KIR Ligands Research Articles

Killer immunoglobulin-like receptor gene repertoire influences viral load of primary human cytomegalovirus infection in renal transplant patients.

Killer cell immunoglobulin-like receptors (KIR) are highly polymorphic members of the immunoglobulin superfamily, which influence the response of natural killer cells and some T-lymphocyte subsets. Analysis of a cohort of previously human cytomegalovirus (HCMV)-negative patients, who developed primary HCMV infection following HCMV-positive renal transplant (n=76), revealed an increase in the frequency of KIR genes located on the telomeric region of B haplotypes (Tel B). The presence of Tel B in combination with the KIR ligand HLA-C2 was significantly more frequent in this subgroup. These genetic factors were associated with resistance to HCMV infection in a second cohort (n=65), where the Tel B genes KIR2DL5, -2DS1, 2DS5 and -3DS1 were all significantly associated with high viral loads. Furthermore, the KIR haplotype Tel A when in combination with the KIR ligand HLA-C1 was significantly protective against the development of severe infection. Our results suggest that KIR are a significant factor in the control of primary HCMV infection, and that determination of KIR gene repertoire may help in detection of renal transplant patients who were most at risk.

KIR diversity in Māori and Polynesians: populations in which HLA-B is not a significant KIR ligand.

HLA class I molecules and killer cell immunoglobulin-like receptors (KIR) form a diverse system of ligands and receptors that individualize human immune systems in ways that improve the survival of individuals and populations. Human settlement of Oceania by island-hopping East and Southeast Asian migrants started ~3,500years ago. Subsequently, New Zealand was reached ~750years ago by ancestral Māori. To examine how this history impacted KIR and HLA diversity, and their functional interaction, we defined at high resolution the allelic and haplotype diversity of the 13 expressed KIR genes in 49 Māori and 34 Polynesians. Eighty KIR variants, including four 'new' alleles, were defined, as were 35 centromeric and 22 telomeric KIR region haplotypes, which combine to give >50 full-length KIR haplotypes. Two new and divergent variant KIR form part of a telomeric KIR haplotype, which appears derived from Papua New Guinea and was probably obtained by the Asian migrants en route to Polynesia. Māori and Polynesian KIR are very similar, but differ significantly from African, European, Japanese, and Amerindian KIR. Māori and Polynesians have high KIR haplotype diversity with corresponding allotype diversity being maintained throughout the KIR locus. Within the population, each individual has a unique combination of HLA class I and KIR. Characterizing Māori and Polynesians is a paucity of HLA-B allotypes recognized by KIR. Compensating for this deficiency are high frequencies (>50%) of HLA-A allotypes recognized by KIR. These HLA-A allotypes are ones that modern humans likely acquired from archaic humans at a much earlier time.

HLA and KIR genotyping correlates with relapse after T-cell-replete haploidentical transplantation in chronic myeloid leukaemia patients.

Background:Conflicting results have been reported regarding the predicative roles of alloreactive natural killer (NK) cells on the outcomes of transplantation in leukaemia patients.Methods:We prospectively analysed the human leukocyte antigen (HLA) typing of donor–recipient pairs and the KIR typing of the donors in 97 CML patients to address the predictive roles of NK cells in relapse undergoing T-cell-replete haploidentical transplantation.Results:Patients with class I ligands for the donor-inhibitory KIR gene exhibited decreased molecular and haematologic relapse rates (P=0.003 and P=0.015, respectively). There was a significantly reduced risk of molecular and haematologic relapse in patients with HLA-C1C2 or C2C2 who accepted donors with KIR2DS1 or in patients with HLA-Bw4 who accepted donors with KIR3DS1 (‘recipient with relevant KIR ligand for donor-activating KIR', n=25), compared with the remaining transplants (n=72, P=0.009 and P=0.009, respectively). In addition, the presence of class I ligand in the recipients of donor-activating KIR contributed to a decreased relapse rate in patients lacking class I ligand in the recipient of donor-inhibitory KIR (P=0.04 and P=0.03, respectively).Conclusions:This study suggests that the presence of class I ligands for the donor-activating or donor-inhibitory KIR gene in the recipient might confer some protection against leukaemic relapse in T-cell-replete haploidentical transplantation.

Oligoclonal band phenotypes in MS differ in their HLA class II association, while specific KIR ligands at HLA class I show association to MS in general

Multiple sclerosis (MS) patients have been reported to have different HLA class II allele profiles depending on oligoclonal bands (OCBs) in the cerebrospinal fluid, but HLA class I alleles and killer cell immunoglobulin-like receptor (KIR) ligands have not been studied. We investigated the association of HLA alleles and KIR ligands according to OCB status in MS patients (n=3876). Specific KIR ligands were associated with patients when compared to controls (n=3148), supporting a role for NK cells in MS pathogenesis. HLA class I alleles and KIR ligands did not differ between OCB phenotypes, but HLA class II associations were convincingly replicated.

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.

