing of tumor cells expressing HLA group C1 (CD158b ligand) and/or C2 (CD158a ligand) antigens. In contrast, killer activating receptors (KARs) were indispensable for the cytolysis of solid pediatric tumors by aAPC-activated NK cells, since the killing was significantly inhibited by fusion proteins binding to the ligands of NKG2D, NK p30, p44, p46, p80 (KARs). About 20-40% inhibition of the killing was accomplished when all four activating receptors were blocked, though other activating receptors have not been well defined. Although acute lymphoblastic leukemia (ALL) blasts were refractory to fresh NK cytotoxicity, 4-1BBL/IL15 activated NK cells demonstrated higher lytic activities (20%-50%) against ALL blasts from either patients or cell lines. ALL blast lysis could be completely or partially inhibited by KAR-blocking fusion proteins, indicating that expression levels of KAR ligands vary among ALL cases and other solid tumors. We conclude that KIR ligand mismatch or absence is not essential for effective NK cytotoxicities on either solid tumors or ALL when fully activated NK cells are utilized. This suggests that adoptive therapy with autologous aAPC-activated NK cells may prove effective in some clinical settings, such as ALL, AML, or certain solid tumors. Further studies to assess the impact of KAR ligand expression on aAPC-activated NK killing of ALL blasts are in progress.Percentage of Activated NK Killings vs. Fresh NK's with/without KAR-Ig Fusion ProteinsActivated NK (E:T=2.5:1)Fresh NK (E:T=25:1)-KAR-Ig Fc+KAR-Ig FcSB tumor (Ewing's)48%30%0.5%HOS (Osteo sarcoma)63%36%0.7%Daudi (B. lymphoma)78%46%0.2%REH (ALL)54%8%3% DisclosuresNo relevant conflicts of interest to declare.

The Contribution of Signaling Endodomains to CD33-Mediated Killing of Acute Myeloid Leukemia by Natural Killer Cells Transiently Modified with Chimeric Antigen Receptor.

Abstract 2463Poster Board II-440Acute myeloid leukemia (AML) is an aggressive malignancy for which current therapy fails to provide durable remission in approximately half of cases. Natural killer (NK) cells, as a key component of innate immunty, have recently shown clinical potential for adoptive immunotherapy against AML, particular when the donor and recipient are KIR mismatched. In addition to patients who do not have a suitable related donor, approximately 30% of patients bear all three families of KIR ligands and therefor can not benefit from KIR mismatch. Thus, the major obstacles for adoptive NK cell immunotherapy are 1) obtaining sufficient numbers of NK cells for effective thereapy and 2) finding a related donor with predicted KIR mismatch. Clinical trials with humanized or engineered mAbs against CD33 have validated this antigen as a target for immunotherapy of AML, but are complicated by side effects such as a hepatotoxicity due to CD33 expression on normal hepatocytes. To address the first hurdle, we developed a method to expand CD3-CD56+ primary NK cells in vitro using artificial APCs expressing membrane-bound IL21, and have validated electroporation as an efficient method for gene modification of these NK cells. To address the second hurdle and expand the therapeutic potential of KIR-matched expanded NK cells, we hypothesized that gene transfer of CD33 Chimeric Antigen Receptor (CAR) could provide additional activation signal to increase the lysis of AML blasts by expanded NK cells, and sought to compare signaling endodomains for this purpose. CD3z is a signal adapter molecule for NKp30, NKp46, and CD16 in NK cells. We developed a CD33CAR composed of a CD33 single-chain variable fragment fused with the CD3z transmembrane domain expressed in Sleeping Beauty transposon vector system, and compared a first generation (CD3z only) endodomain with second generation endodomains (CD3z plus either CD28 or CD137).Transient gene transfer of the CD33CAR DNA into NK cells was accomplished using the Amaxa Nucleofector device. Functional expression of the CAR was determined by binding of a Siglec3-IgG fusion protein to the cell surface followed by secondary staining with anti-IgG-FITC. Cytotoxicity of the NK cells against CD33+ AML cells and CD33-transduced HEK293T cells was determined in a 4h lysis assay using Calcein-AM. While the maximum electroporation efficiency was only 15% at 24h, expression levels as low as 4% significantly increased the cytotoxic activity of NK cells compared to unelectroporated NK cells. Each of the CD33CAR constructs harboring different endodomains yielded an equivalent increase in target cell lysis (Figure). This data supports recent observations that signal transduction through CD3z is sufficient to activate cytotoxic activity in NK cells. However, to increase the percentage of CAR-expressing NK cells we are further evaluating the role of endodomain signaling in CAR-dependent proliferation of NK cells electroporated with both transposon and transposase. Disclosures:No relevant conflicts of interest to declare.

