KIR Ligands Research Articles

KIR specificity and avidity of standard and unusual C1, C2, Bw4, Bw6 and A3/11 amino acid motifs at entire HLA:KIR interface between NK and target cells, the functional and evolutionary classification of HLA class I molecules.

Natural killer (NK) cells make vital contributions to the immune system and the reproductive system. Notably, NK cells of donor origin can recognize and kill residual leukaemic cells and cure malignant patients in hematopoietic stem cell (HSC) transplant setting. NK cell function is regulated by KIRs that recognize cognate HLA class I molecules on target cells, depending on their amino acid residues. In review, we addressed the question of binding capacity and avidity of HLA class I molecules to different killer cell immunoglobulin-like receptors (KIRs) depending on all interacting amino acid residues both on HLA and KIR side. We searched PubMed database and analysed available HLA:KIR crystallographic data for amino acid residues in HLA molecules, those physically involved in binding KIRs (termed here the "entire KIR interface"). Within entire KIR interface, we selected five functional sequence motifs (14-19, 66-76, 77-84, 88-92 and 142-151) and classified them according to the conservation of their amino acid sequences among 8,942 HLA class I molecules. Although some conserved amino acid motifs were shared by different groups of KIR ligands, the HLA motif combinations were exclusive for the ligand groups. In 135 common HLA class I molecules with known HLA:KIR recognition, we found 54 combinations of five motifs in each of the KIR-binding interfaces (C1, C2, Bw4, A3/11) and conserved non-KIR-binding interfaces. Based on the entire KIR interface, this analysis allowed to classify 8,942 HLA class I molecules into KIR specificity groups. This functional and evolutionary classification of entire KIR interfaces provides a tool for unambiguously predicting HLA:KIR interactions for common and those HLA molecules that have not yet been functionally tested. Considering the entire KIR interface in HLA class I molecules, functional interactions of HLA and KIR can be predicted in immune responses, reproduction and allotransplantation. Further functional studies are needed on the HLA:KIR interaction variations caused by the repertoires of peptides presented by HLA molecules and KIR polymorphisms at allelic level.

PB1741 KIR‐LIGAND MISMATCH IS NOT PREDICTIVE FOR LEUKEMIA‐FREE SURVIVAL IN PATIENTS WITH FAVORABLE RISK AML AFTER AUTOLOGOUS STEM CELL TRANSPLANTATION.

Background:Evidence suggests that Natural Killer (NK) Cells may play a role in the prevention of relapse of Acute Myeloid Leukemia after allogeneic stem cell transplantation (alloSCT). In a specific condition being HLA mismatched transplants, NK cells may get activated because subsets of donor NK cells miss inhibitory HLA alleles on the patient's cells. This is called. KIR‐ligand mismatch, were subsets of donor NK cells get activated when the patient's HLA alleles are not fitting to one or two of the inhibitory KIRs expressed on the donor cells (Ruggeri, Science 2002). The prerequisite for effective KIR‐ligand mismatch seems to be extensive AGVHD prevention by CD34‐selection of the stem cell graft or intense immunosuppressive conditioning including ATG. In HLA‐matched sibling alloSCT the same beneficial effect of KIR‐ligand mismatch was observed when intense T cell depletion of the graft had been applied (Hsu et al., Blood 2005). It was shown that early post T‐cell depleted alloSCT normally hyporesponsive NK cells expressing KIR for non‐self HLA temporarily displayed full functional capacity (Hsu, Blood 2009).Aims:We hypothesized that a beneficial effect of KIR‐ligand mismatch effect may be present in autologous SCT (autoHCT) in AML, where GVHD obviously is impossible. This would imply that developing NK cells harboring one or two inhibitory KIR receptors (iKIRs) for which no fitting HLA‐allele is present might temporarily get activated in those patients that do not bear all three relevant groups of HLA‐alleles (Bw4, C1 and C2) for which inhibitory KIRs are almost always there (presence of KIR2DL2/3 (fitting C1), KIR2DL1 (fitting C2) and KIR3DL1 (fitting Bw4) is 100%, 92.7% and 92.1% respectively, Hsu, Blood 2005). Approximately 30% of the population bears all three ligands for these three iKIRs and are not susceptible for KIR‐ligand mismatch.Methods:We typed all autologous transplanted AML patients in our center with genetic favorable risk factors (according to ELN 2017) and in first complete remission for HLA Bw4, C1 and C2. 2‐year leukemia‐free survival (LFS) of those patients not susceptible for KIR‐ligand mismatch was compared with LFS of those being susceptible. It was expected that 33% of the auto‐transplanted patients would relapse (Vellenga, Blood 2011). Assuming a meaningful higher 2‐year LFS of 87% for patients missing at least ligand (i.e. 15% higher than observed for the whole population), 2‐year LFS in those not missing a KIR ligand would have been 32% (3x 72 = 216; 216 – (2x 87) = 216 – 174 = 32). To demonstrate this difference 9 patients per group are needed. However, because of the unequal distribution of the two groups (“missing no KIR ligand” vs. “missing at least 1i KIR ligand”) to ensure that the “not missing a KIR ligand group” contains 9 patients at least 3x9=27 patients are needed.Results:We identified 39 favorable risk auto‐transplanted AML patients. Twelve had all HLA ligands for all three iKIRs present, and 27 lacked at least one ligand. 2‐year LFS was 83% and 83% respectively. For those 11 that missed HLA C1 ligands (for which all individuals have a ligand (i.e. KIR2DL2/3) 2‐year LFS was 81%.Summary/Conclusion:2‐year LFS is very high after autoHCT in first complete remission in favorable risk AML and KIR‐ligand mismatch is not relevant.image

