Inhibitory Killer Cell Immunoglobulin-like Receptors Research Articles

KIR-генетические факторы и ответ на терапию ингибиторами тирозинкиназ при хроническом миелоидном лейкозе

The development of tyrosine kinase inhibitors (TKIs) and their introduction into clinical practice considerably improved the prognosis for chronic myeloid leukemia (CML) patients. About 50 % of patients with achieved deep molecular response are eligible for safe TKI discontinuation. Despite these advances, no reliable biomarkers are known to predict a response and sustaining treatment-free remission after TKI withdrawal. As TKIs do not destroy leukemic stem cells, which can be responsible for relapse, critical importance in CML is attached to natural killers (NK-cells) having antitumor activity. Functional activity of NK-cells is evaluated by expression level and repertoire of killer cell immunoglobulin-like receptors (KIR). Current studies demonstrate that a patient’s KIR genotype affects the probability of achieving early and deep molecular responses to first- and second-generation TKIs, progression-free and overall survivals, and sustaining treatment-free remission. On that ground, KIR-genetic factors can be regarded as promising predictors of response to TKI therapy in CML. Early clinical studies, which dealt with monoclonal antibodies blocking the inhibitory KIR in order to increase NK-cell activity, revealed an acceptable safety profile and efficacy in some hematological diseases (such as acute myeloid leukemia, multiple myeloma, Т-cell lymphoma) if used in combination with cytostatic drugs or antitumor monoclonal antibodies. KIR genotype determination can contribute to the development of effective therapies of this malignant hematological tumor.

Targeting KIR as a novel approach to improve CAR-NK cell function.

Chimeric antigen receptor (CAR) NK cells are demonstrating promising activity in clinical trials and possess a favorable safety profile compared to CAR-T cells. The Killer cell Immunoglobulin-like Receptors (KIR) have a critical role in the control of NK cell function, and recently, this family of activating and inhibitory receptors have been targeted to improve CAR-NK function. These strategies include the utilisation of inhibitory KIR to reduce trogocytosis-associated NK cell fratricide, the downregulation of inhibitory KIR on CAR-NK cells to alleviate HLA mediated suppression, the selection of CAR-NK cell donors enriched for activating KIR, and the use of activating KIR intracellular domains within novel CAR constructs. These pre-clinical studies demonstrate the potential utility of targeting the KIR to improve CAR-NK cell efficacy and patient outcomes.

Killer cell immunoglobulin-like receptor alleles influence susceptibility to occult hepatitis B infection in West African population.

Occult hepatitis B infection (OBI) is a public health problem in Burkina Faso. OBI represents a risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). OBI could be due to mutant viruses undetectable by HBsAg assays or a strong suppression of viral replication and gene expression under the pression of the host immune system. To investigate the role of killer cell immunoglobulin-like receptor (KIR) gene polymorphisms in patients with OBI in Burkina Faso compared to healthy and chronic hepatitis B subjects. A total of 286 participants was recruited, including 42 cases of OBI, 110 cases of chronic hepatitis B and 134 HBV negative subjects. SSP-PCR was performed to search for the presence of KIR genes. The HBV viral load was determined by qPCR. The frequencies of the activator gene KIR2DS5 (P=0.045) and the pseudogene KIR2DP1 (P<0.001) in patients with OBI were higher than those in patients with chronic hepatitis B. These genes are associated with susceptibility of occult hepatitis B infection. The frequencies of the inhibitory KIR gene KIR2DL3 (P=0.01) of patients with occult hepatitis B were lower than those in chronic hepatitis B patients. This gene KIR2DL3 is associated with protection against occult hepatitis B infection. Also, the frequencies of the inhibitory KIR genes KIR2DL2 (P<0.001), KIR2DL3 (P<0.001) and activators KIR2DS2 (P<0.001) in chronic hepatitis B patients were higher compared to the frequencies of the KIR genes in healthy subjects. These genes KIR2DL3, KIR2DL5 (A, B), KIR3DL3, KIR3DS1, KIR2DL2 and KIR2DS2 are thought to be genes associated with the susceptibility to OBI. The KIR2DS5 and KIR2DP1 genes could be associated with susceptibility to OBI. As for the KIR gene KIR2DL3 could be associated with protection against occult hepatitis B infection.

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.

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.

KIR signaling is regulated by electrostatic interaction of its cytosolic tail with the plasma membrane despite being neutral polyampholyte.

