Cell-Extrinsic MHC Class I Molecule Engagement Augments Human NK Cell Education Programmed by Cell-Intrinsic MHC Class I.

The effector potential of NK cells is counterbalanced by their sensitivity to inhibition by "self" MHC class I molecules in a process called "education." In humans, interactions between inhibitory killer immunoglobulin-like receptors (KIR) and human MHC (HLA) mediate NK cell education. In HLA-B(∗)27:05(+) transgenic mice and in patients undergoing HLA-mismatched hematopoietic cell transplantation (HCT), NK cells derived from human CD34(+) stem cells were educated by HLA from both donor hematopoietic cells and host stromal cells. Furthermore, mature human KIR3DL1(+) NK cells gained reactivity after adoptive transfer to HLA-B(∗)27:05(+) mice or bone marrow chimeric mice where HLA-B(∗)27:05 was restricted to either the hematopoietic or stromal compartment. Silencing of HLA in primary NK cells diminished NK cell reactivity, while acquisition of HLA from neighboring cells increased NK cell reactivity. Altogether, these findings reveal roles for cell-extrinsic HLA in driving NK cell reactivity upward, and cell-intrinsic HLA in maintaining NK cell education.

HLA-C-dependent prevention of leukemia relapse by donor activating KIR2DS1.

BACKGROUND: Of the cancers treated with allogeneic hematopoietic stem-cell transplantation (HSCT), acute myeloid leukemia (AML) is most sensitive to natural killer (NK)-cell reactivity. The activating killer-cell immunoglobulin-like receptor (KIR) 2DS1 has ligand specificity for HLA-C2 antigens and activates NK cells in an HLA-dependent manner. Donor-derived NK reactivity controlled by KIR2DS1 and HLA could have beneficial effects in patients with AML who undergo allogeneic HSCT. METHODS: We assessed clinical data, HLA genotyping results, and donor cell lines or genomic DNA for 1277 patients with AML who had received hematopoietic stem-cell transplants from unrelated donors matched for HLA-A, B, C, DR, and DQ or with a single mismatch. We performed donor KIR genotyping and evaluated the clinical effect of donor KIR genotype and donor and recipient HLA genotypes. RESULTS: Patients with AML who received allografts from donors who were positive for KIR2DS1 had a lower rate of relapse than those with allografts from donors who were negative for KIR2DS1 (26.5% vs. 32.5%; hazard ratio, 0.76; 95% confidence interval [CI], 0.61 to 0.96; P=0.02). Of allografts from donors with KIR2DS1, those from donors who were homozygous or heterozygous for HLA-C1 antigens could mediate this antileukemic effect, whereas those from donors who were homozygous for HLA-C2 did not provide any advantage (24.9% with homozygosity or heterozygosity for HLA-C1 vs. 37.3% with homozygosity for HLA-C2; hazard ratio, 0.46; 95% CI, 0.28 to 0.75; P=0.002). Recipients of KIR2DS1-positive allografts mismatched for a single HLA-C locus had a lower relapse rate than recipients of KIR2DS1-negative allografts with a mismatch at the same locus (17.1% vs. 35.6%; hazard ratio, 0.40; 95% CI, 0.20 to 0.78; P=0.007). KIR3DS1, in positive genetic linkage disequilibrium with KIR2DS1, had no effect on leukemia relapse but was associated with decreased mortality (60.1%, vs. 66.9% without KIR3DS1; hazard ratio, 0.83; 95% CI, 0.71 to 0.96; P=0.01). CONCLUSIONS: Activating KIR genes from donors were associated with distinct outcomes of allogeneic HSCT for AML. Donor KIR2DS1 appeared to provide protection against relapse in an HLA-C-dependent manner, and donor KIR3DS1 was associated with reduced mortality. (Funded by the National Institutes of Health and others.).

NK cell tolerance of self-specific activating receptor KIR2DS1 in individuals with cognate HLA-C2 ligand.

