Killer Cell Immunoglobulin-like Receptors Research Articles

Role of the Killer Immunoglobulin-like Receptor and Human Leukocyte Antigen I Complex Polymorphisms in Kaposi Sarcoma-Associated Herpesvirus Infection.

Kaposi sarcoma, caused by the pathogen Kaposi sarcoma-associated herpesvirus (KSHV), is the most common neoplasm for patients with AIDS. Susceptibility to KSHV has been associated with several different genetic risk variants. The purpose of this study was to test whether variants of killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA-I) ligands influence the risk of KSHV infection. A case-control study was performed in Xinjiang, a KSHV-endemic region of China. We recruited 299 individuals with HIV, including 123 KSHV-seropositive persons and 176 KSHV-seronegative controls. We used logistic regression and the MiDAS package to evaluate the association between KIR/HLA-I polymorphisms and KSHV infection. HLA-A*31:01, HLA-C*03:04, and HLA-C*12:03 were found to be associated with KSHV infection, with A*31:01 showing a protective effect under 3 different models (dominant: 0.30 [95% confidence interval {CI}, .08-.82], P = .031; additive: 0.30 [95% CI, .09-.80], P = .030; overdominant: 0.31 [95% CI, .09-.88], P = .042). The effect of A*31:01 might cause the variants of amino acid at HLA-A position 56, with individuals carrying an arginine having a lower KSHV infection risk. The increased homozygous KIR2DL3 was associated with a relatively high KSHV viral load (16.30% vs 41.94%, P = .010). This study provides further insight into the link between HLA-I alleles and KIR genes and KSHV infection, highlighting KSHV-susceptible variants of HLA-I and KSHV replication caused by specific KIR genotype, and revealing a potential role of KIR-mediated natural killer cell activation in anti-KSHV infection.

B7-H7: A potential target for cancer immunotherapy.

Cancer immunotherapy enhances the body's immunity against tumors by mitigating immune escape. Compared with traditional chemotherapy, immunotherapy has the advantages of fewer drugs, a wider range of action and fewer side effects. B7-H7 (also known as HHLA2, B7y) is a member of the B7 family of costimulatory molecules that was discovered more than 20years ago. B7-H7 is mostly expressed in organs such as the breast, intestine, gallbladder and placenta and is detected predominantly in monocytes/macrophages in the immune system. Its expression is upregulated after stimulation by inflammatory factors such as lipopolysaccharide and interferon-γ. B7-H7/transmembrane and immunoglobulin domain containing 2 (TMIGD2) and killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3)-B7-H7 are the two currently confirmed signaling pathways for B7-H7. An increasing number of studies have demonstrated that B7-H7 is widely present in a variety of human tumor tissues, especially in programmed cell death-1 (PD-L1)-negative human tumors. B7-H7 promotes tumor progression, disrupts T-cell-mediated antitumor immunity, and inhibits immune surveillance. B7-H7 also triggers tumor immune escape and is associated with clinical stage, depth of tumor infiltration, metastasis, prognosis, and survival related to different tumor types. Multiple studies have shown that B7-H7 is a promising immunotherapeutic target. Herein, review the current literature on the expression, regulation, receptors and function of B7-H7 and its regulation/function in tumors.

Association of killer cell immunoglobulin-like receptors and their cognate HLA class I ligands with susceptibility to acute myeloid leukemia in Iranian patients