A Reduced Rate of Leukaemia Relapse After HLA-Identical Sibling Stem Cell Transplantation for Acute Myeloid Leukaemia but Not Other Haematological Malignancies Is Associated with Donor KIR Genes 2DL5A, 2DS1 and 3DS1.

Abstract 2282Poster Board II-259Stem cell transplantation (SCT) from a healthy donor can be curative for patients with haematological malignancies resistant to other treatments. Elimination of malignant cells through a graft-versus-leukaemia (GVL) effect involves donor T- and NK-cells but their relative contribution to this process is poorly defined. NK-cell alloreactivity and GVL effects are controlled by the nature of the interaction of NK activation receptors and killer-immunoglobulin-like-receptors (KIR) with major-histocompatibility locus class I antigens on thetarget cell. We performed KIR-genotyping of HLA-matched donors in two hundred and forty eight T-celldepleted SCTs to identify genetic factors affectingtransplant outcome (transplant-related mortality (TRM), leukaemic relapse and survival). Transplant-related factors and donor KIRgenotyping significant in univariate analysis were entered into a multivariateanalysis to identify independent variables predictive of outcome for differentforms of leukaemia.Patients studied were transplanted at a single center between 1993-2008.They included 70 AML, 99 CML, 40 ALL, 39 MDS patients and 13 patients with CLL, NHL or Myeloma. Median age was 36 years (range 9-66). All received aT cell depleted SCT from an HLA identical sibling. Patients were conditioned with totalbody irradiation 12-13.5Gy (4Gy for patients over 55y) and cyclophosphamide120mg/kg +/- fludarabine 125mg/m2. Patients received 0.2-5 × 10 5 CD3 cells /kg and amedian CD34 cell dose of 3 × 10 6/kg. Cyclosporine was used as the sole GVHDprophylaxis.Multivariate analysis confirmed known predictive factors; for survival: highCD34 cell dose(RR 0.51, p <0.001) and standard rather than high risk disease (RR 3.1, p <0.001), for TRM: poor lymphocyte recovery at day 30 (RR 0.4, p < 0.02), for relapse: high risk disease (RR 4.65, p<0.002). In addition, the presence of donor B haplotype KIR genes: 2DL5A, 2DS1 and 3DS1was associated with significantly less relapse in patients with AML (13% vs 57% ) but not inpatients with other myeloid or lymphoid malignancies. AMLpatients receiving SCT from donors with these KIR genesspan style=“font-size: 10pt;”>relapsed five times less frequently than patientstransplanted from donors with other KIR genotypes.No other factors relating to NK alloreactivity including (missing donor KIR ligand, presence of donor haplotype B, total number of inhibitory KIR and total number of activatory KIR) was foundto be predictive for transplant outcomes. Thesefindings suggest specific, genetically determined,interactions between NK-cells and AML cells whichfacilitate the graft-versus-leukaemia effect and haveimplications for donor selection for AML patients. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Novel Monoclonal Antibody Enhances Natural Killer (NK) Cell Cytotoxicity against Multiple Myeloma (MM): Interim Phase 1 Trial Results.