NK cells are regulated by inhibiting and activating cell surface receptors. Most inhibitory receptors recognize MHC class I Ags and protect healthy cells from NK cell-mediated autoaggression. However, certain activating receptors, including the human activating killer cell Ig-like receptor (KIR) 2DS1, also recognize MHC class I. This fact raises the question of how NK cells expressing such activating receptors are tolerized to host tissues. We investigated whether the presence of HLA-C2, the cognate ligand for 2DS1, induces tolerance in 2DS1-expressing NK cells. Anti-HLA-C2 activity could be detected in vitro in some 2DS1 positive NK clones irrespective of the presence or absence of HLA-C2 ligand in the donor. The frequency of anti-HLA-C2 reactivity was high in donors homozygous for HLA-C1. Surprisingly, no significant difference was seen in the frequency of anti-HLA-C2 cytotoxicity in donors heterozygous for HLA-C2 and donors without HLA-C2 ligand. However, donors homozygous for HLA-C2, compared with all other donors, had significantly reduced frequency of anti-HLA-C2 reactive clones. The 2DS1 positive clones that express inhibitory KIR for self-HLA class I were commonly noncytotoxic, and anti-HLA-C2 cytotoxicity was nearly exclusively restricted to 2DS1 single positive clones lacking inhibitory KIR. 2DS1 single positive NK clones with anti-HLA-C2 reactivity were also present posttransplantation in HLA-C2 positive recipients of hematopoietic stem cell transplants from 2DS1 positive donors. These results demonstrate that many NK cells with anti-HLA-C2 reactivity are present in HLA-C1 homozygous and heterozygous donors with 2DS1. In contrast, 2DS1 positive clones from HLA-C2 homozygous donors are frequently tolerant to HLA-C2.

Donor activating KIR3DS1 is associated with decreased acute GVHD in unrelated allogeneic hematopoietic stem cell transplantation.

The natural killer cell receptor KIR3DS1 is associated with improved outcome in malignancies, infections, and autoimmune diseases, but data for the impact of KIR3DS1 in HSCT are inconsistent. Using genomic DNA from the National Marrow Donor Program, we performed donor KIR genotyping for 1087 patients who received an unrelated hematopoietic stem cell transplantation. A total of 33% of donors were KIR3DS1(+). Compared with KIR3DS1(-) donors, donor KIR3DS1 was associated with lower-grade II-IV acute graft-versus-host disease (GVHD; odds ratio = 0.71; 95% confidence interval, 0.55-0.92; P = .009), but not with relapse (hazard ratio = 0.97; 95% confidence interval, 0.73-1.29; P = .82). Furthermore, grade II-IV acute GVHD, overall mortality, and transplantation-related mortality all decreased as the number of copies of donor KIR3DS1 increased (P = .007, P = .03, and P = .02, respectively), with the lowest failure rate occurring among patients homozygous for donor KIR3DS1. Selection of donors with KIR3DS1 may decrease acute GVHD without compromising relapse-free survival, separating the graft-versus-tumor effect from unwanted GVHD.

Killer immunoglobulin-like receptor locus polymorphisms in multiple sclerosis.

OBJECTIVE: The objective of this study was to analyze whether inhibitory and activating killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I alleles defined by their KIR binding motifs are associated with multiple sclerosis (MS) susceptibility or severity. METHOD: We performed a population-based case-control study in 321 patients with clinically isolated syndrome (CIS) and clinically definite MS (CDMS) and 156 healthy blood donors (HD). Inhibitory and activating KIRs and HLA class I alleles were genotyped using polymerase chain reaction (PCR) sequence-specific primers. Allelic frequencies were correlated with prevalence, age of onset, disability and disease duration of CIS and CDMS. RESULTS: The frequency of the inhibitory KIR2DL3 gene was significantly reduced in patients with CIS and CDMS (p = 3.1 × 10(-5)). KIR2DL3-dependent risk reduction remained significant after elimination of patients carrying MS-associated DRB1*15, DRB1*03, DRB1*01 alleles. In addition, individuals carrying two copies for KIR2DL2/KIR2DS2 but lacking KIR2DL3 were overrepresented in the CIS/CDMS cohort. However, both genes did not affect disease risk in presence of KIR2DL3. We did not detect any association between the presence or absence of KIR genes with clinical disease parameters. CONCLUSION: Absence of the inhibitory KIR2DL3 gene is associated with the development of CIS/CDMS. These findings, if confirmed in larger cohorts, suggest that KIR-mediated recognition of HLA class I molecules should be further explored as potential disease mechanism in MS.

A panel of artificial APCs expressing prevalent HLA alleles permits generation of cytotoxic T cells specific for both dominant and subdominant viral epitopes for adoptive therapy.