Acute myeloid leukemia (AML) is one of the most prevalent leukemia in adults. Among the various NK receptors, killer immunoglobulin-like receptors (KIRs) carry out indispensable roles in NK cell development and function through engaging with class I human leukocyte antigens (HLA-I) as their ligands. Besides divergent KIR and HLA loci, KIR/HLA-I combinations have a significant effect on NK cell response. In this case–control study, we aimed to verify the association of KIR/HLA-I combinations with susceptibility to AML in the Southwestern Iranian population. KIR and HLA genotyping was performed with PCR-SSP by some novel primers for 181 patients with AML and 181 healthy controls. According to our results, the frequencies of KIR3DS1 (p = 0.0001, OR = 2.32, 95% CI 1.51–3.58), KIR2DS4fl (p = 0.02, OR = 1.53, 95% CI 1.05–2.21), CxT4 genotypes (p = 0.03, OR = 2.0, 95% CI 1.05–3.82), and T4 gene cluster (p = 0.01, OR = 1.99, 95% CI 1.17–3.41) were significantly higher in patients than controls, while C1/C2 genotype (p = 0.00002, OR = 0.39, 95% CI 0.25–0.61), HLA-A Bw4 (p = 0.02, OR = 0.6, 95% CI 0.38–0.94), and HLA-A*11 (p = 0.03, OR = 0.57, 95% CI 0.34–0.95) alleles were more frequent in controls. In addition, inhibitory (i)KIR/HLA-I combinations analysis revealed higher frequencies of KIR2DL1( +)/HLA-C2( +), KIR2DL2/3( +)/HLA-C1( +), KIR3DL1( +)/HLA-A Bw4( +), and KIR3DL2( +)/HLA-A*03/11( +) in the control group (p = 0.002, OR = 0.49, 95% CI 0.3–0.78; p = 0.04, OR = 0.62, 95% CI 0.39–0.99; p = 0.04, OR = 0.63, 95% CI 0.4–0.99; and p = 0.03, OR = 0.62, 95% CI 0.4–0.95, respectively). Overall, the number of iKIR/HLA-I combinations was more in the control group. Moreover, KIR3DS1( +)/HLA-B Bw4Ile80( +) and the sum of HLA-B Bw4/A Bw4 combined with KIR3DS1 as activating KIR/HLA-I combinations were more frequent among patients than controls (p = 0.01, OR = 1.99, 95% CI 1.14–3.49 and p = 0.005, OR = 1.97, 95% CI 1.22–3.19, respectively). In conclusion, our results postulate that inhibitory combinations play a protective role against AML by developing potent NK cells during education. It is noteworthy that KIR/HLA-I combination studies can be applicable in donor selection for allogeneic NK cell therapy in hematological malignancies.

The genotype list string code syntax for exchanging nomenclature-level genotyping results in clinical and research data management and analysis systems.

The nomenclatures used to describe HLA and killer-cell immunoglobulin-like receptor (KIR) alleles distinguish unique nucleotide and peptide sequences, and patterns of expression, but are insufficient for describing genotyping results, as description of ambiguities and relations across loci require terminology beyond allele names. The genotype list (GL) String grammar describes genotyping results for genetic systems with defined nomenclatures, like HLA and KIR, documenting what is known and unknown about a given genotyping result. However, the accuracy of a GL String is dependent on the reference database version under which it was generated. Here, we describe the GL string code (GLSC) system, which associates each GL String with meta-data describing the specific reference context in which the GL String was created, and in which it should be interpreted. GLSC is a defined syntax for exchanging GL Strings in the context of a specific gene-family namespace, allele-name code-system, and pertinent reference database version. GLSC allows HLA and KIR genotyping data to be transmitted, parsed and interpreted in the appropriate context, in an unambiguous manner, on modern data-systems, including Health Level 7 Fast Healthcare Interoperability Resource systems. Technical specification for GLSC can be found at https://glstring.org.

Polymorphic KIR3DL3 expression modulates tissue-resident and innate-like T cells.