The distribution of KIR‐HLA functional blocks is different from North to South of Italy

The killer cell immunoglobulin-like receptor (KIR)-human leukocyte antigen (HLA) interaction represents an example of genetic epistasis, where the concomitant presence of specific genes or alleles encoding receptor-ligand units is necessary for the activity of natural killer (NK) cells. Although KIR and HLA genes segregate independently, they co-evolved under environmental pressures to maintain particular KIR-HLA functional blocks for species survival. We investigated, in 270 Italian healthy individuals, the distribution of KIR and HLA polymorphisms in three climatic areas (from cold north to warm south), to verify their possible geographical stratification. We analyzed the presence of 13 KIR genes and genotyped KIR ligands belonging to HLA class I: HLA-C, HLA-B and HLA-A. We did not observe any genetic stratification for KIR genes and HLA-C ligands in Italy. By contrast, in a north-to-south direction, we found a decreasing trend for the HLA-A3 and HLA-A11 ligands (P = 0.012) and an increasing trend for the HLA-B ligands carrying the Bw4 epitope (P = 0.0003) and the Bw4 Ile80 epitope (P = 0.0005). The HLA-A and HLA-B KIR ligands were in negative linkage disequilibrium (correlation coefficient -0.1211), possibly as a consequence of their similar function in inhibiting NK cells. The distribution of the KIR-HLA functional blocks was different along Italy, as we observed a north-to-south ascending trend for KIR3DL1, when coupled with HLA-B Bw4 ligands (P = 0.0067) and with HLA-B Bw4 Ile80 (P = 0.0027), and a descending trend for KIR3DL2 when coupled with HLA-A3 and HLA-A11 ligands (P = 0.0044). Overall, people from South Italy preferentially use the KIR3DL1-HLA-B Bw4 functional unit, while those from the North Italy equally use both the KIR3DL2-HLA-A3/A11 and the KIR3DL1-HLA-B Bw4 functional units to fight infections. Thus, only KIR3DL receptors, which exert the unique role of microbial sensors through the specific D0 domain, and their cognate HLA-A and HLA-B ligands are selectively pressured in Italy according to geographical north-to-south distribution.

High-allelic variability in HLA-C mRNA expression: association with HLA-extended haplotypes

Human leukocyte antigen (HLA)-C is a clinically relevant transplantation antigen in unrelated hematopoietic stem cell and cord blood transplantation. Furthermore, HLA-C antigens, as ligands for killer immunoglobulin-like receptors expressed on natural killer cells, have a central role in HIV control. Several studies have reported significant correlations between HLA-C mRNA and cell surface expression with polymorphisms in the 5'- and 3'-regions of the HLA-C locus. We determined HLA-C mRNA in blood donors by using locus as well as allele-specific real-time-PCR and focused the analysis on HLA-extended haplotypes. High inter-individual variability of mRNA expression was disclosed. A lower inter-individual variability for C*07:01 but a higher variability for C*06:02, C*04:01 and C*03:04 alleles were detected. The previously reported associations between HLA-C cell surface expression and -32 kb/-35 kb single nucleotide polymorphisms were not confirmed. Related and unrelated individuals sharing the same two A-B-C-DRB1 or B-C haplotypes show strikingly similar levels of HLA-C mRNA expression in each of the different haplotypic combinations tested. Altogether, our results suggest that HLA-C expression levels best correlate with the extended HLA haplotype rather than with the allotype or with polymorphisms in the 5'-region of the HLA-C locus.

Killer immunoglobulin-like receptors and tumor immunity.

were originally named for their capacity to elicit potent cytotoxicity against tumor cells independent of prior sensitization or gene rearrangement. This process is facilitated through the expression of activating and inhibitory receptors that provide for NK cell "education" and a subsequent ability to survey, recognize, and lyse infected or transformed cells, especially those lacking or possessing mutated MHC class I expression. Since these original observations were made, how NK cells recognize candidate target cells continues to be the topic of ongoing investigation. It is now appreciated that NK cells express a diverse repertoire of activating and inhibitory receptors of which killer immunoglobulin-like receptors (KIR) appear to play a critical role in mediating self-tolerance as well as facilitating cytotoxicity against infected or transformed cells. In addition, in the presence of an activating signal, the absence or mismatch of MHC class I molecules on such targets (which serve as inhibitory KIR ligands) promotes NK cell–mediated lysis. An increasing understanding of the complexities of KIR biology has provided recent opportunities to leverage the NK cell versus tumor effect as a novel avenue of immunotherapy for cancer. The present review summarizes the current understanding of KIR expression and function and highlights ongoing efforts to translate these discoveries into novel NK cell–mediated immunotherapies for cancer.

Influence Of Killer Immunoglobulin-Like Receptor (KIR) and HLA Genotypes On Outcomes After Reduced-Intensity Conditioning Allogeneic Hematopoietic Stem Cell Transplantation For Patients With AML and MDS: A Report From The Center For International Blood and Marrow Transplant Research Immunobiology Working Committee