Role of killer cell immunoglobulin-like receptor (KIR)-ligand interactions to prevent relapse in patients (pts) receiving matched unrelated stem cell transplant (SCT) for acute myeloid leukemia (AML).

7049 Background: NK cells play a pivotal role in cancer immune vigilance. NK cell function is regulated by a balance between activating and inhibitory signals derived from various receptor-ligand interactions. Methods: 390 AML pts received 10/10 unrelated donor SCT. KIR genotype was performed by PCR with sequence specific primers. Hazard ratio (HR) for 2-year relapse was calculated using Fine-Gray regression and adjusted for disease risk index, remission status, pre-SCT MRD, conditioning regimen and presence of HLA-DP mismatch. KIR-ligand (K-L) match was defined as the presence a given KIR in the donor and the presence of its reported ligand in the patient (ex. 2DL1 and HLA-C2). KIRs that have no known ligands were not included in this analysis. The Table shows pt characteristics and K-L matches. Results: There was no correlation between the number of inhibitory or activating KIRs or KIR haplotype (A or B) and the probability of relapse after SCT. However, donor KIRs had a dramatic effect on relapse when they were considered together with the presence of the corresponding ligand in the pt. The 210 pts who had ≥3 inhibitory K-L matches had a significantly higher probability of relapse (HR=1.748, CI=1.147-2.667, p=0.009) than the remaining 180 pts. Similarly, the 96 pts who had at least one known activating K-L match had a lower probability of relapse (HR=0.581, CI=0.345-0.978, p=0.04). When we considered inhibitory and activating K-L matches together, we found that the 168 pts who had ≥3 inhibitory and no activating K-L matches had a significantly higher probability of relapse (HR 2.001, CI=1.376-2.908, p<0.001) than the 222 remaining pts. Conclusions: Donor-pt KIR-ligand matching should be taken into account when choosing unrelated donors for AML pts. [Table: see text]

Diversity of KIR, HLA Class I, and Their Interactions in Seven Populations of Sub-Saharan Africans.

HLA class I and KIR sequences were determined for Dogon, Fulani, and Baka populations of western Africa, Mbuti of central Africa, and Datooga, Iraqw, and Hadza of eastern Africa. Study of 162 individuals identified 134 HLA class I alleles (41 HLA-A, 60 HLA-B, and 33 HLA-C). Common to all populations are three HLA-C alleles (C1+C*07:01, C1+C*07:02, and C2+C*06:02) but no HLA-A or -B Unexpectedly, no novel HLA class I was identified in these previously unstudied and anthropologically distinctive populations. In contrast, of 227 KIR detected, 22 are present in all seven populations and 28 are novel. A high diversity of HLA A-C-B haplotypes was observed. In six populations, most haplotypes are represented just once. But in the Hadza, a majority of haplotypes occur more than once, with 2 having high frequencies and 10 having intermediate frequencies. The centromeric (cen) part of the KIR locus exhibits an even balance between cenA and cenB in all seven populations. The telomeric (tel) part has an even balance of telA to telB in East Africa, but this changes across the continent to where telB is vestigial in West Africa. All four KIR ligands (A3/11, Bw4, C1, and C2) are present in six of the populations. HLA haplotypes of the Iraqw and Hadza encode two KIR ligands, whereas the other populations have an even balance between haplotypes encoding one and two KIR ligands. Individuals in these African populations have a mean of 6.8-8.4 different interactions between KIR and HLA class I, compared with 2.9-6.5 for non-Africans.

HLA and killer cell immunoglobulin-like receptor (KIRs) genotyping in patients with acute ischemic stroke