Most human killer cell immunoglobulin-like receptors (KIR) are expressed by natural killer (NK) cells and recognize HLA class I molecules as ligands. KIR3DL3 is a conserved but polymorphic inhibitory KIR recognizing a B7 family ligand, HHLA2, and is implicated for immune checkpoint targeting. The expression profile and biological function of KIR3DL3 have been somewhat elusive, so we searched extensively for KIR3DL3 transcripts, revealing highly enriched expression in γδ and CD8+ T cells rather than NK cells. These KIR3DL3-expressing cells are rare in the blood and thymus but more common in the lungs and digestive tract. High-resolution flow cytometry and single-cell transcriptomics showed that peripheral blood KIR3DL3+ T cells have an activated transitional memory phenotype and are hypofunctional. The T cell receptor (TCR) usage is biased toward genes from early rearranged TCR-α variable segments or Vδ1 chains. In addition, we show that TCR-mediated stimulation can be inhibited through KIR3DL3 ligation. Whereas we detected no impact of KIR3DL3 polymorphism on ligand binding, variants in the proximal promoter and at residue 86 can reduce expression. Together, we demonstrate that KIR3DL3 is up-regulated alongside unconventional T cell stimulation and that individuals may vary in their ability to express KIR3DL3. These results have implications for the personalized targeting of KIR3DL3/HHLA2 checkpoint inhibition.

Establishing NK-Cell Receptor Restriction by Flow Cytometry and Detecting Potential NK-Cell Clones of Uncertain Significance

Natural killer (NK) cells develop a complex inhibitory and/or activating NK-cell receptor system, including killer cell immunoglobulin-like receptors (KIRs or CD158) and CD94/NKG2 dimers, which are variably combined to generate the individual’s NK-cell receptor repertoire. Establishing NK-cell receptor restriction by flow cytometric immunophenotyping is an important step in diagnosing NK-cell neoplasms, but reference interval (RI) data for interpreting these studies are lacking. Specimens from 145 donors and 63 patients with NK-cell neoplasms were used to identify discriminatory rules based on 95% and 99% nonparametric RIs for CD158a+, CD158b+, CD158e+, KIR-negative, and NKG2A+ NK-cell populations to establish NK-cell receptor restriction. These 99% upper RI limits (NKG2a >88% or CD158a >53% or CD158b >72% or CD158e >54% or KIR-negative >72%) provided optimal discrimination between NK-cell neoplasm cases and healthy donor controls with an accuracy of 100% compared with the clinicopathologic diagnosis. The selected rules were applied to 62 consecutive samples received in our flow cytometry laboratory that were reflexed to an NK-cell panel due to an expanded NK-cell percentage (exceeding 40% of total lymphocytes). Twenty-two (35%) of 62 samples were found to harbor a very small NK-cell population with restricted NK-cell receptor expression based on the rule combination, suggestive of NK-cell clonality. A thorough clinicopathologic evaluation for the 62 patients did not reveal diagnostic features of NK-cell neoplasms; therefore, these potential clonal populations of NK cells were designated as NK-cell clones of uncertain significance (NK-CUS). In this study, we established decision rules for NK-cell receptor restriction from the largest published cohorts of healthy donors and NK-cell neoplasms. The presence of small NK-cell populations with restricted NK-cell receptors does not appear to be an uncommon finding, and its significance requires further exploration.

Killer cell immunoglobulin-like receptor (KIR) genes and their HLA ligands in a Brazilian population.

Killer cell immunoglobulin-like receptors (KIR) exhibit extensive diversity, giving rise to different KIR profiles in populations worldwide. This study aimed to investigate the distribution of KIR genes and HLA ligands in a population from Campinas, southeastern Brazil (n = 292), and to compare their results with other populations. A comprehensive analysis of population-specific genes, genotype, and haplotype frequencies of KIR may facilitate a better understanding of their evolution and role in immunity. The genotyping of 16 KIR genes and HLA class I alleles was performed by the reverse sequence-specific oligonucleotide methodology using the Luminex platform (One Lambda, Inc., Canoga Park, CA). The framework genes were present in all individuals, with the most common non-framework KIR genes detected being KIR2DP1(96.6%), KIR2DL1(95.5%), KIR3DL1(94.5%), KIR2DS4(93.8%) and KIR2DL3(87.3%). KIR2DS1, KIR2DS3, KIR2DS5, and KIR3DS1 presented frequencies below 40%. KIR2DL2, KIR2DL5, and KIR2DS2 showed intermediate frequencies (between53% and 58%). The activating gene KIR2DS5 was the least common in this population (30.8%). Forty-five KIR profiles were found with the commonest being the homozygous A haplotype (27.4%). The distribution of KIR genes in the Brazilian population is similar to Caucasian European and Euro-descendant populations.

