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As new killer-cell immunoglobulin-like receptor (KIR) alleles are discovered, a challenge in KIR typing is maintaining sensitivity and specificity. A single nucleotide polymorphism assay can be used to type functional KIR2DL1 alleles. We improved recently on the earlier method by using a higher-specificity assay. The major modifications include the development of sequence-specific primers to selectively amplify the transmembrane domain of all known KIR2DL1 alleles via polymerase chain reaction with sequence-specific primers (PCR-SSP), and using the PCR products as the template for a revised KIR2DL1 functional allele-typing assay. This modified method allows high-throughput typing with high specificity.

Many malignant cells release the NKG2D ligand ULBP2 from their cell surface to evade immunosurveillance by NK cells and CD8 T cells. Although the shedding mechanism remains unclear, various inhibitors of matrix metalloproteinases have been shown to efficiently block the release of soluble ULBP2. The clinical use of these inhibitors, however, is limited because of adverse side effects. Using high-throughput screening technique, we identified a specific inhibitor of phosphatase of regenerating liver 3 (PRL-3) that could reduce the level of soluble ULBP2 in the culture supernatant of various cancer cell lines. Inhibition or gene knockdown of PRL-3 did not reduce ULBP2 shedding, but rather suppressed posttranslational maturation of ULBP2, resulting in intracellular retention of immature ULBP2. We then found that ULBP2 was constitutively associated with heat shock protein HSP60. Complete maturation of ULBP2 required tyrosine phosphorylation of HSP60 which was mediated by PRL-3.

Killer cell Ig–like receptors (KIRs) on NK cells have been linked to a wide spectrum of health conditions such as chronic infections, autoimmune diseases, pregnancy complications, cancers, and transplant failures. A small subset of effector memory T cells also expresses KIRs. In this study, we use modern analytic tools including genome-wide and multiplex molecular, phenotypic, and functional assays to characterize the KIR+ T cells in human blood. We find that KIR+ T cells primarily reside in the CD56+ T population that is distinctively DNAM-1high with a genome-wide quiescent transcriptome, short telomere, and limited TCR excision circles. During CMV reactivation in bone marrow transplant recipients, KIR+CD56+ T cells rapidly expanded in real-time but not KIR+CD56− T cells or KIR+ NK cells. In CMV+ asymptomatic donors, as much as 50% of CD56+ T cells are KIR+, and most are distinguishably KIR2DL2/3+NKG2C+CD57+. Functionally, the KIR+CD56+ T cell subset lyses cancer cells and CMVpp65-pulsed target cells in a dual KIR-dependent and TCR-dependent manner. Analysis of metabolic transcriptome confirms the immunological memory status of KIR+CD56+ T cells in contrast to KIR−CD56+ T cells that are more active in energy metabolism and effector differentiation. KIR–CD56+ T cells have >25-fold higher level of expression of RORC than the KIR+ counterpart and are a previously unknown producer of IL-13 rather than IL-17 in multiplex cytokine arrays. Our data provide fundamental insights into KIR+ T cells biologically and clinically.

KIR2DL2 and KIR2DL3 segregate as alleles of a single locus in the centromeric motif of the killer cell immunoglobulin-like receptor (KIR) gene family. Although KIR2DL2/L3 polymorphism is known to be associated with many human diseases and is an important factor for donor selection in allogeneic hematopoietic stem cell transplantation, the molecular determinant of functional diversity among various alleles is unclear. In this study we found that KIR2DL2/L3 with glutamic acid at position 35 (E35) are functionally stronger than those with glutamine at the same position (Q35). Cytotoxicity assay showed that NK cells from HLA-C1 positive donors with KIR2DL2/L3-E35 could kill more target cells lacking their ligands than NK cells with the weaker -Q35 alleles, indicating better licensing of KIR2DL2/L3+ NK cells with the stronger alleles. Molecular modeling analysis reveals that the glutamic acid, which is negatively charged, interacts with positively charged histidine located at position 55, thereby stabilizing KIR2DL2/L3 dimer and reducing entropy loss when KIR2DL2/3 binds to HLA-C ligand. The results of this study will be important for future studies of KIR2DL2/L3-associated diseases as well as for donor selection in allogeneic stem cell transplantation.