Disease relapse is a significant cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HCT). In the setting of reduced-intensity conditioning (RIC), a graft-versus-leukemia (GVL) effect is critical for successful outcomes in patients with advanced myeloid malignancies. A GVL effect has been attributed in part to donor-derived alloreactive natural killer (NK) cells, which are regulated by interaction of KIRs with their HLA-class I ligands. Several models of NK reactivity (missing KIR ligand, centromeric haplotype-B content, non-tolerized KIR2DS1) have been associated with improved outcomes following HLA-matched, HLA-mismatched, related, and unrelated donor HCT, particularly for AML patients given myeloablative conditioning. The effect of KIR-HLA combinations on outcomes after RIC HCT, however, is not known. We retrospectively analyzed donor-recipient KIR/HLA genotypes from 929 URD HCTs facilitated by the National Marrow Donor Program for patients with AML (n=624) or MDS (n=305) treated with RIC between 1990 and 2007. 664 donor-recipient pairs were 10/10 HLA-matched and 265 were 9/10 HLA-matched. 332 (37%) patients received ATG and 73 (8%) received alemtuzumab. P-values less than 0.01 were considered significant. We hypothesized that donor-recipient KIR-HLA interactions may be associated with improved post-transplant outcomes following RIC HCT for AML and MDS. Patients lacking the HLA-Bw4 ligand for donor inhibitory KIR3DL1 experienced lower relapse following HCT at 1 year (27 vs 36%, p=0.002) and 5 years (31 vs 42%, p=0.003) compared to patients with the Bw4 ligand (Figure). The lower risk for relapse was confirmed in multivariate analysis (HR 0.71, p=0.005). However, there was no significant association of Bw4 ligand with disease-free survival (HR 0.84, p=0.05) and overall survival (HR=0.97, p=0.70). Risk for acute GVHD was higher among patients lacking KIR ligands after adjusting for other clinical factors. In particular, patients lacking HLA-C2 for donor KIR2DL1 experienced higher grade 2-4 (HR 1.3, p=0.005) and 3-4 acute GVHD (HR 1.5, p=0.002), and patients lacking multiple KIR ligands experienced higher grade 3-4 acute GVHD (HR 1.5, p=0.007). The analysis was then restricted to AML patients, the patient population with greatest reported KIR-HLA effects. Patients whose donors were KIR2DS1+ and HLA-C2C2 (n=33) had higher transplant-related mortality (TRM) (HR, 2.4, p=0.002) compared to all other patients. There was no significant effect of KIR2DS1 with HLA on relapse. In a multivariate analysis, lack of HLA-C2 in AML patients was associated with higher grade 2-4 (HR 1.4, p=0.002) and 3-4 acute GVHD (HR 1.5, p=0.01), and risk for grade 3-4 acute GVHD was higher in patients lacking multiple KIR ligands (HR 1.6, p=0.005). There were no significant associations between donor homozygosity for the centromeric B-haplotype (cenBB) or overall B-haplotype KIR content and RIC HCT outcomes.Overall, these results suggest that in the RIC HCT setting, lack of Bw4 ligand for KIR3DL1 is associated with a lower risk of relapse for AML/MDS. This observation corroborates previous findings in myeloablative HCT transplants. Furthermore, tolerance of donor KIR2DS1 by HLA-C2C2 was associated with worse outcome, as manifested by higher TRM, in AML patients. In contrast, it appears that in RIC HCT homozygosity for the centromeric B-haplotype does not have a significant role in leukemia relapse. The associations of KIR ligands with acute GVHD were not previously observed suggesting that NK cell alloreactivity depends on multiple variables, including RIC. Further elucidation of the biology of NK cell alloreactivity in the RIC setting may provide guidance for future approaches to help optimize conditions for generating GVL reactions without GVHD and less TRM in this transplant population. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Lacking Of Missing Killer-Immunoglobulin-Like Receptor Ligand In Recipients Can Predict Better Prognosis After HLA-Mismatched/Haploidentical Transplantation Without T Cells Depletion In Vitro In Chronic Myeloid Leukemia Patients

IntroductionHLA-mismatched/haploidentical stem cell transplantation (SCT) is a feasible therapeutic option for advanced hematologic malignancies patients who lack an HLA-matched related or unrelated donor. The effect of NK alloreactivity in HLA haploidentcial SCT is still under debate and in particular in transplantation for chronic myeloid leukemia (CML) the data are very limited and with conflicting outcome.The goal of this study was to explorethe predictive roles of missing self model in our HLA-mismatched/haploidentical transplantation without T-cell-depletion in vitro in chronic myeloid leukemia patients, and to develop a simple algorithm on the basis of recipients and donor HLA-C and HLA-Bw4 gene content that can be used today to identify HLA-mismatched donors who will associated withbetter prognosis in T cell¨Creplete transplants. MethodsWe studied the HLA genotype of 78 donor-recipient pairs and the KIR genotype of their donor, who underwent unmanipulatedHLA-mismatched/haploidentical transplantation without T cells depletion in vitro during 2003-2009 in our center. To applythe missing ligand model, the first step was to divide ourdonor-recipient pairs into 2 groups according to the number of KIR ligand indonor and recipient, ie, 3 KIR ligands (“without missing self”) versus fewer than 3(“with missing self”). Meanwhile, to apply the KIR ligand-ligand model, donors who were classified as NK alloreactive against their recipientstermed KIR ligand mismatched donors throughout, possessedHLA class I KIRligand(s) which were missing in the recipients. ResultsAmong the 78 pairs of donor-recipients, 65 and 13 recipients receivedHLA¨Cmismatched/haploidentical transplants from “with missing self (R-L mismatch)” and “without missing self (R-L match)” donors, respectively. Using Ligand-ligand model, 59 and 19 recipients received haploidentical transplantation from “KIR ligand matched (L-L match)” and “KIR ligand mismatched (L-L mismatch)” donors, respectively. In contrast to Perugia's KIR ligand-ligand mismatched model or Handgretinger's KIR missing self model between donor-recipient pairs, we found that the 10-year disease free survival(DFS) rate were higher in patients received transplantation from “without missing self (R-L match)” donorscompared with those from “with missing self (R-L mismatch)” (92.3±7.4% vs. 55.2%±6.2%, p=0.024, Figure1A) , especially in high risk CML patients (100% vs. 37.2%±8.6%, p=0.029, Figure1B). When combined the above missing self model and Ligand-ligand model together, patients were subgrouped as receiving graft from “without missing self and without KIR ligand mismatch (R-L match and L-L match)” (n=13), “with missing self and without KIR ligand mismatch (R-L mismatch and L-L match)” (n=47), and “with missing self and with KIR ligand mismatch (R-L mismatch and L-L mismatch)” (n=18), respectively. Cox regression model showed the 10-yearDFSwas best predicted by the combination of missing self model and Ligand-ligand modelbetween recipients and donors pairs (HR 2.205(1.113-4.368), p=0.023, Figure1C). Meanwhile, donor KIR 2DS5 positive associated with higher DFS post-transplantation (84.2±8.4% vs. 56.8±7.7%, p=0.045) in CML patients. However, donor KIR haplotype B have no effect on DFS and overall survival after allogeneic hematopoietic stem cell transplantation for multiple myeloma [Display omitted] ConclusionsThese data indicate thatpoor prognosis after transplantation is associated with the missing self and KIR ligand mismatch in recipients and T cell alloreaction may play apredominant role in this model.Based on recipients and donor HLA-C and HLA-Bw4 gene content, it could befeasible to identify HLA-mismatched donors who will predict the better prognosis in CML patients post T cell-replete transplant. Disclosures:No relevant conflicts of interest to declare.