Allelic variation of KIR and HLA tunes the cytolytic payload and determines functional hierarchy of NK cell repertoires

AbstractThe functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex–deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.

KIR-HLA interactions extend human CD8+ T cell lifespan in vivo.

BACKGROUNDThere is increasing evidence, in transgenic mice and in vitro, that inhibitory killer cell immunoglobulin-like receptors (iKIRs) can modulate T cell responses. Furthermore, we have previously shown that iKIRs are an important determinant of T cell-mediated control of chronic viral infection and that these results are consistent with an increase in the CD8+ T cell lifespan due to iKIR-ligand interactions. Here, we tested this prediction and investigated whether iKIRs affect T cell lifespan in humans in vivo.METHODSWe used stable isotope labeling with deuterated water to quantify memory CD8+ T cell survival in healthy individuals and patients with chronic viral infections.RESULTSWe showed that an individual's iKIR-ligand genotype was a significant determinant of CD8+ T cell lifespan: in individuals with 2 iKIR-ligand gene pairs, memory CD8+ T cells survived, on average, for 125 days; in individuals with 4 iKIR-ligand gene pairs, the memory CD8+ T cell lifespan doubled to 250 days. Additionally, we showed that this survival advantage was independent of iKIR expression by the T cell of interest and, further, that the iKIR-ligand genotype altered the CD8+ and CD4+ T cell immune aging phenotype.CONCLUSIONSTogether, these data reveal an unexpectedly large effect of iKIR genotype on T cell survival.FUNDINGWellcome Trust; Medical Research Council; EU Horizon 2020; EU FP7; Leukemia and Lymphoma Research; National Institute of Health Research (NIHR) Imperial Biomedical Research Centre; Imperial College Research Fellowship; National Institutes of Health; Jefferiss Trust.

Genotype List String 1.1: Extending the Genotype List String grammar for describing HLA and Killer-cell Immunoglobulin-like Receptor genotypes.

The Genotype List (GL) String grammar for reporting HLA and Killer-cell Immunoglobulin-like Receptor (KIR) genotypes in a text string was described in 2013. Since this initial description, GL Strings have been used to describe HLA and KIR genotypes for more than 40 million subjects, allowing these data to be recorded, stored and transmitted in an easily parsed, text-based format. After a decade of working with HLA and KIR data in GL String format, with advances in HLA and KIR genotyping technologies that have fostered the generation of full-gene sequence data, the need for an extension of the GL String system has become clear. Here, we introduce the new GL String delimiter "?," which addresses the need to describe ambiguity in assigning a gene sequence to gene paralogs. GL Strings that do not include a "?" delimiter continue to be interpreted as originally described. This extension represents version 1.1 of the GL String grammar.

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

Efficient and accurate KIR and HLA genotyping with massively parallel sequencing data.

Killer cell immunoglobulin like receptor (KIR) genes and human leukocyte antigen (HLA) genes play important roles in innate and adaptive immunity. They are highly polymorphic and cannot be genotyped with standard variant calling pipelines. Compared with HLA genes, many KIR genes are similar to each other in sequences and may be absent in the chromosomes. Therefore, although many tools have been developed to genotype HLA genes using common sequencing data, none of them work for KIR genes. Even specialized KIR genotypers could not resolve all the KIR genes. Here we describe T1K, a novel computational method for the efficient and accurate inference of KIR or HLA alleles from RNA-seq, whole-genome sequencing, or whole-exome sequencing data. T1K jointly considers alleles across all genotyped genes, so it can reliably identify present genes and distinguish homologous genes, including the challenging KIR2DL5A/KIR2DL5B genes. This model also benefits HLA genotyping, where T1K achieves high accuracy in benchmarks. Moreover, T1K can call novel single-nucleotide variants and process single-cell data. Applying T1K to tumor single-cell RNA-seq data, we found that KIR2DL4 expression was enriched in tumor-specific CD8+ T cells. T1K may open the opportunity for HLA and KIR genotyping across various sequencing applications.