Many receptors signal upon phosphorylation of tyrosine-based motifs present in their cytosolic tail. Intrinsic disorder appears common among such cytosolic tails. Studies on CD3ζ and CD3ε tails, which are disordered and polybasic, suggested regulation of phosphorylation through accessibility of tyrosines, governed by electrostatic interactions with membrane anionic lipids. We noticed characteristics of intrinsic disorder and previously unappreciated features in tyrosine-based motif-bearing cytosolic tails of, especially, inhibitory receptors. These tails are neutral or acidic polyampholytes, with acidic and basic residues linearly segregated. To explore roles of these electrostatic features, we studied inhibitory killer-cell immunoglobulin-like receptor (KIR). KIR cytosolic tail is disordered neutrally-charged polyampholyte, wherein juxtamembrane and membrane-distal stretches are basic, and the intervening stretch is acidic. Despite lacking net charge, it interacted electrostatically with the plasma membrane. Juxtamembrane stretch was crucial for overall binding, which sequestered tyrosines in membrane, and restrained their constitutive phosphorylation. Ligand binding to KIR was sufficient to release its tail from the plasma membrane, initiating signaling. This tail release occurred independently of KIR polymerization, clustering, and tyrosine phosphorylation, but required the acidic stretch. Signaling strength of KIR was dictated by plasma membrane interaction of its tail. These results revealed an electrostatic protein-lipid interaction that is unusual in being governed by segregated clusters of acidic and basic residues in disordered region of protein. Allowing electrostatic interaction irrespective of net charge, segregated clusters of oppositely charged residues made both binding and unbinding modules inherent to receptor tail, which could enable the interaction to act as an independent signaling switch.

Association of Killer Cell Immunoglobulin-Like Receptors and Their HLA-Ligands with Type 1 Diabetes Among South Indian Population

ABSTRACT Background Type 1 diabetes (T1D) is a multifactorial autoimmune disease, involving strong genetic components with familial predisposition. Killer cell immunoglobulin-like receptors (KIRs) found on the surface of NK cells have ligands of human leukocyte antigen (HLA) class I that are associated with T1D. The present study evaluates the influence of KIR genes and their HLA-ligands in the aetiology of T1D among the South Indian population. Methods A total of 125 T1D patients, along with their parents (n = 126) and siblings (n = 52) were recruited. PCR-based genotyping was performed for KIR genes and HLA class I ligands. The gene frequencies were compared between patients and siblings/parents. Linkage-disequilibrium (LD) analysis was performed to assess the genetic association between KIR gene pairs. Results The results show significant differences in HLA-ligands of KIR genes between patients and parents. The HLA-C1C1 homozygosity was found to be a predisposing risk factor, and HLA-C1C2 heterozygosity was protective towards T1D along with either the activating KIR2DS2 or inhibitory KIRs 2DL1, 2DL2, 2DL3. However, the frequency of inhibitory KIR3DL1 significantly increased in the presence of HLA-B Bw4 Ile80 in parents when compared to patients showing a protective effect on T1D. Two pairs of KIR genes, 2DS4-3DL1 and 2DS1-2DL5, showed strong LD in patients, siblings and parents. Conclusion The KIR-HLA ligand combinations have a significant effect on T1D aetiology among the South Indian population. This study defines a pattern for family-based association studies with genotypic information about KIR genes and their HLA-ligands, providing the first evidence towards T1D among the South Indian population.

The impact of killer cell immunoglobulin-like receptor (KIR) genes and human leukocyte antigen (HLA) class I ligands on predisposition or protection against prostate cancer