NK Cell KIR Ligand Mismatches Influence Engraftment Following Combined Haploidentical and Umbilical Cord Blood (UCB) Transplantation In Patients With Severe Aplastic Anemia (SAA)

BackgroundAllogeneic hematopoietic stem cell transplant (HSCT) is curative for patients (pts) with severe aplastic anemia (SAA). For SAA pts who lack HLA-identical donors, we explored a HSCT approach that co-infuses PBSCs enriched for CD34+ cells from a haplo-identical relative combined with a single umbilical cord blood unit (UCB). Although most pts undergoing this approach had early haplo-myeloid engraftment that was eventually supplanted by the UCB unit, engraftment patterns were highly variable; in some pts full cord myeloid chimerism was delayed greater than one year, while others had loss of cord engraftment with sustained full haplo-donor myeloid chimerism. Here, we investigated the impact of various patient, UCB, and haplo-donor characteristics, as well as NK cell KIR ligand mismatches between graft sources, on engraftment kinetics following haplo-cord HSCT. MethodsPts with SAA or SAA evolved to MDS unresponsive to immunosuppressive therapy with severe neutropenia (ANC<500), who lacked an HLA-matched donor and had an available haploidentical family member, and had at least one ≥ 4/6 matched UCB unit with a minimum TNC dose ≥1.5x107 cells/kg were eligible for HSCT. Pts were conditioned with cyclophosphamide (120 mg/kg), fludarabine (125 mg/m2), equine-ATG (160 mg/kg) and 200 cGy of total body irradiation. On Day 0, pts received a CD34 selected (Miltenyi CliniMacs) G-CSF mobilized haplo-donor allograft combined with a single UCB. Tacrolimus and MMF were given for GVHD prophylaxis. Results16 pts (median age: 18.9 yrs, range: 4.5-27.9) including 13 SAA and 3 with SAA evolved to MDS underwent haplo-cord transplant. Ten pts received a 4/6, and six pts received a 5/6 HLA-matched UCB unit. A median 3.6x107 TNC/kg (range: 1.96-6.93) from the UCB unit, and 3.3x106CD34+ cells/kg (range: 3.0-4.1) from the haplo-donor were transplanted. All 16 pts had sustained engraftment with 15/15 pts evaluable past day 100 having transfusion independence. At a median follow-up of 570 days (range: 55-1826), 14/16 pts survive for an overall survival rate of 81.8%. Two pts died at day 414 and 402 post-HSCT from viral related complications (CMV pneumonitis and limbic encephalopathy). Neutrophil and platelet recovery occurred at a median 10 (range: 9-22), and 21 (range: 10-213) days post-HSCT, respectively. By post-HSCT day 11, 15/16 pts had neutrophil recovery. Cord myeloid engraftment (cord ANC>500, calculated from chimerism data) occurred in 13/16 pts at a median 42 days. 3/16 did not achieve a cord ANC>500 but had sustained haplo-donor engraftment. The cumulative incidence of acute grade II-IV GvHD was 38.1%. Engraftment profiles were highly variable among pts; 12 achieved full cord chimerism in all cell lineages, 2 remained mixed haplo-cord chimeras, and 2 failed to have UCB engraftment but had sustained 100% haplo-donor myeloid chimerism. Higher degrees of HLA matching (out of 10 alleles) between recipient and UCB unit were associated with faster rates of full cord engraftment (p=0.006) and a higher probability of complete loss of haplo-donor chimerism (p=0.018). KIR ligand incompatibility in the haplo vs. cord direction (defined as the presence of a KIR ligand in the haplo-donor graft that is absent in the UCB unit at HLA epitopes Bw4, HLA-C Group 1 & 2, HLA-A3, and HLA-A11) negatively impacted cord myeloid engraftment. 5/5 (100%) pts who failed to achieve full cord myeloid chimerism by post-HSCT day 400 had haplo vs. cord KIR ligand incompatibility. Moreover, both pts who failed to have UCB engraftment and had sustained haplo-donor chimerism had haplo vs. cord KIR ligand incompatibility. In contrast, only 3/11 (27%) pts who achieved full cord myeloid chimerism post-HSCT by day 231 had haplo vs. cord KIR ligand incompatibility (p=0.026). KIR ligand incompatibility in the cord vs. haplo direction showed no significant effect on haplo-donor myeloid engraftment. ConclusionThese results show that haplo-cord HSCT is an effective treatment option for pts with SAA who lack an HLA-matched donor. Further, these results suggest that NK cell alloreactivity, occurring as a consequence of KIR ligand mismatch between the two graft sources, may have a negative impact on cord engraftment when haplo vs. cord KIR ligand incompatibility is present. In summary, this study highlights a novel factor that should be considered during graft selection for haplo-cord transplantation. Disclosures:No relevant conflicts of interest to declare.