Comparative genomics of the Leukocyte Receptor Complex in carnivores.

The mammalian Leukocyte Receptor Complex (LRC) chromosomal region may contain gene families for the killer cell immunoglobulin-like receptor (KIR) and/or leukocyte immunoglobulin-like receptor (LILR) collections as well as various framing genes. This complex region is well described in humans, mice, and some domestic animals. Although single KIR genes are known in some Carnivora, their complements of LILR genes remain largely unknown due to obstacles in the assembly of regions of high homology in short-read based genomes. As part of the analysis of felid immunogenomes, this study focuses on the search for LRC genes in reference genomes and the annotation of LILR genes in Felidae. Chromosome-level genomes based on single-molecule long-read sequencing were preferentially sought and compared to representatives of the Carnivora. Seven putatively functional LILR genes were found across the Felidae and in the Californian sea lion, four to five genes in Canidae, and four to nine genes in Mustelidae. They form two lineages, as seen in the Bovidae. The ratio of functional genes for activating LILRs to inhibitory LILRs is slightly in favor of inhibitory genes in the Felidae and the Canidae; the reverse is seen in the Californian sea lion. This ratio is even in all of the Mustelidae except the Eurasian otter, which has a predominance of activating LILRs. Various numbers of LILR pseudogenes were identified. The structure of the LRC is rather conservative in felids and the other Carnivora studied. The LILR sub-region is conserved within the Felidae and has slight differences in the Canidae, but it has taken various evolutionary paths in the Mustelidae. Overall, the process of pseudogenization of LILR genes seems to be more frequent for activating receptors. Phylogenetic analysis found no direct orthologues across the Carnivora which corroborate the rapid evolution of LILRs seen in mammals.

KIR2DS4 and Its Variant KIR1D in KIR-AA Genotype Donors Showed Differential Survival Impact in Patients with Lymphoid Disease after HLA-Matched Unrelated Hematopoietic Stem Cell Transplantation

Previous studies illustrated associations between presence of activating killer cell immunoglobulin-like receptor (KIR) genes and lower susceptibility to hematologic malignancies in humans. In addition, favorable hematopoietic stem cell transplantation (HSCT) outcomes were reported in patients, who received transplants from donors with KIR genotypes dominant for activating KIR receptors. However, the association of activating KIR genes on an allelic level with disease and their impact on HSCT outcome has been only scarcely investigated. To this end, we genotyped a large transplantation cohort for KIR two Ig domains and short cytoplasmic tail 4 (KIR2DS4) polymorphisms and investigated their association with disease. We next investigated the impact of KIR-AA genotype donor KIR2DS4 polymorphisms (AA/KIR2DS4 versus (vs.) AA/KIR1D), on clinical outcome of HSCT in patient subgroups (myeloid vs. lymphoid). Among 2810 transplant pairs, 68.8% (n=1934) were 10/10 human leukocyte antigen (HLA) matched and 31.2% (n=876) were 9/10 HLA-matched. The distribution of KIR one Ig domain (KIR1D) in patients vs. donors was equal (P value = 0.205). Multivariate analysis in 10/10 HLA-matched patients with lymphoid disease showed improved HSCT outcome (overall survival (OS): hazard ratio (HR) 0.62, P=0.002; disease free survival (DFS): HR 0.70, P=0.011; graft-versus-host disease free and relapse-free survival (GRFS): HR 0.67, P=0.002; non-relapse mortality (NRM): HR 0.55, P<0.001) when they received grafts from AA/KIR1D donors. This effect was not seen in either 9/10 HLA-matched patients with lymphoid disease or patients with myeloid disease. Our study indicates that the presence of KIR1D alleles is not associated with disease in patients and interestingly, using grafts from AA/KIR1D donors translated into a beneficial survival outcome in 10/10 HLA-matched patients with lymphoid disease.