Natural killer (NK) cell development largely depends on killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I ligands. In the current study, we investigated the role of KIR genes, HLA ligands, and KIR-HLA combinations in vulnerability or protection against prostate cancer (PC). To analyze the frequency of 16 KIR genes and 5 HLA ligands, polymerase chain reaction with sequence-specific primers (PCR-SSP) was conducted in 150 PC patients and 200 healthy controls (CNs). KIR2DL5 (p = 0.0346, OR = 0.606, CI = 0.3916–0.9336), KIR2DS5 (p = 0.0227, OR = 0.587, CI = 0.3793–0.9139), HLA-B Bw4Thr80 (p = 0.0401, OR = 0.3552, CI = 0.1466–0.9059), HLA Bw4 (p = 0.0190, OR = 0.4744, CI = 0.2656–0.8521), and T4 gene cluster (including KIR2DS5-2DL5-3DS1-2DS1 genes) (p = 0.0194, OR = 0.5575, CI = 0.3449–0.8938) had a lower frequency in the PC patients compared to the control group. Moreover, a lower frequency of the genotypes contacting activating KIR (aKIR) > inhibitory KIR (iKIR) (p = 0.0298, OR = 0.5291, CI = 0.3056–0.9174) and iKIR + HLA < aKIR + HLA (p = 0.0183, OR = 0.2197, CI = 0.0672–0.7001) in PC patients compared to the CNs implies a protective role for aKIR genes. In the case of KIR-HLA interactions, we detected a significant association between KIR3DS1+ + HLA-A Bw4+ (p = 0.0113, OR = 0.5093, CI = 0.3124–0.8416) and KIR3DL1− + HLA-A Bw4+ (p = 0.0306, OR = 0.1153, CI = 0.0106–0.6537) combinations and resistance to prostate cancer. In contrast, the presence of KIR3DL1 in the absence of HLA-A Bw4 (p = 0.0040, OR = 2.00, CI = 1.264–3.111), HLA Bw4 (p = 0.0296, OR = 2.066, CI = 1.094–3.906), and HLA-Bw4Thr80 (p = 0.0071, OR = 2.505, CI = 1.319–4.703) genes probably predisposes to prostate cancer. Carrying the CxT4 genotype in PC patients was positively associated with lower tumor grades (Gleason score ≤ 6) (p = 0.0331, OR = 3.290, and CI = 1.181–8.395). Altogether, our data suggest a protective role for aKIRs, HLA-B Bw4Thr80, and HLA Bw4 ligands as well as a predisposing role for certain KIR-HLA combinations in prostate cancer. The findings of this study offer new insight into the population's risk assessment for prostate cancer in men. Additionally, predicting immunotherapy response based on KIR-HLA combinations aids in implementing the most effective therapeutic approach in the early stages of the disease.

KIR signaling is regulated by electrostatic interaction of its cytosolic tail with the plasma membrane despite being neutral polyampholyte

Many receptors signal upon phosphorylation of tyrosine-based motifs in their cytosolic tail, with intrinsic disorder as a common feature. Studies on CD3ζ and CD3ε tails, which are disordered and polybasic, suggested regulation of phosphorylation through accessibility of tyrosines, governed by electrostatic interactions with membrane anionic lipids. We noticed characteristics of intrinsic disorder and previously unappreciated features in tyrosine-based motif-bearing cytosolic tails of many, especially, inhibitory receptors. They are neutral or acidic polyampholytes, with acidic and basic residues linearly segregated. To explore roles of these electrostatic features, we studied inhibitory killer-cell immunoglobulin-like receptor (KIR). Its cytosolic tail is a disordered neutrally charged polyampholyte, wherein juxtamembrane and membrane distal stretches are basic, and the intervening stretch is acidic. Despite lacking net charge, it interacted electrostatically with the plasma membrane. The juxtamembrane stretch was crucial for overall binding, which sequestered tyrosines in the lipid bilayer and restrained their constitutive phosphorylation. Human leukocyte antigen-C ligand binding to KIR released its tail from the plasma membrane to initiate signaling. Tail release occurred independently of KIR polymerization, clustering, or tyrosine phosphorylation, but required acidic residues of the acidic stretch. Tail interaction with the plasma membrane dictated signaling strength of KIR. These results revealed an electrostatic protein-lipid interaction that is unusual in being governed by segregated clusters of acidic and basic residues in polyampholytic disordered region of protein. In contrast to previously known, segregated distribution of oppositely charged residues made both binding and unbinding modules inherent to receptor tail, which could make the interaction an independent signaling switch.

Killer Cell Immunoglobulin-like Receptor Profile in Acute Myeloid Leukemia with Heterogenous Cytogenetic Abnormalities and Its Association with Clinical Outcome

