HLA Ligands Research Articles

Inhibitory KIR2DL2 Gene: Risk for Deep Endometriosis in Euro-descendants.

Endometriosis (EDT) is an inflammatory disease characterized by implantation/growth of endometrial tissue, glands, and/or stroma, outside the uterus. Reduced NK cell cytotoxic activity has been implicated in its pathogenesis, together with other immunologic alterations. We investigated the influence of KIR gene polymorphisms and their HLA ligand combinations in deep endometriosis (DE) susceptibility. One hundred sixty women with a histological diagnosis of DE and 202 control women without the disease, who underwent laparoscopy, were enrolled. The DE group was subdivided into initial (I/II; n = 60) and advanced stages (III/IV, n = 100). KIR and HLA class I gene polymorphisms were typed by PCR-SSP and sequence-based-typing (SBT), respectively. We observed a significant association of KIR2DL2, an inhibitory gene of B haplotype, conferring risk for DE in Euro-descendants. Positive associations of Bx haplotype and centromeric AB segments were also found. However, no association with KIR-HLA ligand combination was observed. Our data suggest KIR2DL2 gene to be a relevant factor favoring NK inhibition in DE in Euro-descendants, contributing to the defective NK cytotoxic activity and impaired clearance of ectopic endometrial cells in the disease.

The effect of KIR and HLA polymorphisms on dengue infection and disease severity in northeastern Thais.

Killer cell immunoglobulin-like receptors (KIRs) are cell surface receptors on natural killer (NK) cells and subsets of T cells. The interaction between KIRs and their cognate ligands (Human leukocyte antigen class I molecules, HLA class I) modulates the immune response of NK cells, in particular through clearance of virus-infected cells. Here, we investigated the effect of KIRs and HLA ligands on dengue infections and disease severity. The KIRs and HLA ligands were identified in 235 healthy controls (HC) and 253 dengue patients (DEN) using polymerase chain reaction with sequence specific primer (PCR-SSP); moreover, DEN was classified to 100 dengue fever (DF) and 153 dengue haemorrhagic fever (DHF). Risks were expressed as odds ratios (ORs) and 95% confidence intervals (CIs) with significance set at a two-tailed P value of < 0.05. The Bonferroni correction was applied for multiple comparisons. Twelve significant associations were observed in dengue infections and disease severity; however, two outcomes survived after the Bonferroni correction. Of these, HLA-A11 was associated with an increased risk to develop dengue disease (OR 2.41, 95% CI 1.62-3.60, Pc = 0.004), while KIR3DS1+ Bw4 was a protective genotype to developing DHF (OR 0.28, 95% CI 0.16-0.48, Pc < 0.001). This study revealed an important role of KIR and HLA ligands in innate immune responses to dengue viral infections and, in particular, their effect on clinical outcomes and disease severity.

Cancer testis antigen Cyclin A1 harbors several HLA-A*02:01-restricted T cell epitopes, which are presented and recognized in vivo

Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8+ T cells from two healthy donors with single-peptide-pulsed dendritic cells or monocytes. Intracellular cytokine staining quantified the enrichment of peptide-specific functional T cells. Seven peptides were immunogenic, three of them against both donors. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA-A*02:01-positive AML cell line THP-1. Immunopeptidome analysis based on direct isolation of HLA-presented peptides by mass spectrometry of primary AML and OC samples identified four naturally presented epitopes of Cyclin A1. The immunopeptidome of HeLa cells transfected with Cyclin A1 and HLA-A*02:01 revealed six Cyclin A1-derived HLA ligands. Epitope p410–420 showed high affinity to HLA-A*02:01 and immunogenicity in both donors. It proved to be naturally presented on primary AML blast and provoked spontaneous functional response of T cells from treatment naïve OC and, therefore, warrants further development for clinical application.

Mass spectrometry-based identification of a B-cell maturation antigen-derived T-cell epitope for antigen-specific immunotherapy of multiple myeloma