Adoptive transfer of virus-specific T cells can treat infections complicating allogeneic hematopoietic cell transplants. However, autologous APCs are often limited in supply. In this study, we describe a panel of artificial APCs (AAPCs) consisting of murine 3T3 cells transduced to express human B7.1, ICAM-1, and LFA-3 that each stably express one of a series of six common HLA class I alleles. In comparative analyses, T cells sensitized with AAPCs expressing a shared HLA allele or autologous APCs loaded with a pool of 15-mer spanning the sequence of CMVpp65 produced similar yields of HLA-restricted CMVpp65-specific T cells; significantly higher yields could be achieved by sensitization with AAPCs transduced to express the CMVpp65 protein. T cells generated were CD8(+), IFN-gamma(+), and exhibited HLA-restricted CMVpp65-specific cytotoxicity. T cells sensitized with either peptide-loaded or transduced AAPCs recognized epitopes presented by each HLA allele known to be immunogenic in humans. Sensitization with AAPCs also permitted expansion of IFN-gamma(+) cytotoxic effector cells against subdominant epitopes that were either absent or in low frequencies in T cells sensitized with autologous APCs. This replenishable panel of AAPCs can be used for immediate sensitization and expansion of virus-specific T cells of desired HLA restriction for adoptive immunotherapy. It may be of particular value for recipients of transplants from HLA-disparate donors.

The lytic potential of human liver NK cells is restricted by their limited expression of inhibitory killer Ig-like receptors.

The human liver is enriched in NK cells which are potent effectors of the innate immune system. We have determined that liver NK cells freshly isolated from surgical specimens from patients with hepatic malignancy have less cytolytic activity than autologous blood NK cells. This difference was due to a higher proportion of CD16(-) NK cells in the liver and reduced cytotoxicity by CD16(+) liver NK cells compared with their blood counterparts. CD16(+) liver NK cells had similar expression of activating NK receptors and had similar intracellular granzyme B and perforin content compared with CD16(+) blood NK cells. CD16(+) liver NK cells contained a reduced fraction of cells with inhibitory killer Ig-like receptors specific for self-MHC class I (self-killer Ig-related receptor (KIR)) and an increased fraction of self-KIR(neg)NKG2A(pos) and self-KIR(neg)NKG2A(neg) cells. Using single-cell analysis of intracellular IFN-gamma production and cytotoxicity assays, we determined that CD16(+) liver NK cells expressing self-KIR were more responsive to target cells than those cells that did not express self-KIR molecules. CD16(+) liver NK cells gained cytolytic function when stimulated with IL-2 or cultured with LPS or poly(I:C)-activated autologous liver Kupffer cells. Thus, the human liver contains NK cell subsets which have reduced effector function, but under appropriate inflammatory conditions become potent killers.

HLA alleles determine differences in human natural killer cell responsiveness and potency.

Epidemiological studies have associated certain human disease outcomes with particular killer cell Ig-like receptor (KIR) and HLA genotypes. However, the functional explanation for these associations is poorly understood, because the KIRs were initially described as natural killer (NK) cell inhibitory receptors with specificity for HLA molecules on their cellular targets. Yet resolution of infections is often associated with genotypic pairing of inhibitory KIRs with their cognate HLA ligands. Recent studies in mice indicate a second role for MHC-specific inhibitory receptors, i.e., self-MHC recognition confers functional competence on the NK cell to be triggered through their activation receptors, a process termed licensing. As a result, licensed NK cells with self-MHC-specific receptors are more readily activated as compared with unlicensed NK cells without self-MHC-specific receptors. Such results predict that human NK cells may undergo a similar process. Here, we examined the human NK cell subset expressing KIR3DL1, the only known KIR specific for HLA-Bw4 alleles. The KIR3DL1(+) subset in normal donors with two HLA-B-Bw4 genes displayed increased responsiveness to tumor stimulation compared with the KIR3DL1(+) subset from individuals with only one or no Bw4 genes. By contrast, NK cells lacking KIR3DL1 showed no differences. Therefore, these data indicate that particular KIR and HLA alleles are associated with more responsive NK cells, strongly suggesting that human NK cells are also subjected to NK cell licensing, and providing a potential functional explanation for the influence of KIR and HLA genes in disease as well as interindividual differences in NK cell potency.

HLA class I - associated immunodominance affects CTL responsiveness to an ESO recombinant protein tumor antigen vaccine.

PURPOSE: Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region (ESO 81-110). The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders. EXPERIMENTAL DESIGN: We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL. RESULTS: CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them. CONCLUSIONS: Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness.

Safety and immunogenicity study of NY-ESO-1b peptide and montanide ISA-51 vaccination of patients with epithelial ovarian cancer in high-risk first remission.