CD8 +Helios +T cells: A unique functional T cell subpopulation?

Abstract The zinc finger protein, Helios, is a member of the Ikaros transcription factor family and is expressed in CD4 +T cells, CD8 +T cells and in some NK cells in both mice and humans. While it has been found to have a role in the homeostasis and suppressive function of CD4 +Foxp3 +T regulatory cells (Treg), its function in CD8 +T cells and NK cells is not well characterized. We have demonstrated that ~10–40% of CD8 +T cells from normal donors express Helios. Expression of Helios in CD8 +appears to be downregulated with TCR stimulation alone but is maintained in the presence of excess IL-2 signaling. Thus far, extensive flow cytometry studies have failed to show a correlation between Helios expression and any other surface or intracellular markers. Mouse CD8 +Helios +Ly-49 +T cells have been reported to inhibit the activation of B cells in germinal centers and have been claimed to be the CD8 +counterpart to CD4 +Treg. While these studies have been performed in mice, there is very little data to suggest that human CD8 +Helios +T cells exhibit T suppressor function. In humans, the killer-cell immunoglobulin-like receptors (KIRs), a family of transmembrane glycoproteins, are said to be the functional equivalent of the Ly49 proteins. KIR +CD8 +T cells have previously been said to highly express Helios and have a function in autoimmune conditions. Data we have obtained from a CITE-seq analysis has shown that Heliosexpression in CD8s is highly correlated with many NK-cell associated markers. This, along with data showing that these cells produce cytotoxic effector molecules, leads us to speculate that these cells have a role in immune suppression via cytotoxic killing of self-activated immune cells. This work was supported by the Intramural Research Program of NIAID, NIH. This work was supported by the Intramural Research Program of NIAID, NIH.

Granzymes, IL-16, and poly(ADP-ribose) polymerase 1 increase during wildfire smoke exposure.

Given the increasing prevalence of wildfires worldwide, understanding the effects of wildfire air pollutants on human health-particularly in specific immunologic pathways-is crucial. Exposure to air pollutants is associated with cardiorespiratory disease; however, immune and epithelial barrier alterations require further investigation. We sought to determine the impact of wildfire smoke exposure on the immune system and epithelial barriers by using proteomics and immune cell phenotyping. A San Francisco Bay area cohort (n = 15; age 30 ± 10 years) provided blood samples before (October 2019 to March 2020; air quality index = 37) and during (August 2020; air quality index = 80) a major wildfire. Exposure samples were collected 11 days (range, 10-12 days) after continuous exposure to wildfire smoke. We determined alterations in 506 proteins, including zonulin family peptide (ZFP); immune cell phenotypes by cytometry by time of flight (CyTOF); and their interrelationship using a correlation matrix. Targeted proteomic analyses (n = 15) revealed a decrease of spondin-2 and an increase of granzymes A, B, and H, killer cell immunoglobulin-like receptor 3DL1, IL-16, nibrin, poly(ADP-ribose) polymerase 1, C1q TNF-related protein, fibroblast growth factor 19, and von Willebrand factor after 11 days' average continuous exposure to smoke from a large wildfire (P < .05). We also observed a large correlation cluster between immune regulation pathways (IL-16, granzymes A, B, and H, and killer cell immunoglobulin-like receptor 3DL1), DNA repair [poly(ADP-ribose) 1, nibrin], and natural killer cells. We did not observe changes in ZFP levels suggesting a change in epithelial barriers. However, ZFP was associated with immune cell phenotypes (naive CD4+, TH2 cells). We observed functional changes in critical immune cells and their proteins during wildfire smoke exposure. Future studies in larger cohorts or in firefighters exposed to wildfire smoke should further assess immune changes and intervention targets.

Refining eligibility criteria of unit selection for myeloablative cord blood transplantation in acute leukemia: Real-world experience of a referral center.