Abstract 2880Poster Board II-856 Background:MM is increasing in incidence and remains incurable. .NK cells have modest killing activity against MM tumor cells in part because of inhibitory KIR receptors which recognize HLA class 1 antigens on MM tumor cell targets. However, experimental and clinical data in the allogeneic transplant setting suggest that NK cell stimulation by a mismatch between donor KIR and patient KIR ligand may improve outcomes for MM after a reduction of tumor burden by previously administered treatments. To mimic this effect with a pharmaceutical agent, 1-7F9/IPH2101, a fully human IgG4 anti-KIR mAb specific for KIR2DL1/2/3 (HLA-C specific KIRs) was generated (Romagne et al., Blood June, 2009). 1-7F9/IPH 2101 enhances patient NK cell cytotoxicity against autologous MM tumor cells in vitro. We present the interim results of the human phase I trial of this agent in patients with relapsed/refractory MM. Methods:An open-label, single-agent, dose-escalation, multiple dose safety and tolerability study of IPH2101 is being conducted in heavily pre-treated patients with relapsed/refractory MM. Dose escalation with IPH2101 (0.0003, 0.003, 0.015, 0.075, 0.3, 1, 3 mg/kg as IV infusion) is being studied using a 3+3 scheme. Re-dosing criteria (1/month x 4 months) are based on safety data from previous dosing. KIR occupancy, pharmacokinetics (PK), pharmacodynamics, effects on NK cell maturation, and biological effects of IPH 2101 are being monitored in all patients. Results:Currently, dose escalation is entering the final (3 mg/kg) cohort. Data from the first 22 treated patients are available. No Dose Limiting Toxicity (DLT) has been observed. 1 pt (at DL1) has been replaced and 3 additional pts have been enrolled (at DL4) due to an SAE an acute renal failure possibly related to drug. Related Adverse Events were seen in 4/22 patients (18%). 12/22 pts received at least 2 doses (6pts had 2, 1 pt had 3 and 5 pts had 4 cycles-median 2). KIR full occupancy (> 90%) for at least 3 weeks is reached at 1mg /kg. In accordance with the pre-clinical PK/PD model there is a clear relationship between exposure (Cmax) and KIR occupancy. No deleterious effect on NK cell maturation has been seen. IPH 2101 has been well tolerated to date. In the cohorts accrued to date, two heavily pre-treated patients, both with high-risk cytogenetics, showed evidence to suggest disease stabilization while receiving IPH-2101. Conclusions:IPH 2101 improves autologous NK cell killing of MM tumor cells by blocking inhibitory KIR. In the on-going clinical trial, the antibody appears safe and well tolerated at the doses tested. Updated study results will be presented at the time of the meeting This immunotherapeutic approach may hold promise as treatment for MM and further study is warranted. Disclosures:Squiban:Innate pharma: Employment. Marzetto:Innate Pharma: Employment. Andre:Innate Pharma: Employment. Tollier:Innate Pharma: Employment.

Effect of missing killer-immunoglobulin-like receptor ligand in recipients undergoing HLA full matched, non-T-depleted sibling donor transplantation: a single institution experience of 151 Asian patients

This retrospective analysis studied the impact of natural killer (NK) alloreactivity based on the missing ligand model, for a cohort of recipients undergoing haemopoietic stem cell transplant without T-cell depletion from HLA full-matched sibling donors. All patients received a uniform myeloablative conditioning regimen and prophylaxis for GVHD. A total of 151 patients were studied, including 62 patients with AML or myelodysplastic syndrome, 42 patients with ALL and 47 patients with CML. We found that 81% of patients had at least one missing KIR-ligand (KIR-L), and HLA-C1 allogroup homozygosity is present in 70% of patients. From multivariate analysis, we observed that the only consistently significant factor that was associated with superior survival was disease stage. Missing KIR-L, whether considering HLA-Bw and HLA-C alleles, without or with HLA-A ligands or narrowing to only HLA-C alleles alone to classify the number of missing KIR-L, did not have any impact on OS or relapse-free survival. This negative finding implies that as the KIR-L composition of recipient is not important in this matched non-T-depleted setting, further immunotherapeutic measures involving adoptive NK cell infusions have to be explored to exploit the benefit of NK alloreactivity for such transplants.

The Role of Missing Killer Cell Immunoglobulin-Like Receptor Ligands in T Cell Replete Peripheral Blood Stem Cell Transplantation from HLA-Identical Siblings