PURPOSE: The cancer-testis antigen NY-ESO-1 is expressed by >40% of advanced epithelial ovarian cancers and is a promising immunotherapeutic target. In this study, we describe the effects of vaccination with the HLA-A*0201-restricted NY-ESO-1b peptide on patients with epithelial ovarian cancer in high-risk first remission. EXPERIMENTAL DESIGN: After primary surgery and chemotherapy, high-risk epithelial ovarian cancer patients in first clinical remission received NY-ESO-1b peptide and Montanide every 3 weeks for five vaccinations. Tumor expression was evaluated by immunohistochemistry. Toxicity was monitored using National Cancer Institute Common Toxicity Criteria Scale Version 2. NY-ESO-1 specific humoral immunity (ELISA), T-cell immunity (tetramer and ELISPOT), and delayed-type hypersensitivity were assessed on weeks 0, 1, 4, 7, 10, 13, and 16. RESULTS: Treatment-related adverse events included grade 1 fatigue, anemia, pruritus, myalgias, and hyperthyroidism and grade 2 hypothyroidism. There were no grade 3/grade 4 adverse events. Three of four patients (75%) with NY-ESO-1-positive tumor showed T-cell immunity by tetramer (0.6-9.5%) and ELISPOT (range, 35-260 spots). Four of five patients (80%) with NY-ESO-1-negative tumor showed T-cell immunity by tetramer (1.0-12.1%) and/or ELISPOT (range, 35-400 spots). With a median follow-up of 11.3 months, six of nine patients (67%) have recurred, with a median progression-free survival of 13 months (95% confidence interval, 11.2 months-not reached). Three of nine patients remain in complete clinical remission at 25, 38, and 52 months. CONCLUSION: Vaccination of high-risk HLA-A*0201-positive epithelial ovarian cancer patients with NY-ESO-1b and Montanide has minimal toxicity and induces specific T-cell immunity in patients with both NY-ESO-1-positive and NY-ESO-1-negative tumors. Additional study is warranted.

Infusion of haplo-identical killer immunoglobulin-like receptor ligand mismatched NK cells for relapsed myeloma in the setting of autologous stem cell transplantation.

Killer immunoglobulin-like receptor (KIR)-ligand mismatched natural killer (NK) cells play a key role in achieving durable remission after haplo-identical transplantation for acute myeloid leukaemia. We investigated the feasibility of transfusing haplo-identical, T-cell depleted, KIR-ligand mismatched NK cells, after conditioning therapy with melphalan and fludarabine, to patients with advanced multiple myeloma (MM) followed by delayed rescue with autologous stem cells. No graft-versus-host disease or failure of autologous stem cells to engraft was observed. There was significant variation in the number of allo-reactive NK cells transfused. However, all NK products containing allo-reactive NK cells killed the NK cell target K562, the MM cell line U266, and recipient MM cells when available. Post NK cell infusion there was a rise in endogenous interleukin-15 accompanied by increasing donor chimaerism. Donor chimaerism was eventually lost, which correlated with the emergence of potent host anti-donor responses indicating that the immunosuppressive properties of the conditioning regimen require further optimization. Further, blocking of inhibitory KIR-ligands with anti-human leucocyte antigen antibody substantially enhanced killing of MM cells thus highlighting the potential for modulating NK/MM cell interaction. Encouragingly, 50% of patients achieved (near) complete remission. These data set the stage for future studies of KIR-ligand mismatched NK cell therapy in the autologous setting.

Inhibitory killer Ig-like receptor genes and human leukocyte antigen class I ligands in haematopoietic stem cell transplantation.

Identification of inhibitory killer Ig-like receptor (KIR) genes and their cognate human leukocyte antigen (HLA) class I ligands in donor-recipient pairs for patients undergoing haematopoietic stem cell transplantation (HCT) as treatment for haematopoietic malignancies has recently gained considerable interest. One incentive for these studies is to identify potential donors who are not HLA identical with the recipient but who still could serve as acceptable and potentially preferred donors based on their KIR genotype. It is demonstrated that a majority of individuals have at least one inhibitory KIR gene for which they lack the cognate HLA class I ligand. Therefore, the clinical benefits conferred by 'missing KIR ligand' might not be limited only to HLA mismatched donor-recipient combinations but may be applicable also to HLA identical transplants and even autologous HCT.

Natural killer cell receptors: regulating innate immune responses to hematologic malignancy.

Critical to innate immunity, the natural killer (NK) cell performs its function of immunosurveillance through its recognition of altered or missing self on damaged, infected, or transformed malignant cells. NK cell receptors responsible for detection of human leukocyte antigen (HLA) class I and class I-like proteins on potential target cells transmit inhibitory and activating signals that integrate to determine NK cell function. Advances in the fields of NK cell receptor biology and immunogenetics have enhanced our understanding of NK cell target recognition and may now guide studies to determine NK cell effects in the clinical setting. Analysis of NK cell receptor-ligand relationships, such as the inhibitory killer immunoglobulin-like receptors (KIRs) and their HLA class I ligands, has revealed the potential for NK cell-mediated benefit in allogeneic hematopoietic stem cell transplantation for hematologic malignancies.