IntroductionIn humans, a major component of natural killer (NK) and T cell target recognition depends on the surveillance of human leukocyte antigen (HLA) class I molecules by killer immunoglobulin-like receptors (KIRs).AimsTo implement the knowledge about the immunological genetic background of acute ischemic stroke susceptibility in relation to the frequency of the KIR genes and HLA alleles.MethodsSubjects with acute ischemic stroke and subjects without stroke were genotyped for the presence of KIR genes and of the three major KIR ligand groups, HLA-C1, HLA-C2, and HLA-Bw4, both HLA-B and HLA-A loci.ResultsBetween November 2013 and February 2016, consecutive patients with acute ischemic stroke were recruited. As healthy controls, we enrolled subjects without acute ischemic stroke. Subjects with acute ischemic stroke in comparison with controls showed a higher frequency of 2DL3, 2DL5B, 2DS2, and 2DS4 KIR genes and a lower frequency of HLA-B-Bw4I alleles. Subjects without acute ischemic stroke showed a higher frequency of interaction between KIR 2DS2 and HLAC2. We also observed a higher frequency of 2DL3 and 2 DL4 KIR genes in subjects with atherosclerotic (LAAS) subtype. Multiple logistic regression analysis showed a protective effect towards stroke of HLA-B-Bw4I and interaction between KIR 2DL2 and HLAC1 and 2DS2-HLAC2 and a detrimental effect of 2DL2-HLA-C1_A interactions.ConclusionOur findings of a higher frequency of activating KIR genes seem to be consistent with findings previously reported patients with coronary syndrome. This higher frequency of “proinflammatory” genes in subjects with ischemic stroke could also explain the immunoinflammatory activation of the acute phase of stroke.

KIR Ligand Incompatibility in the Host Versus Graft Direction Predicts Graft Failure and Dominant Graft in Cord Blood Transplantation but Not in Haploidentical Transplantation

Donor-derived natural killer (NK) cell alloreactivity has been shown to improve clinical outcomes following allogeneic stem cell transplant (HSCT). Host versus graft (HvG) directed NK cell alloreactivity can also occur. Several epitopes of HLA-B and HLA-C are ligands for inhibitory killer immunoglobulin-like receptors (KIRs) that regulate NK cell cytotoxicity. These KIR ligands (KIR-L) may be surrogates of NK cell alloreactivity in the HLA-mismatched setting. To determine the impact of donor/recipient KIR-L mismatch (KIR-L-MM) direction on engraftment, we examined cord blood (CBT) and haploidentical (Haplo) HSCT performed at our institution between 2011 and 2018. We computed KIR-L and direction of KIR-L-MM for recipient/ donor pairs using the KIR ligand calculator (https://www.ebi.ac.uk/ipd/kir/ligand.html). Summary data and the relative risk of graft failure were analyzed. In the CBT cohort (N=70), there were KIR-L mismatches (KIR-L-MM) in the HvG and GvH direction in 32 and 31 recipient-cord blood unit pairs respectively. There were no KIR-L-MM in 26 transplants while seven had KIR-L-MM in both GvH and HvG directions. Overall, 16 CBT failed to engraft (23%). There was an increased risk of graft failure in recipients of HvG KIR-L-MM transplants (R.R 2.15; p=0.063) with this trend most significant when the mismatch was in HLA-C ligand (R.R 2.46; p=0.027); Figure 1. Out of 8 double CBTs where one cord unit had HvG KIR-L-MM, the non-mismatched unit fully engrafted in 7 cases (87.5%). In 6 double CBTs where there was KIR-L-MM from one cord unit in the GvH and the GvG (graft versus other graft) direction, 4 (67%) of such units became the dominant graft. Of 8 double CBTs in which both cord units had KIR-L-MM in the HvG direction there was graft failure in 3 (37.5%). Time to neutrophil engraftment and lymphocyte recovery when engraftment occurred was not significantly impacted by KIR-L-MM. In the Haplo cohort (N=26), all of the 5 graft failures (19%) were KIR-L mismatched only in the GvH direction (R.R 9.53; p=0.114). There were 9 HvG KIR-L-MM transplants and they all engrafted. Fourteen transplant pairs in this cohort had KIR-L-MM in the GvH direction; 2 pairs had both HvG and GvH KIR-L-MM while 3 pairs did not have any KIR-L-MM. Time to neutrophil engraftment was not impacted by GvH or HvG KIR-L-MM. This study suggests an increased likelihood of graft failure in CBT when HvG KIR-L-MM exists. There is also a trend in favor of engrafting the GvH/GvG KIR-L-MM unit in double CBT. These factors should be considered when selecting cord units for CBT. A similar graft failure effect of HvG KIR-L-MM is not seen in this small Haplo cohort; on the contrary the data suggests GvH directed KIR-L-MM associates with graft failure. This association merits exploration in a larger database.

Donor Killer Cell Immunoglobulin-Like Receptor Genotype Does Not Improve Graft-versus-Leukemia Responses in Chronic Lymphocytic Leukemia after Unrelated Donor Transplant: A Center for International Blood and Marrow Transplant Research Analysis.