The contribution of natural killer (NK) cells to graft-versus-malignancy (GVM) effects following hematopoietic stem cell transplantation (HSCT) remains uncertain, particularly in the HLA-identical setting. A model considering missing HLA ligands to the donor's inhibitory killer cell immunoglobulin-like receptor (KIR), termed the missing KIR ligand model, has been established in T cell depleted bone marrow transplantation (BMT), but lacks validity in other cohorts with different treatment characteristics. We hypothesized that the impact of missing KIR ligands on relapse-free survival (RFS) and overall survival (OS) in T cell replete peripheral blood SCT (PBSCT) differs from that in the T cell depleted BMT setting, and retrospectively evaluated 100 consecutive, HLA-identical sibling transplantations for hematologic malignancies. In addition to KIR ligand status, we considered the donors' activating KIRs and grafted NK, T, and CD34(+) cell doses. Our findings demonstrate noninferiority for OS (P = .005) and RFS (P = .002) for the heterozygous HLA-C group KIR ligand status (C1/2; n = 47) compared with patients missing either C1 or C2 (n = 53). Similarly, OS (P = .031) and RFS (P = .034) of Bw4-positive patients was noninferior to that of patients missing a Bw4 ligand to KIR3DL1. By multivariate analysis, C1/2 heterozygous patients had a favorable risk ratio (RR) for relapse (RR = 0.28; P = .003), RFS (RR = 0.56; P = .046), and acute graft-versus-host disease grade II-IV (RR = 0.36; P = .05). Following reduced-intensity conditioning (RIC), but not standard-intensity conditioning, myeloablative (MA) transplantation, a grafted NK cell dose above the median (3.4 x 10(7)/kg) was associated with a lower risk of relapse (RR = 0.57; P = .003) and improved survival (RR = 0.78; P = .03). Overall, our findings support a role for NK alloreactivity in HLA-identical HSCT, but argue against a favorable impact of missing KIR ligands in the given setting. We conclude that the mechanism favoring the missing KIR ligand constellation in T cell depleted BMT may not operate in T cell replete PBSCT. The reasons for this differential effect remain unresolved.

Donor KIR3DL1/3DS1 Gene and Recipient Bw4 KIR Ligand as Prognostic Markers for Outcome in Unrelated Hematopoietic Stem Cell Transplantation

Given their antileukemic activity, natural killer (NK) cells can alter the outcome of hematopoietic stem cell transplantation (HSCT). The physiologic functions of NK cells are regulated by the interaction of killer immunoglobulin-like receptors (KIR) with specific HLA class I ligands. In the literature, different models based on HLA class I and/or KIR donor (D)/recipient (R) gene disparities are considered as predictors of NK cell alloreactivity. In this retrospective and multicentric French study, we analyzed the clinical impact of the different NK-alloreactivity models in 264 patients who underwent T repleted unrelated HSCT. First, we did not observe that the "KIR ligand-ligand" model had a significant clinical impact on unrelated HSCT outcome, whereas the "missing KIR ligand" model had a significant but limited effect on unrelated HSCT, because only the absence of C1 ligand in patients with myelogenous diseases was associated with a decreased overall survival (OS) (hazard ratio=2.17, P=.005). The "KIR receptor-receptor" and the "KIR receptor-ligand" models seemed the most capable of predicting NK alloreactivity because they had a significant impact on acute graft-versus-host disease (aGVHD) occurrence, OS, and relapse incidence in D/R unrelated pairs. In particular, KIR3DL1 gene mismatches in the GVH direction (D(+)R(-)) and the D KIR3DL1(+)/3DS1(+) and R Bw4(-) combination were respectively correlated with the lowest OS in HLA identical pairs (HR=1.99, P =.02) and the highest incidence of relapse in HLA nonidentical D/R unrelated pairs (HR=4.72, P =.03). Overall, our results suggest a detrimental effect of KIR3DL1(+)/3DS1(+) donor NK cells transplanted into HLA-Bw4(-) patients in the absence of an educational process via KIR3DL1/HLA-Bw4 interactions.

Reassessing the Impact of Donor HLA-C Genotype on Long-Term Liver Transplant Survival

HLA-C is the major inhibitory ligand for killer immunoglobulin-like receptors (KIRs) that are expressed on natural killer (NK) cells. Based on their KIR specificity, HLA-C alleles can be divided into two groups, termed HLA-C1 and HLA-C2. Donor HLA-C group has recently been identified by Hanvesakul et al. (Am J Transplant 2008) as a critical determinant of clinical outcome following liver transplantation: Possession of at least one HLA-C group 2 allele by the donor was associated with significantly improved long-term graft and patient survival, presumably due to an inhibition of host NK cell function. To verify this study, we performed genotyping of 913 deceased liver donors for the relevant KIR epitopes of HLA-C and correlated the presence or absence of donor HLA-C2 genotype with graft and patient survival. In our study, donor HLA-C2 genotype had no impact on 10-year graft or patient survival. We cannot confirm a major role of donor HLA-C2 genotype on long-term allograft survival after liver transplantation.