Killer immunoglobulin-like receptors (KIRs) play an essential role in the regulation of natural killer cell functions. KIR genes are highly polymorphic in nature, showing both haplotypic and allelic variations among people. We demonstrated in both in vitro and in vivo models a significant heterogeneity in function among different KIR2DL1 alleles, including their ability to inhibit YT-Indy cells from degranulation, interferon γ production, and cytotoxicity against target cells expressing the HLA-Cw6 ligand. Subsequent experiments showed that the molecular determinant was an arginine residue at position 245 (R245) in its transmembrane domain that mechanistically affects both the efficiency of inhibitory signaling and durability of surface expression. Specifically, in comparison with R245-negative alleles, KIR2DL1 that included R245 recruited more Src-homology-2 domain-containing protein tyrosine phosphatase 2 and β-arrestin 2, showed higher inhibition of lipid raft polarization at immune synapse, and had less down-regulation of cell-surface expression upon interaction with its ligand. Thus, our findings provide novel insights into the molecular determinant of KIR2DL1 and conceivably a fundamental understanding of KIR2DL1 allelic polymorphism in human disease susceptibility, transplant outcome, and donor selection.

In transmembrane (TM) domains, tetraspanin KAI1/CD82 contains an Asn, a Gln, and a Glu polar residue. A mutation of all three polar residues largely disrupts the migration-, invasion-, and metastasis-suppressive activities of KAI1/CD82. Notably, KAI1/CD82 inhibits the formation of microprotrusions and the release of microvesicles, while the mutation disrupts these inhibitions, revealing the connections of microprotrusion and microvesicle to KAI1/CD82 function. The TM polar residues are needed for proper interactions between KAI1/CD82 and tetraspanins CD9 and CD151, which also regulate cell movement, but not for the association between KAI1/CD82 and alpha3beta1 integrin. However, KAI1/CD82 still efficiently inhibits cell migration when either CD9 or CD151 is absent. Hence, KAI1/CD82 interacts with tetraspanin and integrin by different mechanisms and is unlikely to inhibit cell migration through its associated proteins. Moreover, without significantly affecting the glycosylation, homodimerization, and global folding of KAI1/CD82, the TM interactions maintain the conformational stability of KAI1/CD82, evidenced by the facts that the mutant is more sensitive to denaturation and less associable with tetraspanins and supported by the modeling analysis. Thus, the TM interactions mediated by these polar residues determine a conformation either in or near the tightly packed TM region and this conformation and/or its change are needed for the intrinsic activity of KAI1/CD82. In contrast to immense efforts to block the signaling of cancer progression, the perturbation of TM interactions may open a new avenue to prevent cancer invasion and metastasis.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). To identify recipient risk factors, a genome-wide study was performed including 481,820 single-nucleotide polymorphisms (SNPs). Two GVHD susceptibility loci (rs17114803 and rs17114808) within the SUFU gene were identified in the discovery cohort (p = 2.85 × 10−5). The incidence of acute GVHD among patients homozygous for CC at SUFU rs17114808 was 69%, which was significantly higher than the 8% rate observed in CT heterozygous patients (p = 0.0002). In an independent validation cohort of 100 patients, 50% of the patients with the CC genotype developed GVHD compared to 8% of the patients with either CT or TT genotype (p = 0.01). In comparison to CC dendritic cells, those from CT expressed higher levels of SUFU mRNA and protein, had lower levels of surface HLA-DR and induced less allogeneic mixed leukocyte response (MLR). Ectopic expression of SUFU in THP-1 derived DCs reduced HLA-DR expression and suppressed MLR, whereas silencing of SUFU enhanced HLA-DR expression and increased MLR. Thus our findings provide novel evidence that recipient SUFU germline polymorphism is associated with acute GVHD and is a novel molecular target for GVHD prevention and treatment.

The fixation of molecular O2 by the oxygenase activity of Rubisco leads to the formation of phosphoglycolate in the chloroplast that is further metabolized in the process of photorespiration. The initial step of this pathway is the oxidation of glycolate to glyoxylate. Whereas in higher plants this reaction takes place in peroxisomes and is dependent on oxygen as a co-factor, most algae oxidize glycolate in the mitochondria using organic co-factors. The identification and characterization of a novel glycolate dehydrogenase in Arabidopsis thaliana is reported here. The enzyme is dependent on organic co-factors and resembles algal glycolate dehydrogenases in its enzymatic properties. Mutants of E. coli incapable of glycolate oxidation can be complemented by overexpression of the Arabidopsis open reading frame. The corresponding RNA accumulates preferentially in illuminated leaves, but was also found in other tissues investigated. A fusion of the N-terminal part of the Arabidopsis glycolate dehydrogenase to red fluorescent protein accumulates in mitochondria when overexpressed in the homologous system. Based on these results it is proposed that the basic photorespiratory system of algae is conserved in higher plants.