Allogeneic hematopoietic cell transplantation (alloHCT) remains the sole curative therapy for patients with chronic lymphocytic leukemia (CLL), leading to 40% to 45% long-term survival. The impact of donor killer immunoglobulin-like receptor (KIR) genotype on outcomes of unrelated donor (URD) alloHCT for CLL is unknown. We examined 573 adult URD CLL recipient pairs. KIR genotype (presence/absence) was determined for each donor, and comprehensive modeling of interactions with recipient HLA class I loci (KIR ligands) was used to evaluate their effect on relapse and survival. Recipients had a median age of 56 years, and most were not in remission (65%). Both 8/8 HLA-matched (81%) or 7/8 HLA matched grafts (19%) were studied. Factors associated with improved overall survival (OS) were reduced-intensity conditioning (hazard ratio [HR] of death, .76) and good performance status (HR, .46), whereas alloHCT in nonremission (HR, 1.96) and mismatched donors (HR, 2.01) increased mortality. No models demonstrated a relationship between donor KIR genotype and transplant outcomes. Cox regression models comparing donors with A/A versus B/x KIR haplotypes and those with KIR gene content scores of 0 versus 1 versus ≥2 yielded similar rates of nonrelapse mortality, relapse, acute graft-versus-host disease (GVHD), and chronic GVHD and the same progression-free survival and OS. Relapse risk was not different for grafts from donors with KIR3DL1 transplanted into HLA C1/1 versus C2 recipients. This large analysis failed to demonstrate an association between URD KIR genotype and transplant outcome for patients with CLL, and thus KIR genotyping should not be used as a donor selection criterion in this setting.

The Direction of KIR Ligand Incompatibility Is Associated with Graft Failure and May Predict the Dominant Donor Following Double Umbilical Cord Blood Transplantation

While there is plausible evidence suggesting the benefit of natural killer (NK) cell alloreactivity in post-transplantation outcomes, there is no consensus on how such alloreactivity can be best predicted when selecting unrelated graft donors. Epitopes of HLA Class I molecules HLA-B and HLA-C are the ligands for inhibitory killer immunoglobulin-like receptors (KIRs) that regulate NK cell cytotoxicity. Based on the hypothesis that NK cell licensing in the bone marrow selects for circulating NK cells with corresponding inhibitory KIRs for self KIR ligands (KIR-L), these ligands, as determined by HLA-B/C antigens have been used as surrogates to predict NK cell alloreactivity in mismatched transplants. There is also the potential for alloreactivity by NK cells in the host versus graft (HvG) direction particularly in cord blood transplantation where reduced intensity conditioning is common. We examined cord blood transplantations at our institution to determine the impact of donor/recipient KIR ligand mismatch, and its direction, on graft failure.Methods: We analyzed the Stem Cell Transplant database of all cord blood transplants (CBT) performed at University Hospitals Seidman Cancer Center between December 2010 and April 2018. Data extracted from electronic medical record review included demographics, indication for transplant, disease status at time of transplant, conditioning regimen, graft versus host disease (GVHD) prophylaxis including the use of antithymocyte globulin (ATG) and post transplantation outcomes. For the cohort we computed KIR ligands for transplant recipients and cord blood unit(s), then predicted direction of alloreactivity using the KIR ligand calculator available online (https://www.ebi.ac.uk/ipd/kir/ligand.html). We determined the relative risk of developing graft failure as well as summary statistics of time to neutrophil engraftment and achieving a stable lymphocyte count of 500/ul or more in those that engrafted.Results: We identified 67 patients that received cord blood transplants for which donor and recipient KIR-L could be adequately computed. Three of these patients required a second cord blood transplant for graft failure, resulting in a total of 70 CBTs. Demographics, disease characteristics and treatment regimens are outlined in Table 1. There were KIR-L mismatches (MM) in the HvG and GvH direction in 32 and 31 recipient-cord blood unit pairs respectively. While there were no KIR-L MM in 26 transplants, 7 transplants had KIR-L mismatches in both GvH and HvG directions. There was an increased risk of graft failure in recipients of HvG-directed KIR-L MM transplants (R.R 2.15; p=0.063) with this trend most significant when the mismatch was in HLA-C ligand (R.R 2.46; p=0.027); Table 2. Out of 8 double CBTs where 1 cord unit had HvG-directed KIR-L MM, the non-mismatched unit fully engrafted in 7 (75%) cases. In 1 case the non-mismatched unit shared donor chimerism with the KIR-L MM unit at 85% and 15% respectively till 10 months post-transplant. In 6 double CBTs where there was KIR-L MM from only 1 cord unit in the GvH-direction, 4 (67%) of such units became the dominant graft. Of 8 double CBTs in which both cord units had KIR-L MM in the HvG direction there was graft failure in 3 (37.5%). The time to neutrophil engraftment and lymphocyte recovery in the patients that engrafted was not significantly impacted by KIR-L incompatibility (Table 3).Conclusions: From this study we see an increased likelihood of graft failure when KIR-ligand incompatibility exists in the host versus graft direction for CBT. There is also a trend in favor of engrafting the cord unit that is KIR-L mismatched in the graft versus host direction against the second cord in double CBT. These factors should be considered when selecting cord units for double CBT as well as in making single cord decisions where multiple options are available. [Display omitted] DisclosuresLittle:Hemex: Patents & Royalties: Patent, no honoraria; Doris Duke Charitable Foundations: Research Funding; PCORI: Research Funding; NHLBI: Research Funding. Malek:Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

KIR 2DS4 May Influence Autologous and Cord Blood(CB) Natural Killer (NK)Cell Mediated in Vitro Cytotoxicity Against Freshly Isolated Human Bone Marrow Myeloma Plasma Cells and Cell Lines