Personalized immunotherapy: a siren myth?

10.2217/PME.09.39 © 2009 Future Medicine Ltd “...the antigenic characteristics of tumor cells can be perceived by the innate and cognate immune systems, which represent the extrinsic barriers against tumor progression, or at least a substantial component of the host–tumor equilibrium.” Antoine Tesniere1,2,3 1U848 INSERM, ‘Cancer, Apoptosis & Immunity, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Universite Paris Sud, Villejuif, France Guido Kroemer1,2,3 1U848 INSERM, ‘Cancer, Apoptosis & Immunity, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Universite Paris Sud, Villejuif, France Thomas Tursz Departement de medecine, Institut Gustave Roussy, Villejuif, France

Receptor systems controlling natural killer cell function are genetically stratified in Europe

Natural killer (NK) cells are components of the innate immune system that function in identifying and destroying aberrant or pathogen-infected cells. These functions are largely controlled by killer cell immunoglobulin-like receptors (KIRs). KIRs inhibit and activate NK cell functions through interactions with their ligands, epitopes encoded by human leukocyte antigen (HLA) class I genes (HLA-C1, C2 and Bw4). Genes that encode KIR and their HLA ligands vary in frequency across human populations. Here, we characterize two Irish populations for KIR and HLA and determine the spatial distribution of functionally important KIR:HLA systems in Europe, a region known for its considerable underlying genetic stratification. We find that Southern Europe is a region characterized by higher frequencies of activatory KIR and strong inhibitory HLA ligand systems (2DL1:HLA-C2 and 3DL1:Bw4). A lower frequency of activatory KIR and the predominance of a comparatively weaker inhibitory ligand system (2DL3:HLA-C1) are observed northwards. Despite contrasting KIR:HLA systems in Northern and Southern Europe, there is a clear balance between inhibitory and activatory repertoires, and their ligands in both regions. These findings show 'functional stratification' of the epistatic KIR:HLA receptor system in Europe, the presence of which will likely affect NK cell-mediated immunity across different populations.

Killer immunoglobulin‐like receptor ligand HLA‐Bw4 protects against multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system. A human leukocyte antigen (HLA) class II association is well established (DRB1*1501-DQB1*0602), but more recently HLA class II-independent associations with HLA class I variants have also been reported. The HLA class I (HLA-A, -B, -C) molecules serve as ligands for both T-cell receptors and killer immunoglobulin-like receptors (KIRs). We investigated the HLA class I alleles defined by their KIR binding motifs and the KIR genes to evaluate whether these genes could influence MS susceptibility or severity, alone or in combination. We typed Norwegian MS patients (n = 631) and controls (n = 555) for HLA-A, -B, -C and -DRB1 alleles as well as the presence or absence of genes encoding inhibitory (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3) and activating (KIR2DS1, KIR2DS2, KIR2DS3, KIR2DL4, KIR2DS4, KIR2DS5, KIR3DS1) KIRs. The frequency of the HLA-Bw4 specificity, which is the ligand for the inhibitory KIR3DL1, was significantly reduced in MS patients as compared with controls (41.4% vs 55.1%, p(uncorrected (uc)) = 4.6 x 10(-6)). Also after stratifying for known HLA class II associations, the HLA-Bw4 association was seen (p(uc) = 0.002). No significant differences in gene carrier frequencies of inhibitory and activating KIRs were observed. However, our data indicate that MS patients who carry the activating KIR2DS2 and the inhibitory KIR2DL2 genes have more severe disease than patients not carrying these genes. Carriage of the ligand of the inhibitory KIR3DL1 receptor, HLA-Bw4, was found to protect against MS in an HLA-DRB1 independent manner.

What Is the Role for Donor Natural Killer Cells after Nonmyeloablative Conditioning?