Purpose: Not all natural killer (NK) cells are equally cytotoxic against leukemia because of differences in receptor gene content and surface expression. We correlated NK cell genotype and phenotype at diagnosis of childhood acute lymphoblastic leukemia (ALL) with minimal residual disease (MRD) after induction chemotherapy. Experimental Design: The NK cells and leukemia blasts of 244 patients were analyzed at diagnosis by killer-cell immunoglobulin-like receptor (KIR) typing and immunophenotyping. The results were correlated statistically with postinduction MRD status. Results: The odds of being MRD positive in patients with KIR telomeric (Tel)-A/B genotype were 2.85 times the odds in those with Tel-A/A genotype (P = 0.035). MRD-positive patients were more likely to have KIR2DL5A (P = 0.006) and expressed less activating receptor NKp46 and FASL on their NK cells (P = 0.0074 and P = 0.029, respectively). The odds of being MRD positive increased by 2.01-fold for every percentage increase in NK cells expressing KIR2DL1 in the presence of HLA-C2 ligand (P = 0.034). The quantity of granzyme B inhibitor PI-9 in the leukemia blasts was greater in patients who were MRD positive (P = 0.038). Collectively, five NK cell–related factors (Tel-B–associated KIR2DL5A, NKp46, FASL, granzyme B, and PI-9) are strongly associated with MRD positivity at the end of induction with 100% sensitivity and 80% specificity. Conclusions: Our data support the hypothesis that NK cells with a strong effector phenotype in the setting of decreased leukemia resistance are associated with better leukemia control. Clin Cancer Res; 20(23); 5986–94. ©2014 AACR.

Purpose Killer-cell immunoglobulin-like receptors (KIRs) that regulate natural-killer cells are highly polymorphic. Some KIR2DL1 alleles encode receptors that have stronger signaling function than others. We tested the hypothesis that the clinical outcomes of allogeneic hematopoietic stem-cell transplantation (HSCT) could be affected by donor KIR2DL1 polymorphism. Patients and Methods All 313 pediatric patients received allogeneic HSCT at a single institution. Donor KIR2DL1 functional allele typing was retrospectively performed using single nucleotide polymorphism assay. Results Patients who received a donor graft containing the functionally stronger KIR2DL1 allele with arginine at amino acid position 245 (KIR2DL1-R245) had better survival (P = .0004) and lower cumulative incidence of disease progression (P = .001) than those patients who received a donor graft that contained only the functionally weaker KIR2DL1 allele with cysteine at the same position (KIR2DL1-C245). The effect of KIR2DL1 allelic polymorphism was similar in patients with acute myeloid leukemia or acute lymphoblastic leukemia among all allele groups (P ≥ .71). Patients who received a KIR2DL1-R245–positive graft with HLA-C receptor-ligand mismatch had the best survival (P = .00003) and lowest risk of leukemia progression (P = .0005) compared with those who received a KIR2DL1-C245 homozygous graft. Conclusion Donor KIR2DL1 allelic polymorphism affects recipient outcomes after allogeneic HSCT. These findings have substantial implications for prognostication and donor selection.