Background and Aim: NK cell effects are mediated by Killer Immunoglobulin-like Receptor (KIR) inhibition. There are few reports on the protective role of KIRs against relapse in myeloma(MM) (Gabriel et al Blood 2010, Kröger et al leukemia 2011). However, which NK cells or for whom will be more effective and the role of individual differences in this mechanism is indefinite. In this study we aimed to accomplish cytotoxicity against both MM cells and human myeloma cell lines (HMCL) and to compare the cytotoxicity achieved with CB versus autologous NK cells as well as to investigate the influence of NK cell KIR geno/phenotypes.Patients and Methods: A total of 18 patients with refractory/relapsed MM were included in this study.Cell culturing: CD138+ PC isolated from fresh bone marrow aspirates of relapsed MM patients or HMCL (U266, RPMI8226 and H929) using RosetteSep™ Human Multiple Myeloma Cell Enrichment Cocktail (StemCell Technologies) were subjected to cytotoxicity assays with CB derived or autologous PB derived NK cells expanded in the presence of IL-15 (10ng/ml) for 2-8 days.Phenotyping: Effector cells (autologous or CB-NK) were analyzed by flow cytometry using CD45, CD3, CD16, CD56, CD158a (KIR2DL1), CD158i (KIR2DS4) and CD158b1/b2 (KIR 2DL2/2DL3) monoclonal antibodies.Cytotoxicity assays:3,3 dioctadecyloxacarbocyanine perchlorate (DiO) dye was used to distinguish target cells (PC) from effector cells (NK). PC and NK cells were mixed at an 1:10 target: effector cell ratio and inoculated with Propidium Iodide (PI) in order to counter stain the dead cells. Naive PC were used as controls. Following 120 mins of incubation, PC death was evaluated using flow cytometry. After incubation of target cells (PC) with effector cells (NK cells), cytotoxic effects were determined by subtraction of NK mediated PC death from spontaneous PC death.Results: Following IL-15 expansion NK cells were found to express the CD16+56+3- phenotype. Eighteen MM patient PCs and six different HMCL samples were used in cytotoxicity assays(Table 1). In vitro PC viability was less than 45% in four samples and could not be analyzed in the study. Following incubation of PC wo NK cells, spontaneous PC death ratio was found to be 22.6% (min-max: 3%-42.3%). Twelve cytotoxicity assays targeting six MM PC were performed with autologous and CB-derived NK cells. Autologous NK and CB NK mediated cytotoxicity median rates were found to be 19.0% (range: 1.4% - 43.6%) and 31.3% (range: 2.4% - 51.2%), respectively. Additionally, CB-NK cells were evaluated against HMCL samples (four U266, one RPMI8226 and one H929). Median cytotoxicity against HMCL was 27.4% (range: 5.6% - 52.1%). Highest cytotoxicity against MM PC (MM-9) and HMCL (H929) were achieved by CB NK cells (51.2% and 52.1 % respectively). There was no cytotoxic effect against PCs of patients MM6 and MM10 (Figure 1-2).Flow-cytometric phenotyping of effector NK cells showed consistency (10/13) with genotypes. Since only one (CB19) with 19% CD158i positivity was PCR negative, a %20 cut-off rate was chosen. Both CD158a (KIR2DL1) and CD158b1/b2 (KIR 2DL2/2DL3) were positive among all patients and no significant correlation with cytotoxic effects was detectable. However out of five high cytotoxic reactions three NK cells were 2DS4 (+). While out of eight assays which resulted with low cytotoxicity only three NK cells were positive for the activating KIR 2DS4. When we analyzed CB NK cells against HMCL U266, three CB NK cells two of which were 2DS4(+) showed high cytotoxicity, while the 2DS4(-) CB NKs did not. The only assay targeting RPMI-8226 which is known to be a NK resistant HMCL, KIR2DS4(+) (CB17) derived NK cells were not sufficiently cytotoxic.Conclusion: Allogeneic CB NK cells were able to induce higher cell kill than autologous PB-derived NK cells. KIR types of autologous NK cells were not predictive for cytotoxic efficacy. Lacking KIR2DS4 in CB NK cells was correlated with less cytotoxicity. Additionally, KIR2DS4(+) CB NK cells were found to be highly cytotoxic against both PC and HMCL. NK cells may be an effective alternative due to its profound clinical advantages such as matching for HLA or KIR ligand is not required. Furthermore, if confirmed by others, certain KIR haplotypes (ie Haplotype A and KIR2DS4 +) may offer better therapeutic potential.Acknowledgment: The study has been supported by Scientific and Research Council of Turkey (TUBITAK grant no: 115S579) and Turkish Academy of Sciences. [Display omitted] DisclosuresBeksac:Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen,Janssen-Cilag,Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Fine-tuning CD8+ T cell responses

Immunogenetics Human cytotoxic CD8+ T cells are important for defense against viral infections. Boelen et al. investigated whether inhibitory killer cell immunoglobulin-like receptors (iKIRs) carried by patients with chronic viral infections affected the efficacy of their CD8+ T cell responses. Possession of an iKIR gene along with a gene encoding a KIR ligand enhanced protective and detrimental human leukocyte antigen (HLA) class I associations for HIV-1, hepatitis C virus, and human T cell leukemia virus type 1. Analysis of virus dynamics, in vitro survival assays, and mathematical modeling suggested that iKIR ligation enhances HLA associations by increasing T cell survival. In contrast to many reported iKIR-disease associations, these observations applied to all iKIRs and the three viral infections studied. Sci. Immunol. 3 , eaao2892 (2018).