The algorithm for cord blood (CB) unit selection is still somewhat ambiguous. We retrospectively analyzed 620 cases of acute leukemia between 2015 and 2020, who were treated with myeloablative single-unit umbilical CB transplantation (UCBT). We found that, when human leukocyte antigen (HLA) mismatch was ≤3/10, CD34+ cell dosage<0.83 × 105/kg-considerably lower than prevalent guidelines-was permissible without affecting survival. Moreover, synergy between donor killer-cell immunoglobulin-like receptors (KIR) haplotypes-B and donor-recipient HLA-C mismatch protected against relapse-related mortality. We submit that minimum required CD34+ cell dosage can possibly be relaxed to broaden access to UCBT, and donor KIR genotyping should be considered during unit selection.

Abstract 887: Killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2) immune checkpoint modulates CAR-T cell effector function

Abstract Significance: A modulatory role of killer immunoglobulin-like receptor 2DL2 (KIR2DL2) during T cell receptor (TCR) late signaling events has been proposed. However, its role in CAR-T cell effector function has not yet been studied. Our work aims to understand the differential modulatory effect of KIR2DL2 immune checkpoint on the effector function of therapeutic T cells used for the treatment of liquid and solid tumors. Methods: We generated CAR-T cells, with or without KIR2DL2 overexpression, using a bicistronic retroviral vector based on the MSGV1 backbone. HLA-C1-deficient tumor cells (HLA-C1−/−) were generated by CRISPR/Cas-based knockout of the B2M gene. We designed gRNAs targeting KIR2DL2, validated their cleavage efficiency and proved the feasibility to abrogate its expression in primary T cells. We tested the inhibitory role of KIR2DL2 in vitro in HLA-C1+ or HLA-C1−/− tumor cells using impedance- and bioluminescence-based cytotoxic assays at different effector:target ratios. We evaluated cytokine production after coculture of CAR-T and target cells using the ELLA system. To assess the modulatory role of KIR2DL2 in vivo, we generated HLA-C1+ or HLA-C1− subcutaneous xenografts of human pancreatic adenocarcinoma (HPAC) cells, then treated the mice with KIR2DL2+ or KIR2DL2− PSCA-CAR-T cells. We assessed the effect of KIR2DL2 in a lymphoblastic leukemia (ALL) xenograft model (NALM6 cells), treated with either KIR2DL2+ or KIR2DL2-deficient CD19-CAR-T cells (i.v.). Results: In vitro, KIR2DL2+ CAR-T cells were significantly less cytotoxic in an HLA-C1-dependent manner, regardless of their target antigen. Cytokine analysis after coculture showed that overexpression of KIR2DL2 in CAR-T cells impaired their ability to produce IFN-γ, IL-2 and TNF-α in presence of HLA-C1. In vivo, KIR2DL2-overexpressing PSCA-CAR-T cells injected in NSG mice harboring PSCA+/HLA-C1+ pancreatic tumors were not able to eliminate tumors. In contrast, KIR2DL2-overexpressing CAR-T cells transferred to mice harboring PSCA+/HLA-C1−/− tumor cells performed as well as their KIR2DL2− counterparts. Two gRNAs targeting KIR2DL2 were validated in primary T cells by means of an enzymatic cleavage assay and flow cytometry. Current efforts are focused on characterizing the impact of KIR2DL2 ablation on the anti-leukemia activity of CAR-T cells in vivo. Conclusions: KIR2DL2 impairs CAR-T cell effector function and cytokine secretion in vitro in both pancreatic and ALL tumor models, and in vivo in the pancreatic model. We implemented abrogation of KIR2DL2 by CRISPR/Cas9 during CD19-CAR-T cell manufacturing. Ongoing efforts focus on testing the impact of KIR2DL2 ablation on the in vivo performance of CAR-T cells. These results strengthen the notion of KIR2DL2 as an immune checkpoint with a relevant role in T cell immunosurveillance and may constitute an actionable target to enhance adoptive cell therapies. Citation Format: Miguel Gomez Fontela, Sebastian Snedal, Elena Martinez Planes, Daniel Abate-Daga. Killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2) immune checkpoint modulates CAR-T cell effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 887.