We investigated the impacts of the tempo of early (days 14, 28, and 42) donor T cell and natural killer (NK) cell engraftment, missing recipient killer cell immunoglobulin-like receptor (KIR) ligands, and numbers of donor inhibitory and activating KIR genes on hematopoietic cell transplantation (HCT) outcomes in 282 patients with hematologic malignancies given nonmyeloablative conditioning. Modeling chimerism levels as a continuous linear variable, we found that high early donor T cell chimerism was significantly associated with acute graft-versus-host disease (aGVHD) ( P = .01), whereas high donor NK cell chimerism levels had no such association ( P = .38). Conversely, high donor NK cell chimerism levels were significantly associated with low relapse risk ( P = .0009), whereas no significant association was seen with high donor T cell chimerism ( P = .10). The qualitative associations between donor T cell and NK cell chimerism levels and GVHD and relapse did not change after adjustment for the presence of recipient KIR ligands or numbers of donor inhibitory or activating KIR genes. Our data indicate that prompt engraftment of donor NK cells correlated with lessened risks of relapse, but not with GVHD, whereas the converse was true for T cells.

Peripheral NK Cell Activation and Expansion by Artificial Antigen Presenting Cells, 2D11 (41.25)

Abstract Natural killer (NK) cells play an important role in early immune defenses against both nonself cells and autologous cells infected with viruses, bacteria, or parasites or in malignant transformation. NK cells mediate direct cytotoxic attack without prior sensitization on their targets through activating receptors, NKG2D, NCR, TRAIL, and inhibitory receptors ("absence of self"), and/or produce cytokines and chemokines that contribute to cytotoxicity and immunoregulation. A number of studies have reported that activation/expansion enhances NK cytotoxicity, but optimal methods for activating and expanding NK cells have not been well defined. Here we demonstrate that artificial APCs (2D11), previously reported and used to expand antigen-specific T cells, also efficiently expand NK cells via NKG2D and 4-1BB signaling mediated by MICA/B and 4-1BBL expression respectively on 2D11 aAPCs. In the presence of exogenous IL15, which is trans-presented by IL-15Rα on 2D11, robust expansion of highly lytic activated NK cells occurs. Compared to resting NK cells, IL15/2D11-activated NK cells upregulated TRAIL and NKp30, 44, 46 expression, and exerted enhanced cytotoxicity against K562, Ewing's tumor cell lines, including autologous tumors, ALL, as well as NK-resistant Daudi cells. In previous studies, expression of KIR ligands on tumor cells has been put forth as a mechanism of tumor immune escape from NK cell mediated killing. Our preliminary results demonstrate that expression of KIR ligands on tumor targets no longer inhibits killing by NK cells activated with 2D11. Ongoing studies are underway to test the hypothesis that NK cells activated by IL15/2D11 will prove more effective therapeutics due to breaking up of KIR-mediated inhibition of tumor cell killings.

Exploration of the Genetic Basis of GVHD by Genetic Association Studies

Graft-versus-host disease (GVHD), as well as graft-versus-leukemia effect (GVL), are essentially allo-immune reactions, which are induced by the engrafted donor T cells that recognize the host-derived allo-antigens presented on their targets (Figure 1). In HLA-matched transplantation, these antigens are called minor histocompatibility antigens (mHags), and are typically defined by the host single nucleotide polymorphisms (SNPs) that are not shared by the donor and therefore considered to be genetically mismatched between the donor and the recipient [1–3]. Thus, the development of both allo-reactions absolutely depends on the presence of 1 or more mismatched mHags, although these reactions could be further modified by other genetic as well as environmental factors, including, cytokine polymorphisms and GVHD prophylaxis. So, in view of better preventing GVHD and specifically targeting allo-immunity to the tumor component, central questions are what mHAgs are responsible for the development of GVHD or GVL and what genetic factors can influence the overall reactions, which are the plausible targets of genome-wide association studies (GWAS) [4-8]. To identify the genetic basis of GVHD, we conducted GWAS by genotyping more than 500,000

Receptores ker de células natural killer

NKC (natural killer cells) are a population of lymphocytes that play an essential role in innate immunity. KIR molecules are receptors expressed on the surface of these cells with an inhibitory or activating function that contributes to the regulation of NK cells. The KIR genes are located on chromosome 19q13 at the Leukocyte Receptor Complex, and exhibit high polymorphism. The KIR ligands are HLA class I molecules. NK cell functions are related to the variation of the expression of these molecules on the surface of the target cells – especially infected, allogeneic and tumor cells. The aim of this work was to make a review about KIR receptors. The Pubmed/medline and ScienceDirect online databases were accessed, using receptor, NK and KIR as keywords. KIR molecular structure, nomenclature and classification, gene diversity, allelic and haplotypic variability and its ligands were described. Regulation of KIR genes expression and NK cell function were also presented.