[Killer Ig-like receptor gene content diversity and haplotype analysis in Chinese Han population in Shanghai].

OBJECTIVE: To detect the diversity of killer Ig-like receptor(KIR) gene content and the combination of haplotypes in Chinese Han population in Shanghai area. METHODS: DNA samples from 87 randomly unrelated healthy individuals in Shanghai Han population were genotyped with SSP/PCR method. RESULTS: (1) Frequencies of KIR genes: All of 18 known KIRs genes, such as 2DL1-5, 2DS1-5, 3DL1-3, 3DS1, KIR1D and the pseudogenes X, Xv and Z(KIR2DP1) were observed in Shanghai Hans. All individuals contain 3DL3, 2DL4, 3DL2 and 3DL1; the most common genes were 2DL3, Z, 2DL1 and X; the following were 2DS4, 1D, 2DL5, 2DS1, 3DS1 and 2DS5; the next were 2DS2, 2DL2, 2DS3 and Xv. (2) Frequencies of KIR gene haplotypes; there were 13 haplotypes detected in 87 Han individuals, among them, the most frequent one was type 2 (haplotypeA-2DS4). (3) Frequencies of KIR genotypes: 18 kinds of the combinations of the haplotypes were observed; the most frequent ones were AJ(2,2), AF (1,2). Also, In this study were identified five new genotypes FZ1 2 9 , FZ2 1 16 , FZ3 6 17 , FZ4 4 13 and FZ5 2 6 ,which had not been observed in Caucasians so far. CONCLUSION: These findings suggest that there are distinctive frequencies of KIR gene content, haplotype as well as genotype in Chinese Han population in Shanghai area.

Unlicensed NK cells target neuroblastoma following anti-GD2 antibody treatment.

Survival outcomes for patients with high-risk neuroblastoma (NB) have significantly improved with anti-disialoganglioside GD2 mAb therapy, which promotes NK cell activation through antibody-dependent cell-mediated cytotoxicity. NK cell activation requires an interaction between inhibitory killer cell immunoglobulin-like receptors (KIRs) and HLA class I ligands. NK cells lacking KIRs that are specific for self HLA are therefore "unlicensed" and hyporesponsive. mAb-treated NB patients lacking HLA class I ligands for their inhibitory KIRs have significantly higher survival rates, suggesting that NK cells expressing KIRs for non-self HLA are mediating tumor control in these individuals. We found that, in the presence of mAb, both licensed and unlicensed NK cells are highly activated in vitro. However, HLA class I expression on NB cell lines selectively inhibited licensed NK cell activity, permitting primarily unlicensed NK cells to mediate antibody-dependent cell-mediated cytotoxicity. These results indicate that unlicensed NK cells play a key antitumor role in patients undergoing mAb therapy via antibody-dependent cell-mediated cytotoxicity, thus explaining the potent "missing KIR ligand" benefit in patients with NB.

Vaccination with recombinant NY-ESO-1 protein elicits immunodominant HLA-DR52b-restricted CD4+ T cell responses with a conserved T cell receptor repertoire.

PURPOSE: ESO is a tumor-specific antigen with wide expression in human tumors of different histologic types and remarkable spontaneous immunogenicity. We have previously shown that specific T(H)1 and antibody responses can be elicited in patients with no detectable preexisting immune responses by vaccination with rESO administered with Montanide ISA-51 and CpG ODN 7909. The purpose of the present study was to characterize vaccine-induced ESO-specific CD4(+) T cell responses. EXPERIMENTAL DESIGN: We generated CD4(+) T cell clones from patient C2, who had the highest CD4(+) T cell response to the vaccine, and analyzed their fine specificity and HLA class II restriction to determine the recognized epitope. We then assessed the response to the identified epitope in all vaccinated patients expressing the corresponding HLA class II allele. RESULTS: We found that ESO-specific CD4(+) T cell clones from patient C2 recognize peptide ESO(119-143) (core region 123-137) presented by HLA-DR52b (HLA-DRB3*0202), a MHC class II allele expressed by about half of Caucasians. Importantly, following vaccination, all patients expressing DR52b developed significant responses to the identified epitope, accounting for, on average, half of the total CD4(+) T cell responses to the 119-143 immunodominant region. In addition, analysis of ESO-specific DR52b-restricted CD4(+) T cells at the clonal level revealed significant conservation of T cell receptor usage among different individuals. CONCLUSIONS: The identification of a DR52b-restricted epitope from ESO that is immunodominant in the context of vaccine-elicited immune responses is instrumental for the immunologic monitoring of vaccination trials targeting this important tumor antigen.