Introduction: Acute myeloid leukemia (AML) is associated with heterogeneous cytogenetic abnormalities which contributes towards biology and prognosis of the disease. Natural killer (NK) cells are the first line of defense against tumor cells. NK cells interact with AML cells via multiple receptors including killer cell immunoglobulin-like receptor (KIR). Evidence of susceptibility of AML cells to NK cells in the context of KIR-ligand mismatch NK immunotherapy has suggested a role of KIR repertoire in the development and clinical outcome of AML. The aim of this study is to determine the KIR profile of AML patients with different cytogenetic abnormalities and to evaluate its association with clinical outcome. Methods: Peripheral blood was collected from patients diagnosed with AML after informed consent in accordance with Declaration of Helsinki and approved from Ethical Review Board of Aga Khan University. Peripheral blood mononuclear cells were isolated and subjected to genomic DNA extraction. KIR genotyping was performed by polymerase chain reaction using sequence-specific primers (Vilches et al, Tissue Antigens 2007). Relationship between KIR loci in AML and healthy individuals was compared using chi-square test. Statistical analysis was performed using SPSS statistics (version 26) and a p value of <0.05 was considered statistically significant. Results: KIR genotyping was performed on 35 cases of AML with mean age of 39 years (range:16-58). Number of male and female patients was 25 and 10 respectively. We found 3 (8.57%) patients with AA KIR haplotype while 33 (91.42%) patients showed Bx haplotype (Figure 1). All the patients with AA haplotype harbored t(15;17) (q22; q21) which has a favorable prognosis while patients with Bx KIR haplotype showed cytogenetic abnormalities with favorable, intermediate, and adverse prognosis. The B-content score of patients with Bx KIR haplotype (n=32) was scored from 1 to 4 based on the total sum of B loci (Cooley et al, Blood 2010). We found that majority of cases had a B-content score of 2 (53.12%) while 7 (21.87%), 6(18.75%) and 2 (6.25%) patients had a score of 1, 3 and 4 respectively. Cytogenetic profile associated with favorable, intermediate, and adverse prognostic risk was found in all groups of patients irrespective of B-content score (Table 1). A standard induction treatment was given to 26 patients with Bx KIR haplotype. Around 67% of patients with B-content score of 1 to 3 showed complete remission (CR) while 100% of patients with B-content score of 4 showed CR after first induction chemotherapy. Estimated KIR gene frequency (gf) in these AML patients was compared with 78 normal individuals recruited from Aga Khan University in Karachi (Norman et al, Gene and Immunity 2002). We found no difference in gf of KIR locus 2DL3 (0.55 vs 0.70), 3DL1 (0.75 vs 0.56), 2DL2 (0.52 vs 0.42), 2DL5 (0.42 vs 0.53), 2DS1 (0.44 vs 0.31), 2DS2 (0.42 vs 0.31),2DS5 (0.28 vs 0.28) and 3DS1 (0.28 vs 0.34) in AML cases vs normal individuals. However, we found significant difference in gene frequency of KIR2DL1 (0.42 vs 0.68) [p=0.002], 2DS4 (0.75 vs 0.47) [p=0.007] and 2DS3 (0.07 vs 0.26) [p=0.002] in AML cases compared to healthy individuals. Conclusion: We found that the majority of patients diagnosed with AML harboring heterogenous cytogenetic abnormalities exhibit a Bx KIR haplotype with B-content score of 2. We did not find any specific cytogenetic abnormality to be associated with a particular B-content score. As we look at our data closely, it seems that it is the cytogenetic abnormality and not the B-content score that dictates the outcome after first induction chemotherapy. We found that patients with AML have decrease in gene frequency of 2DL1 (inhibitory KIR) and 2DS3 (activating KIR) while an increase in gene frequency of 2DS4 (activating KIR) compared to healthy individuals. Further studies are required to determine if variable gene frequency of these 3 KIR loci contributes towards the development of AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Haploidentical Hematopoietic Stem Cell Transplant Plus Add-Back NK Cells in Pediatric High-Risk ALL