Aim A number of transplant centers currently use killer-cell immunoglobulin-like receptor (KIR) genotyping as criteria for donor selection for hematopoietic stem cell transplant (HSCT) and natural killer (NK) cell protocols. Several factors contribute to donor selection, including presence or absence of multiple inhibitory and stimulatory KIR, centromeric vs. telomeric KIR haplotypes, “A” vs. “B” content KIR haplotypes, and NK cell education or licensing. Recent studies at our institution demonstrated that amino acid residue 245 of the KIR2DL1 protein is important in determining the signaling capacity, and had a significant effect on the overall outcome of HSCT. Patients who received a donor graft containing KIR2DL1 alleles encoding an arginine at position 245 (KIR2DL1-R 245 ) had better survival and lower incidence of disease progression than the patients who received a donor graft containing KIR2DL1 alleles encoding a cysteine at the same position (KIR2DL1-C 245 ). The goal of this work was to validate a lab-developed clinical test to distinguish the KIR2DL1 R 245 /C 245 allelic polymorphism and to facilitate donor selection based on this criterion. Methods Our research laboratory developed a real-time PCR single nucleotide polymorphism (SNP) assay that specifically detects alleles of the KIR2DL1 gene encoding arginine or cysteine at position 245, and avoids the interference of 2DL2 alleles that share the same sequence flanking this region. The HLA laboratory used the backbone of this assay to validate it for clinical use as a lab-developed companion test to current KIR genotyping. Results The SNP assay performed according to the required qualifications, including accuracy, precision, sensitivity, and specificity. Samples tested showed 100% concordance with the expected genotyping of the “Gold Standard,” cell lines with sequenced KIR2DL1 alleles from the CIBMTR Repository. Conclusions This SNP assay was validated for clinical use in the HLA laboratory, and should be in use in the near future as a companion test to the KIR genotyping currently used for donor selection in HSCT. P. Arnold: Grant/Research Support; Company/Organization; American Lebanese Associated Charities (ALSAC). P. Chou: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC). R. Bari: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC), Assisi Foundation of Memphis. 7. Other (Identify); Company/Organization; Dr. Bari is named as an inventor on patent applications claiming a SNP assay used for KIR allele typing, which is owned by SJCRH and is licensed to a commercial entity. W. Leung: Grant/Research Support; Company/Organization; American Lebanese Syrian Associated Charities (ALSAC), Assisi Foundation of Memphis. 7. Other (Identify); Company/Organization; Dr. Leung is named as an inventor on patent applications claiming a SNP assay used for KIR allele typing, which is owned by SJCRH and is licensed to a commercial entity.

Abstract 3292 Previous studies have demonstrated that many T cell subsets possess NK-like features, including the CD56+ and KIR+ populations. Collectively, these studies showed that these NK-like T cells are predominantly αβ+ CD8+ with memory phenotype and could recognize HLA-E associated viral peptides after expansion upon TCR engagement. However, their clonality, transcriptome, regulation, specificity, and memory response in human have not been fully elucidated. We hypothesize that these NK-like T cells are phenotypically and functionally distinct from conventional T and NK cells and they play unique roles in virus and cancer control. Herein, we extensively characterized the CD56+ T cells and the KIR+ T subset by analysis of TREC, TCRVβ spectrum, telomere length, surface biomarkers, genome-wide transcriptome, multi-analyte cytokine profiling, cancer cell susceptibility, tetramer staining, and real-time response to CMV reactivation in stem cell transplant recipients. In contrast to CD56– T cells, CD56+ T cells are limited in TREC, TCRVβ, telomere length, cytokine secretion, transcription of metabolic genes stx6, nnt, galnt2, hvcn1, tyms, rpa1 tmf1, ecop, and tspan3, and are mostly KIR+, CD8+, DNAM1+, NKG2D+, CD44+, NKp46– and CD25–. Compared to KIR– CD56+ T cells, KIR+ CD56+ T cells are even more limited in TREC, TCRVβ, telomere length, and cytokine secretion, but have elevated transcription of NK cytotoxicity-related genes arrb1, ppp3cc, and lamc3, higher degranulation after activation by IL-2/IL-15 and CD3/CD28 antibodies, better killing of cancer cells after cytokine priming, and are mostly KIR2DL2/3+, NKG2D+, NKp46+, CD16+, NKG2C+, CD57+, and 2B4+. Importantly during CMV reactivation after stem cell transplantation, the percentage of KIR+ CD56+ T cells in the patient's blood increased dramatically and was significantly higher (p=0.0021) than in those without viral reactivation. Ex vivo, KIR+ CD56+ T cells demonstrate CMVpp65 tetramer staining, memory response to CMV peptides, and potent lysis of CMVpp65-pulsed target cells dependent on both KIR and TCR specificity. Furthermore, we identified for the first time that KIR– CD56+ T cells are Rorc+ IL-13-secretor. In conclusion, both CD56+ and KIR+ NK-like T-cell subsets are unique in biological and clinical properties and have distinct roles in cancer and infection control. Disclosures: No relevant conflicts of interest to declare.