Negative Impact Of KIR/HLA Interactions On NK Cell Activity Can Be Overcome By The Glycoengineered Antibody GA101 (Obinutuzumab)

Antibody dependent cellular cytotoxicity (ADCC) is one of the mechanisms by which therapeutic antibodies mediate tumor cell killing. The anti-CD20 antibody rituximab is the current standard of care in the treatment of B-cell lymphomas. GA101, a novel anti-CD20 antibody, contains a glycoengineered Fc-portion allowing approximately 10-fold greater affinity to FcgR3A, the Fc-IgG receptor expressed on the majority of natural killer (NK) cells.NK cell function is also regulated by inhibitory killer-cell immunoglobulin-like receptors (KIR), which interact with HLA class I antigens (2DL1-HLA-C2; 2DL2/3-HLA-C1, 3DL1-HLA-Bw4). The KIR/HLA interaction during NK cell development leads to the acquisition of full effector function in the “licensing” process, but also provides one of the main mechanisms of NK cell tolerance. The present study analyzed how KIR/HLA interactions influence ADCC, and whether there are differences between conventional and glycoengineered antibodies. We analyzed the activation (in terms of the degranulation measured by the CD107a expression) and killing capacity of KIR-positive NK cells induced by rituximab, GA101, and the parental non-Fc modified (wild-type) GA101wt. Target cells included HLA-negative B-cell lymphoma lines or B-cell lines expressing one or more HLA molecules.We confirmed previous observations that the licensing status affects the potential for rituximab-induced ADCC (degranulation against HLA-deficient 721.221 in licensed cells 35 ± 4% versus 19 ± 3% of unlicensed cells, p<0.01); and that KIR/HLA interactions strongly and selectively inhibit the response to targets expressing cognate HLA ligands (e.g. CD107a expression in KIR3DL1+ NK cells 17 ± 3% against 721.221-Bw4 cells, compared to 32 ± 4% against 721.221, p<0.01). Next, we analyzed rituximab-induced NK cell activation in donors expressing one, two, or three KIR ligands after co-incubation with target B-cell lines expressing corresponding HLA molecules. These experiments showed that the inhibitory effect during target cell encounter dominates over the activating effect of NK cell licensing, which leads to unlicensed NK cells being the strongest effectors of ADCC with rituximab (Figure, Panel A).We next compared the effect of the KIR/HLA interaction on rituximab-, GA101wt- and GA101-induced ADCC. GA101 largely compensated the hyporesponsiveness of unlicensed cells and NK cell activation was independent of the presence of HLA KIR ligands on target cells (Figure, Panel A). Finally, we addressed the question of how levels of NK cell degranulation correspond to target cell elimination. Correlation between CD107a expression and target cell elimination was excellent for all antibodies (Figure Panel B). GA101 induced the highest level of activation and the most effective target elimination. In contrast to rituximab and GA101wt, no negative impact of KIR/HLA interaction on degranulation or target cell elimination could be detected for GA101. [Display omitted] In summary, we show that KIR/HLA interactions are relevant for ADCC with rituximab, with the negative impact during target cell encounter dominating over the positive effect of licensing. In contrast, the novel glycoengineered GA101 antibody overrides the negative signals derived from the KIR/HLA interaction and activates all NK cell subsets. These data suggest that the Fc-modification to enhance ADCC can be an effective strategy to augment the efficacy of therapeutic monoclonal antibodies by recruiting NK cells irrespective of their inhibitory KIR expression. Disclosures:Terszowski:Roche: Research Funding. Klein:Roche Glycart AG: Employment. Stern:Roche: Research Funding.

KIR Ligation During Ex Vivo Expansion Allows Molding Of The KIR Repertoire

NK Cell subpopulations express tremendous diversity through polymorphisms, haplotypes, differential expression, and licensing of the Killer Immunoglobulin-like Receptors (KIR). KIR diversity affects both the predisposition to cancer, and the response to therapies such as hematopoietic stem cell transplantation. Clinical trials that take advantage of the anti-cancer properties of NK cells have been limited to choosing donors on the basis of KIR genotypes and/or HLA haplotypes. Moreover, adoptive immunotherapy approaches have been limited by low NK cell doses. The latter hurdle has been recently mitigated by methods for expanding clinical grade NK cells ex vivo. These approaches for growing large numbers of cells now enable investigation into selecting more potent NK cell subsets for increased therapeutic efficacy.We hypothesized that the desired KIR repertoire could be molded through inhibition of undesirable KIR populations by crosslinking with relevant anti-KIR antibodies during expansion with our previously described method, which produces a mean 30,000-fold expansion of NK cells in 3 weeks.First, we determined that maximum inhibition was obtained when anti-KIR antibodies were applied to previously activated NK cells, crosslinked with secondary antibody, and then restimulated for proliferation.Robust reduction of targeted KIR-positive populations could be achieved for each inhibitory KIR (Fig. 1). When pre-activated with anti-KIR2DL1 for one stimulation cycle, NK cells expressing this KIR were decreased by a median of 70.4% ± 19.3%. Similarly, KIR2DL2/3+ NK cells could be reduced by 56% ± 17.5%, and KIR3DL1+ NK cells could be reduced by 53.5% ± 16.3%. When anti-KIR antibodies were combined, similar suppression of multiple-KIR subpopulations was observed. Other NK cell receptors were not significantly affected during targeted KIR inhibition.We then assessed the resulting NK cell populations for degranulation responses to targets with selected HLA as KIR ligands. Inhibition of KIR-expressing subpopulations during expansion resulted in NK cell populations with enhanced degranulation against tumor cells expressing the HLA ligand of the targeted KIR. Importantly, the cytotoxicity of the bulk NK cell population against HLA-negative targets remained.These results indicate that KIR crosslinking during NK cell propagation enables significant reduction in the targeted KIR subpopulations, resulting in an NK cell population with a selective decrease in KIR inhibition. By utilizing antibody-controlled expansion for molding of the KIR repertoire according to patient HLA type, a personalized NK cell product may be produced with enhanced potency, improving NK cell immunotherapy.▪ Disclosures:No relevant conflicts of interest to declare.