Introduction: Natural killer (NK) lymphocytes are cells of innate immunity that protect against tumor and infections. NK cells recognize self and non-self HLA class I ligands, are regulated by a balance between activator and inhibitory signals through surface receptors, and can interact with HLA class I molecules on target cells. Killer-cell Immunoglobulin-like Receptors (KIRs) are inhibitory molecules expressed on NK cells and belong to the immunoglobulin superfamily. KIRs function to "educate" NK cells in effective defense against unhealthy self/non-self cells and tolerance against healthy self cells. NKs are thought to play a key role in preventing early relapse of hematologic malignancies, virus infection and graft versus host disease (GvHD) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT).In B-/T-cell alpha/beta depleted haplo-HSCT, donor-versus-recipient allo-reactive NK cells can exert an efficient graft versus leukemia (GvL) effect, mediated by inhibitory KIRs where the HLA ligand is missing on recipient cells. Donor NK cells after haplo-HSCT are "immature" and not effective as the mature NKs. These immature NK cells are CD56bright, are derived from CD34+ stem cells, and mature to a CD56dim phenotype. The mature CD56dim NK cells are characterized by acquisition of KIRs, CD16 and CD57 in mature stages, which have functional significance in targeting and medicating their cytolytic activity. Based on these considerations, we performed haplo-HSCT with donor NK cell add-back to provide mature, functional immunological surveillance against tumor (MRD) and virus reactivation, which can frequently occur in manipulated haplo-HSCT for pediatric acute lymphoblastic leukemia (ALL). Methods: We evaluated the alloreactive effect of donor-derived NK cells in three pediatric high-risk ALL who underwent B-/T-cell alpha/beta depleted haplo-HSCT followed by donor NK infusion. Patients' characteristics are summarized in the Table. All 3 patients were male; median age was 13 months. Patients 1 and 2 had infant ALL, and patient 3 had refractory-relapsed (r/r) ALL. All patients received a median of 3 lines chemotherapy. Patient 1 received a prior allo-HSCT. Patients 1 and 2 received chemo-based conditioning regimens consisting of thyotepa, and fludarabine; In addition, patient1received treosulfan, and patient 2 received busulfan. Treosulfan was given to patient 1, due to less toxicity profile given prior allo-HSCT. Patient 3 received Total Body Irradiation (TBI), thyotepa, and fludarabine. All patients received rituximab and letermovirfor EBV and CMV prophylaxis, respectively, for all patients. Mother was Donor for all patients. Median CD34+, CD19+, CD3+-alpha/beta cell dose was 8.8, 0.05, 0.41 x 106/kg, respectively. NK cell allo-reactivity was tested by evaluating KIR genotyping and KIR/KIR-ligand mismatch in graft-versus-host direction. All three patients present KIR/KIR-ligand mismatch. Results: Engraftment was day +13, +14 and +19 for ANC, and +14, +18 and +17 for PLT (for both >20K and >50K), for patients 1, 2 and 3, respectively. NK cell number were monitored on day +7, +14, +21 and +28 (data pending). Patients received NK cell infusion on day 30 +/- 2 days. No infusion reactions were observed. MedianNK infused were 256x106/kg. None of the patients developed CMV, EBV or ADV reactivation. Patient 2 developed Grade 2 acute GvHD (skin) treated with steroids. No Grade GvHD 3-4 was observed. Median follow up was 58 days. All patients had full donor chimerism at day 30 ±2. All patients are currently in hematological CR; patient 1 converted to positive MRD at nine months from transplant. Conclusion: Alloreactive NK cell add-back infusion after B-/T-cell alpha/beta depleted haplo-HSCT could represent an effective option to improve transplant outcome and prevent virus reactivation. These NK cells may improve efficacy by enhancing GvL and could contribute to maintain the "disease control" through immunologic surveillance in high-risk ALL. Indeed, NK killing, which is higher in mature NK cells, depends on acquisition of sequential phenotypic maturation. Thus, infusion of mature donor NK cells may help to maintain control of MRD and virus reactivation through immunologic surveillance. Although our preliminary observations seem to be promising, this hypothesis should be confirmed in clinical trial of a larger number of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Donor selection for KIR alloreactivity is associated with superior survival in haploidentical transplant with PTCy.

With the continuous increase in the use of haploidentical donors for transplantation, the selection of donors becomes increasingly important. Haploidentical donors have been selected primarily based on clinical characteristics, while the effects of killer cell immunoglobulin-like receptors (KIRs) on outcomes of haploidentical-hematopoietic stem cell transplantation (haplo-HSCT) with post-transplant cyclophosphamide (PTCy) remain inconclusive. The present study aimed to thoroughly evaluate the effect of KIRs and binding ligands assessed by various models, in addition to other patient/donor variables, on clinical outcomes in haplo-HSCT. In a cohort of 354 patients undergoing their first haplo-HSCT, we found that a higher Count Functional inhibitory KIR score (CF-iKIR) was associated with improved progression-free survival (adjusted hazard ratio [HR], 0.71; P = .029) and overall survival (OS) (HR, 0.66; P = .016), while none of the other models predicted for survival in these patients. Moreover, using exploratory classification and regression tree analysis, we found that donor age <58 years combined with cytomegalovirus-nonreactive recipient was associated with the best OS, whereas donor age >58 years was associated with the worst OS. In the rest of our cohort (80%), cytomegalovirus-reactive recipients with a donor <58 years old, a higher CF-iKIR was associated with superior OS. The 3-year OS rates were 73.9%, 54.1% (HR, 1.84; P = .044), 44.5% (HR, 2.01; P = .003), and 18.5% (HR, 5.44; P <.001) in the best, better, poor, and worse donor groups, respectively. Our results suggest that KIR alloreactivity assessed by CF-iKIR score can help optimize donor selection in haplo-HSCT.