Molecular evolution of elements controlling HLA-C expression: Adaptation to a role as a killer-cell immunoglobulin-like receptor ligand regulating natural killer cell function.

The regulatory elements controlling the transcription of the HLA-A, HLA-B, and HLA-C genes have been extensively studied and compared. However, few studies have considered regulatory differences in the HLA genes from the perspective of their role as ligands for the killer-cell immunoglobulin-like receptor (KIR) family of HLA receptors expressed by natural killer (NK) cells. HLA-C is the most recently evolved gene, and there is considerable evidence pointing to its emergence as a specialized KIR ligand playing a major role in the missing-self recognition system of NK cells. Here I evaluate gene-specific differences in regulatory elements of the HLA genes, showing alterations that are consistent with the adaptation of HLA-C to a role in NK cell regulation.

NK cell development in a human stem cell niche: KIR expression occurs independently of the presence of HLA class I ligands

AbstractThe development of mature natural killer (NK) cells expressing killer cell immunoglobulin-like receptors (KIRs) depends on cell contact–dependent signals from nonhematopoietic cells. So far, detailed studies of this process have been hampered by the lack of an appropriate in vitro model. Here, human bone marrow–derived mesenchymal stem cells (MSCs), generated under good manufacturing practice (GMP) conditions, are established as a supportive niche for in vitro NK cell differentiation. In the presence of MSCs, cord blood and bone marrow–derived hematopoietic stem and progenitor cells (HSPCs) effectively and reproducibly differentiated into mature KIR-expressing NK cells. Notably, the novel in vitro differentiation assay enabled us to analyze the impact of HLA class I ligands on KIR repertoire development. To this end, a panel of MSC lines divergent for expression of the major KIR ligands C1, C2, and Bw4 was used for NK cell differentiation. The resulting NK cell repertoires were independent of the presence of specific KIR ligands on MSCs and were, in fact, invariably dominated by expression of the C1-specific inhibitory KIR2DL3. Similarly, short hairpin RNA–mediated knockdown of HLA class I ligands on MSCs did not delay or change the course of KIR expression. Our data suggest that the initial acquisition of KIRs during NK cell development is biased toward recognition of C1 ligands, irrespective of the presence of self-ligands. Altogether, the MSC/HSPC model constitutes a novel platform to study NK cell development in a human stem cell niche. Moreover, the system constitutes a promising GMP-compliant platform to develop clinical-grade NK cell products from cord blood HSPCs.

Successful engraftment after cord blood transplantation from an HLA‐homozygous donor (homo‐to‐hetero cord blood transplantation) in a primary myelofibrosis patient with broad HLA antibodies

Donor-specific human leukocyte antigen (HLA) antibodies are a significant risk factor for graft failure in cord blood transplantation (CBT). Although there are several treatments to decrease HLA antibodies, such as platelet transfusion, plasma exchange, rituximab, and bortezomib, their effectiveness has not been established. We herein report the case of a primary myelofibrosis (PMF) patient with broad HLA antibodies who underwent CBT from an HLA-homozygous donor in which the alleles were matched only in the host-versus-graft direction (homo-to-hetero CBT). The cord blood was killer cell immunoglobulin-like receptor (KIR) ligand matched. She received a reduced-intensity conditioning regimen. We used tacrolimus and mycophenolate mofetil as prophylaxis against graft-versus-host disease (GVHD). The neutrophils engrafted on Day 31. A chimerism analysis with fluorescence in situ hybridization of peripheral blood cells showed 99.9% donor type on Day 33. She developed only mild acute skin GVHD and chronic skin GVHD. This case indicates the usefulness of homo-to-hetero CBT in a patient with broad HLA antibodies with a strong mean fluorescence intensity, which is a significant risk factor for graft failure. Further studies are necessary to determine the risk of GVHD and to elucidate the association between KIR ligand incompatibility and graft failure in homo-to-hetero CBT.