The B-cell maturation antigen (BCMA) is currently being evaluated as promising tumor-associated surface antigen for T-cell-based immunotherapy approaches, such as CAR T cells and bispecific antibodies, in multiple myeloma (MM). Cytotoxic T cells bearing BCMA-specific T-cell receptors might further allow targeting HLA-presented antigens derived from the intracellular domain of BCMA. By analyzing a mass spectrometry-acquired immunopeptidome dataset of primary MM samples and MM cell lines for BCMA-derived HLA ligands, we identified the naturally presented HLA-B*18-restricted ligand P(BCMA)B*18. Additionally, P(BCMA)B*18 was identified on primary CLL samples, thereby expanding the range for possible applications. P(BCMA)B*18 induced multifunctional BCMA-specific cells de novo from naïve CD8+ T cells of healthy volunteers. These T cells exhibited antigen-specific lysis of autologous peptide-loaded cells. Even in the immunosuppressive context of MM, we detected spontaneous memory T-cell responses against P(BCMA)B*18 in patients. By applying CTLA-4 and PD-1 inhibition in vitro we induced multifunctional P(BCMA)B*18-specific CD8+ T cells in MM patients lacking preexisting BCMA-directed immune responses. Finally, we could show antigen-specific lysis of autologous peptide-loaded target cells and even MM.1S cells naturally presenting P(BCMA)B*18 using patient-derived P(BCMA)B*18-specific T cells. Hence, this BCMA-derived T-cell epitope represents a promising target for T-cell-based immunotherapy and monitoring following immunotherapy in B-cell malignancy patients.

Identification of HCMV-derived T cell epitopes in seropositive individuals through viral deletion models.

In healthy individuals, immune control of persistent human cytomegalovirus (HCMV) infection is effectively mediated by virus-specific CD4+ and CD8+ T cells. However, identifying the repertoire of T cell specificities for HCMV is hampered by the immense protein coding capacity of this betaherpesvirus. Here, we present a novel approach that employs HCMV deletion mutant viruses lacking HLA class I immunoevasins and allows direct identification of naturally presented HCMV-derived HLA ligands by mass spectrometry. We identified 368 unique HCMV-derived HLA class I ligands representing an unexpectedly broad panel of 123 HCMV antigens. Functional characterization revealed memory T cell responses in seropositive individuals for a substantial proportion (28%) of these novel peptides. Multiple HCMV-directed specificities in the memory T cell pool of single individuals indicate that physiologic anti-HCMV T cell responses are directed against a broad range of antigens. Thus, the unbiased identification of naturally presented viral epitopes enabled a comprehensive and systematic assessment of the physiological repertoire of anti-HCMV T cell specificities in seropositive individuals.

Donor and Host Coexpressing KIR Ligands Promote NK Education Post Allogeneic Hematopoietic Stem Cell Transplantation