Recipient HLA-C1 Enhances The Clinical Advantage Of Killer-Cell Immunoglobulin-Like Receptor B haplotype Donors In Myeloablative Unrelated Transplantation For Acute Myelogenous Leukemia

The interactions of variable killer-cell immunoglobulin-like receptors (KIR) with polymorphic HLA class I ligands form an extraordinary immunogenetic system that influences NK cell biology, human susceptibility to disease, and the outcome of hematopoietic cell transplantation (HCT). The independent segregation of KIR and HLA genes, on chromosomes 19 and 6, respectively, increases the functional diversity of the system. Previously we found that patients undergoing unrelated donor (URD) HCT for acute myelogenous leukemia (AML) had superior leukemia-free survival (LFS) and less relapse with KIR B/x genotype donors than with KIR A/A donors. At higher resolution we identified the significant protective effect of donors with “Better” (≥2 B-motifs) and “Best” (≥2 B-motifs with Cen-B/B) KIR gene content compared to “Neutral” (0 or 1 B-motif) donors, supporting a donor selection strategy that is being tested in an ongoing prospective multicenter trial. The heterogeneity of transplant cohorts (preparative regimens, graft source, T cell depletion, HLA match status) has complicated the evaluation of the mechanisms underlying the beneficial effects mediated by NK cells. We analyzed 2404 URD transplants for AML and found a strong interaction between the protection associated with KIR B donors and the conditioning intensity (myeloablative [MA] vs. reduced intensity [RI]) for relapse (p=0.0002) and LFS (p=0.043). “Better” and “Best” donors were associated with significantly enhanced LFS and relapse protection in MA transplants, but appears to have an opposite effect in RI transplants. Therefore, to further explore the mechanisms of NK cell mediated protection, we focused our evaluation on a cohort of 1007 MA, T cell replete URD transplants for AML. We evaluated the interaction of donor KIR with recipient and donor HLA C1, C2 and Bw4. Superior LFS (RR 0.78 [0.66-0.92], p=0.0024) and relapse protection (RR 0.50 [0.38-0.67], p<0.0001) were observed with “Better+Best” donors (n=299) compared to “Neutral” donors (n=656). The benefit associated with “Better+Best” donors was enhanced in recipients expressing 1 or 2 HLA-C allotypes carrying the C1 epitope (C1/x (n=245) vs. C2/C2(n=51)) (RR 0.65 [0.41-1.01); p=0.05). No significant beneficial interactions were found between donor KIR and donor HLA-C group KIR ligands or with recipient or donor Bw4. Importantly, we did not identify any specific KIR B-haplotype defining gene as associated for the LFS advantage and relapse protection observed in C1/x recipients compared to C2/C2 recipients. Individual analyses for KIR B/x donors with or without 2DS1, 2DS2, 2DS3, 2DS5, 2DL2, 2DL5, 3DS1 showed equally significantly improved outcome (RR ranged from 0.71-0.80, p=0.002-0.03 for LFS and RR 0.56-0.63; p=0.0003-0.0008 for relapse). There was no clinical advantage of any KIR B genes in C2/C2 recipients. Importantly, the components of the relapse protection associated with interactions between “Better+Best” donors and C1/x recipients may depend upon the HLA-match status of the transplant. (P=0.018 for the interaction). In the <10/10 HLA-matched transplants (n=513), the “Better+Best” donors conferred potent relapse protection in C1/x (n=122) vs. C2/C2 (n=28) recipients (RR 0.35 [0.16-.76]; P <0.01) (Figure 1; right). This relapse protection, of equivalent strength in HLA-C or other HLA mismatches, resulted in improved LFS (RR 0.55 [0.34-.90]; P=0.02). Conversely, in the 10/10 HLA-matched transplants (n=494), treatment related mortality (TRM) was reduced when “Better+Best” donors could engage recipient HLA-C1 (n=123, RR 0.40 [0.20-.0.81]; P=0.01) although LFS was not statistically different, due in part to a lack of enhanced relapse protection (RR 3.00 [0.69-13.01]; P=0.14)(Figure 1; left). Interactions between KIR B-haplotype donors and homozygous C1/C1 in the recipient were associated with less acute GVHD when all transplants were analyzed. In summary, analysis in this homogeneous cohort demonstrates that the outcome of URD transplantation for AML is improved by interactions between recipient HLA-C1 and “Better+Best” KIR donors, which reduce relapse in <10/10 HLA-matched and reduce TRM in 10/10 HLA-matched transplants. This protection is observed in MA but not RI conditioning, suggesting that NK cell reconstitution and function differs in these settings.Can't read 3B2 tag because stream don't exist.Tag: Figure_12 Disclosures:No relevant conflicts of interest to declare.