The impact of donor and recipient KIR genes and KIR ligands on the occurrence of acute graft-versus-host disease and graft survival after HLA-identical sibling hematopoietic stem cell transplantation

After allogeneic hematopoietic stem cell transplantation (allo-HSCT), donor natural killer (NK) cells trigger alloreactions against potential recipient cells by their killer immunoglobulin-like receptors (KIRs). This study investigated whether KIR/HLA genotypes and KIR haplotypes of donors and recipients exhibit a critical function in the prevalence of acute graft-versus-host disease (aGVHD) and persistence of the graft after HLA-identical sibling allo-HSCT for patients with hematological malignancies. We studied KIR and HLA genotypes in 115 related donors and recipients (56 patients with AML and 59 patients with ALL) who had received allo-HSCT from HLA-matched sibling donors. We evaluated 17 KIR genes and some alleles, including their ligands, using the PCR-SSP assay. KIR gene frequency results between donors and recipients showed that donors had more activating KIR than their recipients. Chi-square comparison of KIR genotype frequencies in donors versus recipients revealed a significant difference (P < 0.001). We found a survival association between the donor lacking and the recipient having group B KIR haplotypes, although this was not statistically significant. This study suggests that we could exploit NK cell alloreactivity as a part of the optimization of donor selection and potential immunotherapeutic regimens to help facilitate good engraftment and reduce the risk of aGVHD incidence after allo-HSCT.

The combination of maternal KIR-B and fetal HLA-C2 is associated with decidua basalis acute atherosis in pregnancies with preeclampsia

Acute atherosis is an arterial lesion most often occurring in pregnancies complicated by preeclampsia, a hypertensive pregnancy disorder. Acute atherosis predominates in the maternal spiral arteries in the decidua basalis layer of the pregnant uterus. This layer forms the fetal-maternal immunological interface, where fetal extravillous trophoblasts interact with maternal immune cells to promote decidual spiral artery remodeling and maternal immune tolerance towards the fetus. Of the classical polymorphic class I HLAs, extravillous trophoblasts express only HLA-C. HLA-C is a ligand for killer immunoglobulin-like receptors (KIR) on NK- and T-cells. Genetic combinations of fetal HLA-C and maternal KIRs affect pregnancy outcome. However, the role of HLA and KIR genes in acute atherosis is unknown.We hypothesized that specific genetic combinations of fetal HLA and maternal KIR are associated with the presence of acute atherosis lesions in the decidua basalis. We genotyped HLA class-I and II loci in paired fetal and maternal DNA samples from 166 pregnancies (83 preeclamptics, 83 controls). Acute atherosis was identified in 38 of these. Maternal KIR-loci were also genotyped.We found that the combination of maternal KIR-B haplotype and fetal HLA-C2 was significantly associated with acute atherosis in preeclampsia. In preeclamptic pregnancies with acute atherosis, 60% had this combination, compared to 24.5% in those without acute atherosis (p = 0.001). We suggest that interactions between fetal HLA-C2 and activating KIRs on maternal decidual NK-cells or T-cells may contribute to the formation of acute atherosis by promoting local decidual vascular inflammation.

The Presence of HLA-A Bw4-80I KIR Ligands Could Predict “Difficult-to-Treat” Psoriasis and Poor Response to Etanercept

Psoriasis is an immune-mediated dermatosis with a wide genetic predisposition. The immunogenetic background, specifically interactions between human leukocyte antigen (HLA) class I ligands and killer-cell immunoglobulin-like receptor (KIRs), have functional significance in modulating natural killer (NK) cells and can influence susceptibility and response to biological therapy. The main aim of this study was to correlate HLA-A and -B KIR ligands with response to biological therapy in patients with psoriasis. HLA-A and -B polymorphisms were determined in 48 patients (35 males and 13 females), with a mean of 22years of disease (range 8-55). All patients were treated with biological therapy (adalimumab, etanercept, infliximab, or ustekinumab) for at least 6months. This study identifies, with statistical significance, the presence of at least one ligand HLA-A Bw4-80I in the "poor-responder" population (patients who needed two or more biologics) compared with the "responder" population (patients with good response after a single biological drug) (47.62 vs. 11.11%; p = 0.006) as well as in "non-responders to etanercept" compared with "responders to etanercept" (52.63 vs. 5%; p = 0.001). Our preliminary results suggest that at least one ligand HLA-A Bw4-80I could be associated with "difficult-to-treat" psoriasis and that this ligand may reduce the probability of response to etanercept, producing more tumor necrosis factor (TNF)-α and neutralizing NK activity through a predominance of activating KIR. The ab initio identification of genetic markers of response to biologic therapy could improve the efficacy and economic impact of these agents.

Killer cell immunoglobulin-like receptor ligand mismatching and outcome after haploidentical transplantation with post-transplant cyclophosphamide.