Background:Educated NK cells prevent autoreactive behavior but also permit cytotoxicity against target cells that have down-regulated HLA class I expression. When and how the process of education occurs has not been clearly discerned. Several groups reported that both the donor and host MHC could influence NK cell education in mouse models. In humans, Dulphy et al demonstrated that NK-cell education is shaped by donor HLA genotype. Moreover, our previous study found NK-cell education was shaped by host HLA genotype post allo-HSCT. However, due to the lack of single-KIR+ NK cells, functional analysis limited the full evaluation of the interaction between donor/host HLA and donor inhibitory KIR, so the contribution of donor HLA could not be excluded. Aims: In this research, we have investigated the relative contributions of donor and recipient HLA to NK cells education, the interplay between functional reconstitution and the involvement of donor/host HLA interaction in NK cell control of leukemia cells. Methods: Two cohorts of patients were enrolled in this study. We first prospectively enrolled 114 patients undergoing haplo-SCT between May 2016 and April 2017 to explore NK cell phenotypes and functional reconstitution. From June 2012 to April 2016, 276 AML/MDS patients that underwent haploidentical transplantation were enrolled in the second cohort to analyze the effect of donor-host KIR-HLA combinations on relapse post transplantation. Molecular HLA typing and KIR genotyping were performed according to the manufacturer's instructions (One Lambda, Canoga Park, CA, USA). Peripheral blood mononuclear cells of each sample were analyzed by 15-colors flow cytometry. The cytotoxicity and cytokine secretion of NK cells was determined using CD107a expression and IFN-γ production against the K562 cell line. Single-KIR+ NK cells were grouped into the following groups: (A) nsKIR: where both hosts and donors lacked HLA ligands for one donor KIR; (B) d-rsKIR, where donors and hosts, encoded HLA ligands for donor KIRs; (C) dsKIR, where donors, but not hosts, encoded HLA ligands for donor KIR; and (D) rsKIR, where hosts, but not donors, encoded HLA ligands for donor KIR. Results: 1. Donor KIR ligated by both donor and host HLA is associated with better single-KIR+ NK cell education among the same patients. KIR2DL2/L3 single+ NK cell exhibited higher reactivity compared to KIR2DL1 single+ NK cell in pairs of donors C1C1 or C1C2 and host C1C1. KIR2DL2/L3 single+ NK cell exhibited higher reactivity than KIR3DL1 single+ NK cell in pairs of donors Bw4C1Cx and host C1Cx. KIR2DL1 single+ NK cell exhibited comparable reactivity with KIR2DL2/L3 single+ NK cell in pairs of donor Bw4C1C2 and host Bw4C1C2. 2. Donor KIR ligated by both donor and host HLA contribute to better single-KIR+ NK cell education among the same single-KIR+ NK cells. KIR2DL2/L3 single+ NK cell in the group of d-rsKIR (C1Cx-C1Cx) exhibit higher reactivity compared with other groups (dsKIR (C1Cx-C2C2), rsKIR (C2C2- C1Cx)). KIR2DL1 single+ NK cells in group of d-rsKIR (C2Cx-C2Cx) exhibited higher reactivity compared with other groups (nsKIR (C1C1-C1C1), dsKIR (C2Cx-C1C1), rsKIR (C1C1-C2Cx)). KIR3DL1 single+ NK cells in groups of d-rsKIR (Bw4Bwx-Bw4Bwx) exhibited higher reactivity compared with other groups (nsKIR (Bw6Bw6-Bw6Bw6), dsKIR (Bw4Bwx-Bw6Bw6), rsKIR (Bw6Bw6-Bw4Bwx)). 3. Both donor and host HLA must coexist for maximum education of NK cells given donor 3 inhibitory KIRs. When both of donor and host presenting all HLA (Bw4C1C2), we showed a remarkable hierarchy of responses among NK populations. NK cells with two inhibitory KIRs for self-HLA exhibited higher NK responsiveness (CD107α and IFN-γ) compared with single KIR+ NK cells. NK cells with 3 inhibitory KIRs for self-HLA exhibited maximum responsiveness. 4. Both donor and host exhibiting all HLA (Bw4C1C2) for donor 3 inhibitory KIRs contributes to least relapse following haploidentical allo-HSCT. In the second cohort, the lowest relapse rate was found in d-rsKIR group (n=31, 0%) compared with rsKIR group (n=55 ,0% vs. 10.0±4.9%, P=0.115), dsKIR group (n=33, 0% vs 14.9%±7.0%, P=0.039), or nsKIR group (n=156, 0% vs. 18%±3.5%, P=0.022). Summary: This study demonstrated that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells would achieve better functional education and contribute to least relapse for the patients. Disclosures No relevant conflicts of interest to declare.

The Education of NK Cells Determines Their Responsiveness to Autologous HIV-Infected CD4 T Cells.

Several studies support a role for specific killer immunoglobulin-like receptor (KIR)-HLA combinations in protection from HIV infection and slower progression to AIDS. Natural killer (NK) cells acquire effector functions through education, a process that requires the interaction of inhibitory NK cell receptors with their major histocompatibility complex (MHC) class I (or HLA class I [HLA-I]) ligands. HLA-C allotypes are ligands for the inhibitory KIRs (iKIRs) KIR2DL1, KIR2DL2, and KIR2DL3, whereas the ligand for KIR3DL1 is HLA-Bw4. HIV infection reduces the expression of HLA-A, -B, and -C on the surfaces of infected CD4 (iCD4) T cells. Here we investigated whether education through iKIR-HLA interactions influenced NK cell responses to autologous iCD4 cells. Enriched NK cells were stimulated with autologous iCD4 cells or with uninfected CD4 cells as controls. The capacities of single-positive (sp) KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1 NK cells to produce CCL4, gamma interferon (IFN-γ), and/or CD107a were assessed by flow cytometry. Overall, we observed that the potency of NK cell education was directly related to the frequency of each spiKIR+ NK cell's ability to respond to the reduction of its cognate HLA ligand on autologous iCD4 cells, as measured by the frequency of production by spiKIR+ NK cells of CCL4, IFN-γ, and/or CD107a. Both NK cell education and HIV-mediated changes in HLA expression influenced NK cell responses to iCD4 cells.IMPORTANCE Epidemiological studies show that natural killer (NK) cells have anti-HIV activity: they are able to reduce the risk of HIV infection and/or slow HIV disease progression. How NK cells contribute to these outcomes is not fully characterized. We used primary NK cells and autologous HIV-infected cells to examine the role of education through four inhibitory killer immunoglobulin-like receptors (iKIRs) from persons with HLA types that are able to educate NK cells bearing one of these iKIRs. HIV-infected cells activated NK cells through missing-self mechanisms due to the downmodulation of cell surface HLA expression mediated by HIV Nef and Vpu. A higher frequency of educated than uneducated NK cells expressing each of these iKIRs responded to autologous HIV-infected cells by producing CCL4, IFN-γ, and CD107a. Since NK cells were from non-HIV-infected individuals, they model the consequences of healthy NK cell-HIV-infected cell interactions occurring in the HIV eclipse phase, when new infections are susceptible to extinction.