KIR genotype and haplotype repertoire in Kuwaiti healthy donors, hematopoietic cell transplant recipients and healthy family members.

The gene complex located on chromosome 19q13.4 encodes the Killer-cell Immunoglobulin-like Receptors (KIRs), which exhibit remarkable polymorphism in both gene content and sequences. Further, the repertoire of KIR genes varies within and between populations, creating a diverse pool of KIR genotypes. This study was carried out to characterize KIR genotypes and haplotypes among 379 Arab Kuwaiti individuals including 60 subjects from 20 trio families, 49 hematopoietic cell transplantation (HCT) recipients and 270 healthy Kuwaiti volunteer HCT donors. KIR Genotyping was performed by a combination of reverse sequence specific oligonucleotide probes (rSSO) and/or Real Time PCR. The frequencies of KIR genes in 270 healthy Kuwaiti volunteer donors were compared to previously reported frequencies in other populations. In addition, we compared the differences in KIR repertoire of patients and healthy donors to investigate the reproducibility of previously reported significant differences between patients with hematological malignancies and healthy donors. The observed frequencies in our cohort volunteer HCT donors was comparable to those reported in neighboring Arab populations. The activating genes KIR2DS1, KIR2DS5 and KIR3DS1 and the inhibitory gene KIR2DL5 were significantly more frequent in patients compared to healthy donors, however, none of the previously reported differences were reproducible in our Kuwaiti cohort. This report is the first description of KIR gene carrier frequency and haplotype characterization in a fairly large cohort of the Kuwaiti population, which may have implications in KIR based HCT donor selection strategies.

Presence of KIR2DL2/S2, KIR2DL5, and KIR3DL1 Molecules in Liver Transplant Recipients with Alcoholic Cirrhosis Could Be Implicated in Death by Graft Failure

Background: The second-most frequent diagnosis among patients receiving liver transplants (LTs) is alcoholic liver disease. The multifactorial pathophysiology of alcoholic liver disease depends on the innate immune system and the inflammatory cascade. According to recent studies on these receptors, killer-cell immunoglobulin-like receptors (KIRs) may be involved in sepsis, liver rejection, and virus relapse. We aimed to investigate the impact of preclinical issues like ascites and encephalopathy and KIR genetic traits on death from sepsis, multiorgan failure (MF), and graft failure (GF) in AC patients undergoing LTs. Methods: We retrospectively reviewed 164 consecutive and deceased Caucasian AC patients who underwent LTs. Pre-transplant complications, cause of death, and patient survival were analyzed. Genomic DNA was taken from peripheral blood, and PCR-SSO was used for genotyping KIR. Results: Compared to GF patients, there was a statistically significant increase in the frequency of KIR2DL2+ (75.8% vs. 51.2%; p = 0.047). Another increase in frequency was also observed in KIR2DS2+ in sepsis compared to the GF group (51.2% vs. 43.7%; p = 0.018). In patients who passed away from MF, a decrease in KIR2DL5+ was observed in AC patients with and without encephalopathy (p = 0.018). The frequency of KIR3DL1+ in the AC patients significantly increased the mortality from sepsis (p = 0.045), which was confirmed by multivariate logistic regression. The frequency of KIR3DL1+ in the AC patients significantly increased the mortality from sepsis (p = 0.012) and was confirmed by multivariate logistic regression. KIR2DS1+ and KIR2DS4+ showed increased mortality due to GF compared to patients without these genes (p = 0.011 and 0.012, respectively). However, this fact was confirmed only for KIR2DS1+ by multivariate logistic Cox regression. Conclusions: The presence of the KIR2DL2/S2+, KIR2DL5+, and KIR3DL1+ genes increases the frequency of death from multiple organ failure or graft failure. Our findings highlight the AC patient's vulnerability to a LT during hospitalization. Following the transplant and outside of it, we adopt essential preventive measures to create a routine healthcare screening to enhance and modify treatments to increase survival.