Selective Expansion of NKG2C+ Adaptive NK Cells Using K562 Cells Expressing HLA-E.

Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.

Blockade of inhibitory killer cell immunoglobulin-like receptors and IL-2 triggering reverses the functional hypoactivity of tumor-derived NK-cells in glioblastomas

Killer cell immunoglobulin-like receptors (KIRs) comprise a group of highly polymorphic inhibitory receptors which are specific for classical HLA class-I molecules. Peripheral blood and freshly prepared tumor cell suspensions (n = 60) as well as control samples (n = 32) were investigated for the distribution, phenotype, and functional relevance of CD158ab/KIR2DL1,-2/3 expressing NK-cells in glioblastoma (GBM) patients. We found that GBM were scarcely infiltrated by NK-cells that preferentially expressed CD158ab/KIR2DL1,-2/3 as inhibitory receptors, displayed reduced levels of the activating receptors CD335/NKp46, CD226/DNAM-1, CD159c/NKG2C, and showed diminished capacity to produce IFN-γ and perforin. Functional hypoactivity of GBM-derived NK-cells persisted despite IL-2 preactivation. Blockade with a specific KIR2DL-1,2/3 monoclonal antibody reversed NK-cell inhibition and significantly enhanced degranulation and IFN-γ production of IL-2 preactivated NK-cells in the presence of primary GBM cells and HLA-C expressing but not HLA class-I deficient K562 cells. Additional analysis revealed that significant amounts of IL-2 could be produced by tumor-derived CD4+ and CD8+CD45RA- memory T-cells after combined anti-CD3/anti-CD28 stimulation. Our data indicate that both blockade of inhibitory KIR and IL-2 triggering of tumor-derived NK-cells are necessary to enhance NK-cell responsiveness in GBM.

KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19.

In this work, we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8+ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

Inhibitory KIR2DL2 receptor and HHV-8 in classic or endemic Kaposi sarcoma

KIR2DL2, an inhibitory Killer cell Immunoglobulin-like Receptor (KIR), has been shown to predispose to the development of several herpesvirus-associated diseases by inhibiting the efficiency of Natural Killer (NK) cells against virus-infected cells. The aim of this observational study was to assess the prevalence of KIR2DL2 and Human Herpes Virus 8 (HHV8) in patients affected with classical and endemic Kaposi sarcoma (KS), as well as in controls. Blood samples collected from 17 Caucasian, HIV-negative, immunocompetent patients affected with classical KS (c-KS), 12 African, HIV-negative patients with endemic KS (e-KS), 83 healthy subjects and 26 psoriatic patients were processed for genotypization by PCR for two KIR alleles, such as KIR2DL2 and KIR2DL3 and analyzed for HHV-8 presence. The totality of both c-KS and e-KS patients presented HHV-8 infection, whereas HHV8 was found in 26.9% of psoriatic subjects and 19.3% of healthy subjects. KIR2DL2 was found in the 76.5% of c-KS subjects, while the receptor was found in 41.7% of the e-KS group, 34.6% of psoriatic patients and 43.4% of healthy controls (p < 0.0001). A significantly higher prevalence of KIR2DL2 in c-KS patients than in all the other subjects was also confirmed comparing age-matched groups. Based on these results, the inhibitory KIR2DL2 genotype appears to be a possible cofactor which increases the risk of developing c-KS in HHV8-positive, immunocompetent subjects, while it seems less relevant in e-KS pathogenesis.

Activating receptor KIR2DS2 bound to HLA-C1 reveals the novel recognition features of activating receptor.

Killer cell immunoglobulin-like receptors (KIRs) are important receptors for regulating the killing of virus-infected or cancer cells of natural killer (NK) cells. KIR2DS2 can recognize peptides derived from hepatitis C virus (HCV) or global flaviviruses (such as dengue and Zika) presented by HLA-C*0102 to activate NK cells, and has shown promising results when used for cancer immunotherapy. Here, we present the complex structure of KIR2DS2 with HLA-C*0102 at a resolution of 2·5Å. Our structure reveals that KIR2DS2 can bind with HLA-C*0102 and HLA-A*1101 in two different directions. Moreover, Tyr45 (in activating receptor KIR2DS2) and Phe45 (in inhibitory KIRs) distinguish the two different binding models and binding affinity between activating KIRs and inhibitory KIRs. The conserved 'AT' motif of the peptide mediates recognition and determines the peptide specificity of recognition. These structural characteristics shed light on how KIRs activate NK cells and can provide a molecular basis for immunotherapy by NK cells.