A protective effect conferred by KIR3DL1 and its cognate ligand against cervical cancer among ethnic Han Chinese population and its potential mechanism

To explore the role of inhibitory KIR (iKIR) and its cognate HLA ligand in the occurrence and development of cervical cancer among ethnic Han Chinese and its potential mechanism. Peripheral blood samples from 265 Han Chinese patients with cervical intraepithelial neoplasia (CIN)/cervical cancer and 200 ethnically matched healthy controls were collected. The results of KIR PCR-SSP, HLA PCR-rSSO and KIR3DL1 PCR-SBT, together with cervical cancer data from the TCGA database, were used to assess the association of iKIR genes, receptor-ligand gene combinations, iKIR transcription level in the tumor tissue and the KIR3DL1 alleles with the occurrence and development of cervical cancer. Among the four iKIR genes (KIR2DL1, 2DL2/3, 3DL1 and 3DL2), the frequencies of KIR3DL1 and KIR3DL1-HLA-Bw4 genes among controls were significantly higher than those of the cervical cancer group (96.5% vs. 87.0%, P = 0.018; 81.5% vs. 64.8%, P=0.009). The survival rate of cervical cancer patients with a high transcription level of KIR3DL1 in tumor tissues was significantly higher than those with a low/medium transcription level (P=0.028). The frequency of strong-inhibitory and high-expression KIR3DL1*01502 allele among the healthy population was significantly higher than that of the cervical cancer group (76.0% vs. 59.3%, P =0.015). Combined KIR3DL1 and KIR3DL1-HLA-Bw4 can confer a protective effect against the development of cervical cancer, which may be attributed to the strong-inhibitory and high-expression allele of KIR3DL1*01502.

Family-based Association Study of Killer Cell Immunoglobulin-Like Receptor Genes with Leukemia

Abstract NK cell function is controlled by the cell expression of killer immunoglobulin-like receptors (KIRs) and their ligation with the corresponding HLA ligands. Various malignancies have been associated with certain KIRs surface cell expression and various KIR/HLA ligand combinations. Prior research using case/control study design demonstrates the role of KIR and KIR HLA ligands as genetic factor involved in tumor susceptibility. The objective of this study was to investigate the family-based association of KIRs, HLA class I ligands and KIR/ligand combinations with leukemia diagnosis in families having a leukemia diagnosed child. Sixty-seven families that met the index leukemia case criteria (acute lymphoblastic leukemia, ALL, n = 45; acute myeloid leukemia, AML, n = 13; chronic myeloid leukemia, CML, n = 9; first degree healthy relatives n = 159) were examined. Our study consisted of two phases. In Phase1 case-control study, we primarily compared patients to their healthy siblings to asses if a marker or genotype may be associated with leukemia, excluding the impact of the environment. Phase 2 consisted of a secondary family-based association study. KIR genotyping was performed by PCR-SSP method. KIR HLA ligands were defined by direct method using PCR-SSP method and/or indirect base on high resolution typing of HLA-A, -B, -C alleles. Results of phase 1 showed an increase in the frequency of KIR genotype (with a ratio = 0.57; higher frequency for inhibitory KIRs vs. activating KIRs) among leukemia patients compared to healthy siblings. Results of the phase 2 familial study observed an association between HLA-C1+/BBw4+/ABw4+ haplotype (a mediator of inhibitory signals) and leukemia. Also, we concluded that the absence of HLA-ABw4 alleles was related to leukemia development.

Computational Analysis of HLA-presentation of Non-synonymous Recipient Mismatches Indicates Effect on the Risk of Chronic Graft-vs.-Host Disease After Allogeneic HSCT.