Specific KIR and HLA Genotypes Affect Outcomes Of Single-Agent Anti-CD20 Immunotherapy Of Follicular Lymphoma

Lymphoma immunotherapy with anti-CD20 antibody (rituximab) acts primarily through Antibody Dependent Cellular Cytotoxicity (ADCC), implying a role for NK cells. The activation and inhibition of NK cells are influenced by Killer Immunoglobulin-like Receptors (KIRs) and their HLA ligands. To evaluate the involvement of NK cells in response to immunotherapy, we investigated the role of both KIR and HLA polymorphisms in response to single agent rituximab. We have analyzed the relationship between patient genotype and clinical parameters to determine if there are beneficial KIR/HLA interactions that can lead to improved clinical outcome. Methods204 follicular lymphoma (FL) patients participating in the Eastern Cooperative Oncology Group (ECOG) RESORT trial (E4402) were treated with rituximab. The patients who showed an initial response were randomized into two different rituximab treatment regimens. Approximately 70% of FL patients showed initial response to 4 weekly doses of rituximab, and were randomized between arm A: therapy “as needed” (4 weekly doses, only upon progression) or arm B: scheduled maintenance (1 dose every 13 weeks). Clinical outcomes analyzed included duration of response, time to rituximab failure (TTRF), time to cytotoxic therapy (TCT) and decrease in tumor size. The data presented here represent both treatment arms combined, i.e. arms A+B, for all clinical data parameters mentioned. Genomic DNA samples were used for genotyping patients for the presence of 15 KIR loci using a Real Time PCR-based method as previously published (Alves, Tissue Antigens, 2008). HLA specificities corresponding to KIR ligands (HLA-C1, C2, Bw4 and ABw4) were typed using a combination of PCR-SSP methods (Olerup and Invitrogen). ResultsThose patients homozygous for HLA-C1 (C1C1) showed a trend towards a longer duration of response (p=0.06) as compared to those that were heterozygous (C1C2) or homozygous for HLA-C2 (C2C2). A similar difference was observed where patients that were HLA-C1 positive [C1+ (C1C1 or C1C2)] showed a trend towards longer TTRF (p=0.071), as well as a significantly longer TCT (p=0.003), compared to C2C2 patients. Further analyses revealed that the influence of these HLA C specificities on clinical outcome could be related to the presence or absence of inhibitory and/or activating KIR genes. Specifically, HLA-C1 is a ligand for the inhibitory KIR genes 2DL2 and 2DL3. Among the patients who had the KIR2DL2 inhibitory gene (KIR2DL2+), those that were C1+ showed a prolonged duration of response (p=0.049), TCT [Figure 1A, (p=0.003)] and TTRF (p<0.001)] compared to KIR2DL2+ patients who were C2C2 and thus lacked the C1 cognate ligand. Similarly, KIR2DL3+ patients that were C1+ had a significantly longer TCT (p=0.007) than patients that were C2C2, but significant effects were not observed for these KIR2DL3+/C1+ patients with respect to the three other analyzed clinical parameters. Therefore, further studies are required to confirm that KIR2DL3 has an effect independent of other KIRs. [Display omitted] [Display omitted] HLA-C2 alleles are ligands for an activating KIR gene, KIR2DS1. The observed HLA-C genotype effect on clinical outcome could potentially be conveyed by the interaction of HLA-C2 with KIR2DS1. Among the C2C2 patients, KIR2DS1+ patients had a significantly shorter TTRF than those without KIR2DS1 (p=0.023), as well as a significantly smaller decrease in tumor size (p=0.039), implying that the interaction of the activating KIR receptor (KIR2DS1) with its cognate ligand (HLA-C2) conveys a disadvantage to patient outcome. ConclusionSingle-agent rituximab immunotherapy of FL reveals differences in outcomes based on KIR and HLA polymorphisms. The HLA-C2 homozygous individuals seem to be at a disadvantage. This may relate to 2 significant observations highlighting diverse effects of inhibitory and activating KIR-HLA interactions leading to NK cell “licensing” and “hyporesponsiveness”, respectively. Specifically, the data from this study are consistent with: A) a beneficial effect from inhibitory KIR2DL2 and KIR2DL3 interaction with HLA-C1, and B) a detrimental effect due to the contribution of activating KIR2DS1 interacting with its ligand HLA-C2.BG, AKE and WW are co-first authors. Disclosures:Horning:Genentech: Employment, Equity Ownership. Kahl:Genentech: Consultancy, Research Funding.

Effects of KIR ligand incompatibility on clinical outcomes of umbilical cord blood transplantation without ATG for acute leukemia in complete remission

To clarify the effect of killer cell immunoglobulin-like receptor (KIR) ligand incompatibility on outcomes of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients in complete remission after single cord blood transplantation (CBT), we assessed the outcomes of CBT registered in the Japan Society for Hematopoietic Cell Transplantation (JSHCT) database. A total of 643 acute leukemia (357 AML and 286 ALL) patient and donor pairs were categorized according to their KIR ligand incompatibility by determining whether or not they expressed HLA-C, Bw4 or A3/A11 by DNA typing. A total of 128 patient–donor pairs were KIR ligand-incompatible in the graft-versus-host (GVH) direction and 139 patient–donor pairs were incompatible in the host-versus-graft (HVG) direction. Univariate and multivariate analyses showed no significant differences between the KIR ligand-incompatible and compatible groups in the GVH direction for both AML and ALL patients of overall survival, disease-free survival, relapse incidence, non-relapse mortality and acute GVH disease. However, KIR incompatibility in the HVG direction ameliorated engraftment in ALL patients (hazard ratio 0.66, 95% confidence interval 0.47–0.91, P=0.013). Therefore, there were no effects of KIR ligand incompatibility in the GVH direction on single CBT outcomes for acute leukemia patients without anti-thymocyte globulin use. However, it is necessary to pay attention to KIR incompatibility in the HVG direction for engraftment.