Haploidentical stem cell transplantation with T cell-replete grafts and post-transplant cyclophosphamide (PTCy) is increasingly used with encouraging outcome. Natural killer (NK) cell alloreactivity, predicted by missing killer cell immunoglobulin-like receptor (KIR) ligands in the recipient that are present in their donor improves outcome of T cell-depleted haploidentical transplants. We explored the role of KIR ligand mismatching in 444 acute leukemia patients after T cell-replete transplants with PTCy. Thirty-seven percent of all patients had KIR ligand mismatching. Patients were in first remission (CR1) (39%), second remission (CR2) (26%), or active disease (35%). Stem cell source was peripheral blood (PBSC, 46%) or bone marrow (54%). The 2-year relapse, non-relapse mortality (NRM), and survival rates were 36.0% (95% confidence interval (CI), 31.4-40.7), 23.9% (20.0-28.0), and 45.9% (40.8-51.0), respectively. Multivariate analysis identified acute myeloid leukemia compared with acute lymphoblastic leukemia (hazard ratio (HR) 0.55, P = 0.002), female gender (HR 0.72, P = 0.04), and good performance status (HR 0.71, P = 0.04) as factors associated with better survival, while advanced age (HR 1.13, P = 0.04), active disease (HR 3.38, P < 0.0001), and KIR ligand mismatching (HR 1.41, P = 0.03) as associated with worse survival. KIR ligand mismatching was associated with a trend for higher relapse but not with graft-versus-host disease or NRM. The KIR ligand-mismatching effect was more prominent in patients given PBSC. In conclusion, there is no evidence that KIR ligand mismatching results in better outcome in the PTCy setting.

In situ delivery of allogeneic natural killer cell (NK) combined with Cetuximab in liver metastases of gastrointestinal carcinoma: A phase I clinical trial

ABSTRACTDespite successful introduction of NK-based cellular therapy in the treatment of myeloid leukemia, the potential use of NK alloreactivity in solid malignancies is still elusive. We performed a phase I clinical trial to assess the safety and efficacy of in situ delivery of allogeneic NK cells combined with cetuximab in liver metastasis of gastrointestinal origin. The conditioning chemotherapy was administrated before the allogeneic NK cells injection via hepatic artery. Three escalating doses were tested (3.106, 8.106 and 12.106 NK cells/kg) following by a high-dose interleukin-2 (IL-2). Cetuximab was administered intravenously every week for 7 weeks. Nine patients with liver metastases of colorectal or pancreatic cancers were included, three per dose level. Hepatic artery injection was successfully performed in all patients with no report of dose-limiting toxicity. Two patients had febrile aplasia requiring a short-term antibiotherapy. Grade 3/4 anemia and thrombopenia were also observed related to the chemotherapy. Objective clinical responses were documented in 3 patients and among them 2 occurred in patients injected with cell products harboring two KIR ligand mismatches and one in a patient with one KIR ligand mismatch. Immune monitoring revealed that most patients presented an increase but transient of IL-15 and IL-7 cytokines levels one week after chemotherapy. Furthermore, a high expansion of FoxP3+regulatory T cells and PD-1+ T cells was observed in all patients, related to IL-2 administration. Our results demonstrated that combining allogeneic NK cells transfer via intra-hepatic artery, cetuximab and a high-dose IL-2 is feasible, well tolerated and may result in clinical responses.

Donor Selection Based on Killer Cell Immunoglobulin–Like Receptor (KIR) Genotype May Improve Outcome After T-Cell–Replete Haploidentical Transplantation

BackgroundIn haploidentical stem cell transplantation (SCT), the “ideal donor” selection is not performed in a standardized way according to killer cell immunoglobulin–like receptor (KIR) genotype expressed by potential donors. The aim of this study was to evaluate the relevance of KIR genotype in a series of patients submitted to haploidentical SCT in our center. MethodsWe retrospectively analyzed 30 patients that were prepared with the use of a conditioning regimen including thiotepa-busulfan-fludarabine with high doses of post-transplantation cyclophosphamide (CyPT) and tacrolimus as graft-versus-host disease (GVHD) prophylaxis. We analyzed the impact of the KIR genotype variables (donor AA/Bx haplotype, donor B content, KIR inhibitor mismatches, and mismatching in KIR ligands in the graft-versus-host direction and the host-versus-graft direction) on overall survival, GVHD-free survival, and event-free survival. ResultsStatistical significance was found for the presence of mismatches on KIR ligands in the graft-versus-host direction in relation to the diagnosis of chronic GVHD (54% vs 100%; P = .004). Significance was also found for the effect of the donor presence AA or Bx haplotype in relation to the diagnosis of chronic GVHD (50% vs 86%; P = .033). ConclusionsKIR genotyping can provide useful information that can help us with the right donor choice for haploidentical SCT without T-cell depletion with high doses of CyPT. Donors with Bx haplotype that do not show KIR ligand incompatibilities in the graft-versus-host direction may provide a lower risk of GVHD.

KIR and HLA ligands demonstrate genetic inheritance diversity in Japanese descendants from Paraná, Brazil

In order to investigate killer immunoglobulin-like receptors (KIR) and their ligands, human leukocyte antigen (HLA), diversity in the Brazilian population influenced by migrations, unrelated Brazilian Japanese descendants were selected and genotyped for the KIR genes and HLA class I allele groups. Genetic heterogeneity in Brazil Paraná Japanese was observed for KIR genes, whose frequency distributions demonstrated similarity with mixed Brazilian populations and with the Japanese population, suggesting gene flow. The data contributed to the identification of the genetic constitution of the Brazilian population influenced by immigrations and two new genotypes were defined.