A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors

While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed via surface plasmon resonance, and removal of heparan sulfate proteoglycans from cell surfaces abolished KIR3DS1-Fc binding. Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain (KIR3DS1, KIR3DL1, KIR3DL2, KIR2DL4, and KIR2DL5) bound to heparan sulfate, while those without a D0 domain (KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4) did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition.

Recipient HLA-B Leader Genotype, and Its Relationship to Total KIR Missing Ligand, Informs Relapse and Survival Following Haploidentical Transplantation Using Post-Transplant Cyclophosphamide

▪In addition to donor T cells, natural killer (NK) cells are proposed to play a significant role in the graft-versus-leukemia (GVL) effect following haploidentical donor transplantation (HIDT). Following HIDT, donor NK cells express activating (NKG2C) receptors (Ciurea, Leukemia 2021), whose ligand is the non-classical human leukocyte antigen, HLA-E. For surface expression, HLA-E requires binding of leader peptides derived from class I HLA molecules. The rs1050458C/T dimorphism at position -21 of exon 1 of HLA-B gives rise to leader peptides with either methionine (M) or threonine (T) at the second residue of the processed leader peptide. M-containing HLA-B leader peptides promote higher HLA-E expression than T-leaders, potentially favoring more robust natural NK cell recognition of HLA-E-expressing tumor cells. Alternatively, donor NK cells can be activated through inhibitory killer cell immunoglobulin-like receptors (iKIR: 2DL1, 2DL23, 3DL1, 3DL2), if they fail to engage a corresponding class I HLA ligand on recipient leukemia cells (missing ligand (ML) paradigm). We hypothesized that M-containing B-leaders (either MM or MT genotype), and potentially its association with KIR ML, may inform relapse and survival after HIDT using post-transplant cyclophosphamide (PTCy). A total of 322 patients with acute leukemia, MDS, lymphoma, CLL or CML, receiving a HIDT-PTCy from a single institution were evaluated with a median follow-up time of 45 months [range 6, 184]. Baseline characteristics included a median age of 50 years [19, 80], 47% non-white, HCT-CI ≥3 in 50%, PBSC graft in 80%, and myeloablative conditioning in 49%. M-containing B-leader genotype (either MM or MT) was seen in 42% and 44% of recipients and donors, respectively. The B-leader on the unshared donor-recipient haplotypes was matched (either T or M) in 61% of transplants. ML for iKIR 2DL1, 2DL23, 3DL1, and 3DL2 was noted in 29%, 20%, 24% and 72% respectively. Total ML was 0, 1, 2 and 3 in 10%, 43%, 40% and 7% respectively. In univariate analysis, an M-containing recipient B-leader genotype [R(M+)] improved OS and DFS compared to a recipient TT genotype [R(M-)] (75 vs. 53%, p<0.001; 66 vs. 47%, p<0.001), which was primarily due to a lower risk of relapse/progression (24% vs. 38%, p=0.012). In regard to total iKIR ML, the presence of 2-3 ML was associated with better overall (OS) and disease-free (DFS) survival compared with 0-1 (67% vs. 57%, p=0.08; 62% vs. 48%, p=0.019), which was due to lower relapse/progression (25% vs. 38%, p=0.015). There was no association of either recipient B-leader or total iKIR ML with the incidence of NRM, acute or chronic GVHD. When recipient B-leader and ML were combined, the detrimental effect of a R(M-) genotype was seen exclusively in patients with ML 0-1 (see figure). DFS for R(M-)/ML(0-1), R(M+)/ML(0-1), R(M-)/ML(2-3), R(M+)/ML(2-3) was 36%, 68%, 65% and 60%, respectively (p<0.001). Corresponding relapse/progression rates were 49%, 22%, 26% and 25%, respectively (P<0.001). In multivariate analysis, controlling for patient age/sex/race, disease risk index, donor age, HLA-DR mm, HLA-DP npmm and year of transplantation, both recipient B leader and total iKIR ML were independently associated with DFS (HR 0.57, p=0.002 and HR 0.67, p=0.026) and relapse/progression (HR 0.55, p=0.006 and HR 0.55, p=0.007). In summary, a recipient M-containing B-leader genotype (MM or MT) reduces relapse and improves survival following HIDT-PTCy, presumably through HLA-E-mediated effector cell engagement. Furthermore, the negative consequences of low total ML for iKIR, in terms of increased relapse risk and lower DFS, can be mitigated when a R(M+) B-leader is present. In addition to the clinical importance of this novel finding for optimizing HIDT-PTCy, it further supports the role of alloreactive donor NK cells for optimal GVL in this context. [Display omitted] DisclosuresSolh: Partner Therapeutics: Research Funding; Jazz Pharmaceuticals: Consultancy; ADCT Therapeutics: Consultancy, Research Funding; BMS: Consultancy.