Chimerism in Children With Primary Immunodeficiencies is Influenced by Number of Activating Killer Cell Immunoglobulin-Like Receptor Genes in the Donor and/or Killer Cell Immunoglobulin-Like Receptor Ligand Mismatch

BackgroundStem cell transplantation (SCT) is a curative treatment for children with primary immunodeficiencies. MethodsThe present retrospective analysis describes the long-term outcomes at a median follow-up of 9 years of 29 patients with immunodeficiency after SCT; 5 sibling and 24 alternative donor transplantations. T-cell engraftment emphazed on thymic dependent signal-joint T-cell receptor excision circles (sjTREC) generation and donor chimerism in relation to killer cell immunoglobulin-like receptor genes and their ligands. ResultsAll children except two were reconstituted successfully from grafted material, including 9 and 18 cases of mixed chimerism (MC) and complete chimerism (CC), respectively. Univariate analyses showed that the number of activating KIR genes or HLA-C1/C2 ligand mismatches (P = .048) and possibly transplantation from an alternative donor (P = .054) facilitated CC development. Multivariate analysis showed that the presence of donor KIR haplotype B or incompatibility within C1/C2 ligands (relative risk, 6.1; 95% confidence interval, 1.08–34.69; P = .025) were significantly associated with the development of CC. ConclusionsThese results suggested that the donor-activating KIR gene repertoire affected successful engraftment.

NK-KIR Gene Repertoire and Outcome of Patients with Acute Myeloid Leukemia after Allogeneic Hematopoietic Cell Transplantation from Unrelated Donors

In this retrospective study, the influence of donor and recipient KIR-gene content and their respective ligands including clinical parameters as potential confounding variables on the outcome of 150 acute myeloid leukemia (AML) patients undergoing allogenic hematopoietic cell transplantation (HCT) from unrelated donors was systematically investi- gated. There was no significant influence of KIR ligand mismatching and of donor/recipient KIR haplotype combinations on overall survival (OS), disease free survival (DSF), non-relapse mortality (NRM) and relapse. Isolated effects of KIR haplotypes, were detected for acute, chronic Graft versus Host Disease (aGvHD and cGvHD) as well as for the cumula- tive incidence of non-relapse mortality and relapse. The incidence of non-relapse mortality was evaluated in donor and re- cipient pairs harbouring KIR AA homozygosity (AA/Bx: p=0.038, HR=0.73, 95% CI=0.35-1.46 and AA/AA: p=0.043, HR=0.64, 95% CI 0.53-1-17). Our data suggest that KIR genotyping may be useful in patients in whom several HLA- identical unrelated donors can be identified but is probably not necessary for the primary donor selection algorithm.

Irradiated and Activated Autologous PBMCs Induce Expansion of Highly Cytotoxic Human NK Cells In Vitro

Adoptive cell transfer of ex vivo-activated natural killer (NK) cells is a promising therapy for cancer treatment. Because of inhibitory signaling through killer immunoglobulin-like receptor (KIR)-KIR ligands, KIR-mismatched allogeneic NK cell transfer is considered to be a more effective strategy than is autologous transfer. However, purified NK cells do not expand well enough in vitro with good manufacturing practice-compliant components for clinical use. Some investigators have developed selective expansion of NK cells from peripheral blood mononuclear cells, but these cells have the risk of graft-versus-host disease in allogeneic settings because of T cells contamination. In this study, we developed a novel method for NK cell activation and expansion. Using only good manufacturing practice-compliant components and autologous feeder cells, once purified NK cells were effectively expanded (2500-fold at day 17). The expanded cells were highly purified NK cells, and the use of these cells is suitable for allogeneic transfer without the risk of graft-versus-host disease induction. Importantly, the expanded NK cells also showed enhanced cytotoxicity compared with NK cells conventionally expanded by recombinant human interleukin 2. Finally, induction of NKG2D ligand expression on feeder cells implies that the NKG2D-NKG2DL interaction may play a role in NK cell expansion. In conclusion, this method can be used to obtain NK cells for more successful allogeneic NK cell adoptive transfer for use in antitumor immune therapy.

Polymorphisms of killer immunoglobulin-like receptors (KIRs) and HLA ligands in northeastern Thais

Killer cell immunoglobulin-like receptors (KIRs) are cell surface receptors on natural killer (NK) cells and subsets of T cells. The functions of NK cells are partly regulated by interactions between KIRs and HLA ligands on target cells. In this study, the presence or absence of 17 KIR genes and their known HLA ligands have been investigated in 235 unrelated individuals living in northeastern Thailand (NET). Subtypes of KIR2DS4 including full length (KIR2DS4F) and deleted forms (KIR2DS4D) have also been determined. Framework genes (KIR2DL4, 3DL2, 3DL3, and 3DP1) were found in all individuals and KIR genes belonging to the A haplotype (KIR2DL1, 2DL3, 3DL1, and 2DS4) were present in more than 90% of NET. KIR2DS4D (61.7%) was more common than KIR2DS4F (52.8%). A total of 33 different KIR genotypes were observed. Of these, three new genotypes were identified. The most common genotype (AA) was observed in 35.7% of NET, and HLA-C alleles bearing the C1 epitope (HLA-C1) had the highest frequency (97%). All individuals had at least one inhibitory KIR and its corresponding HLA ligand; 40.9% of NET had three pairs of receptor-ligand combinations, and 18.3% had all three receptor-ligand combinations of KIR2DL3+C1, 3DL1+Bw4, and 3DL2+A11. Surprisingly, the patterns of KIR gene frequencies in NET are more similar to those of Caucasians than Japanese, Korean, and Chinese. This is the first report on complete analysis of KIR and known HLA ligands in Thais. These data provide basic knowledge on KIR for further studies on disease associations and transplantation in northeastern Thais.