Association of Ikir-Mismatching and Donor Akirs with Better Outcomes in Haploidentical Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia

Objectives: Natural killer (NK) cells are the first donor-derived lymphocytes to be reconstituted after transplantation and play a critical role in improving transplantation outcomes. Their surface receptor, killer cell immunoglobulin like receptor (KIR), can trigger the alloreactivity of NK cells and have been shown to be protective for acute and chronic graft-versus-host disease (aGVHD, cGVHD) while retaining graft-versus-leukemia (GVL) effect. However, different results have been reported about KIR matching models and KIR alleles based on patient, donor and transplant characteristics, resulting in significant controversy about the best donor selection strategy. Here, we investigated the potential influence of KIR matching and KIR alleles on GVHD prophylaxis, overall survival (OS) and relapse rate (RR) of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) in acute myeloid leukemia (AML) patients. Methods：Data from 79 patients with AML treated with haplo-HSCT between May 2015 and May 2021 in the transplantation unit of the Fujian Medical University Union Hospital were retrospectively analyzed. The cohort included 49 male patients (62.0%) and 30 female (38.0%), with a median age of 25 years (1-68 years). KIR genotyping was performed by the PCR-SSO method. The amplicons were quantified on the Luminex 200 flow analyzer and analyzed using the Quick-Type for Lifecodes software for generating KIR data. HLA-A and -B alleles were typed by polymerase chain reaction using sequence-specific primers (PCR-SSP) (TBG, Taipei, Taiwan). HLA-C genotyping was determined by reverse hybridization with sequence specific oligonucleotides probes (rSSO) Line probe assay. Cox proportional hazards model and Kaplan-Meier survival curves were used for analysis. Results：At the time of transplantation, 49 cases (62.0%) were at CR1, while 30 (38.0%) were not. aGVHD occurred in 16 patients (20.3%) and recurrence arose in 10 patients (12.7%), 4 (5.1%) cases cGVHD was observed. After adjusting for age, disease-risk, disease-status, HLA-match, donor gender, conditioning regimen intensity and type of post-grafting GVHD prophylaxis, Cox regression analysis revealed that both KIR ligand-ligand mismatching (KLM) and KIR receptor-ligand matching (RLM) was associated with an decreased risk of aGVHD and relapse compared to KIR ligand-ligand matching and receptor-ligand matching group, respectively (aGVHD: KLM: p=0.047, RLM: p＜0.001; RR: KLM: p=0.049, RLM: p=0.017). Furthermore, RLM shows more accurate in prediction of relapse and aGVHD compared with KLM in both aGVHD and relapse. (aGHVD: p=0.009; RR: p=0.039). After taking activating KIR (aKIR) into consideration to compensate the defect of only inhibitory KIR (iKIR) dominant model, we found that patients with more donor activating KIRs can reach the lower incidence of aGVHD and relapse, and the benefit can gradually increase parallel with the increase in donor activating KIRs. (aGVHD：p=0.019；RR：p=0.037). Patients with both receptor-ligand mismatch and the most donor aKIRs can reach the lowest relapse, lowest incidence of aGVHD and best overall survival (OS). Conclusions: KIR-mismatch, both KLM and RLM significantly reduced the risk of aGVHD and relapse after halpo-HSCT in AML patients and RLM show more superiority in the prediction of HSCT outcome. The synergistic effects of receptor-ligand mismatch and more donor aKIRs can provide a better clinically applicable donor selection strategy to improve haplo-HSCT outcome in AML patients. DisclosuresHu: Astellas Pharma, Inc.: Research Funding.