Genetic mismatches in protein coding genes between allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipient and donor can elicit an alloimmunity response via peptides presented by the recipient HLA receptors as minor histocompatibility antigens (mHAs). While the impact of individual mHAs on allo-HSCT outcome such as graft-vs.-host and graft-vs.-leukemia effects has been demonstrated, it is likely that established mHAs constitute only a small fraction of all immunogenic non-synonymous variants. In the present study, we have analyzed the genetic mismatching in 157 exome-sequenced sibling allo-HSCT pairs to evaluate the significance of polymorphic HLA class I associated peptides on clinical outcome. We applied computational mismatch estimation approaches based on experimentally verified HLA ligands available in public repositories, published mHAs, and predicted HLA-peptide affinites, and analyzed their associations with chronic graft-vs.-host disease (cGvHD) grades. We found that higher estimated recipient mismatching consistently increased the risk of severe cGvHD, suggesting that HLA-presented mismatching influences the likelihood of long-term complications in the patient. Furthermore, computational approaches focusing on estimation of HLA-presentation instead of all non-synonymous mismatches indiscriminately may be beneficial for analysis sensitivity and could help identify novel mHAs.

Acute myeloid leukemia immunopeptidome reveals HLA presentation of mutated nucleophosmin.

Somatic mutations in cancer are a potential source of cancer specific neoantigens. Acute myeloid leukemia (AML) has common recurrent mutations shared between patients in addition to private mutations specific to individuals. We hypothesized that neoantigens derived from recurrent shared mutations would be attractive targets for future immunotherapeutic approaches. Here we sought to study the HLA Class I and II immunopeptidome of thirteen primary AML tumor samples and two AML cell lines (OCI-AML3 and MV4-11) using mass spectrometry to evaluate for endogenous mutation-bearing HLA ligands from common shared AML mutations. We identified two endogenous, mutation-bearing HLA Class I ligands from nucleophosmin (NPM1). The ligands, AVEEVSLRK from two patient samples and C(cys)LAVEEVSL from OCI-AML3, are predicted to bind the common HLA haplotypes, HLA-A*03:01 and HLA-A*02:01 respectively. Since NPM1 is mutated in approximately one-third of patients with AML, the finding of endogenous HLA ligands from mutated NPM1 supports future studies evaluating immunotherapeutic approaches against this shared target, for this subset of patients with AML.

The relation between activator and inhibitor killer-cell immunoglobulin-like receptors and hepatotoxicity in oncological treatment.

The molecular interactions between killer-cell immunoglobulin-like receptors (KIRs) and their related HLA class I ligands play a central role in regulating the responses of natural killer (NK) cells. Our study aim was to determine the role played by KIR genes and their HLA ligands in the genetic predisposition for the development of hepatotoxicity in children treated with chemotherapy for an oncological process. The study group was composed of 22 children with cancer, being treated with chemotherapy at the Unit of Pediatric Oncology of the Maternity Hospital Virgen de las Nieves (Granada, Spain) and presenting signs of drug-induced liver injury (DILI). Twenty-four children receiving similar treatment but presenting no signs of DILI were selected as a control group. The children with the KIR K2DS2 were four times more likely to have hepatotoxicity (OR=4.08, P=0.034, 95% CI: 1.1-15). The patients with 2DS2 and the C1 ligand were ten times more likely to undergo an episode of hepatotoxicity (P=0.007). KIRs may be risk factors for susceptibility to hepatotoxicity following chemotherapy.

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

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

Multi-omics discovery of exome-derived neoantigens in hepatocellular carcinoma

BackgroundAlthough mutated HLA ligands are considered ideal cancer-specific immunotherapy targets, evidence for their presentation is lacking in hepatocellular carcinomas (HCCs). Employing a unique multi-omics approach comprising a neoepitope identification pipeline, we assessed exome-derived mutations naturally presented as HLA class I ligands in HCCs.MethodsIn-depth multi-omics analyses included whole exome and transcriptome sequencing to define individual patient-specific search spaces of neoepitope candidates. Evidence for the natural presentation of mutated HLA ligands was investigated through an in silico pipeline integrating proteome and HLA ligandome profiling data.ResultsThe approach was successfully validated in a state-of-the-art dataset from malignant melanoma, and despite multi-omics evidence for somatic mutations, mutated naturally presented HLA ligands remained elusive in HCCs. An analysis of extensive cancer datasets confirmed fundamental differences of tumor mutational burden in HCC and malignant melanoma, challenging the notion that exome-derived mutations contribute relevantly to the expectable neoepitope pool in malignancies with only few mutations.ConclusionsThis study suggests that exome-derived mutated HLA ligands appear to be rarely presented in HCCs, inter alia resulting from a low mutational burden as compared to other malignancies such as malignant melanoma. Our results therefore demand widening the target scope for personalized immunotherapy beyond this limited range of mutated neoepitopes, particularly for malignancies with similar or lower mutational burden.

Abstract B018: Landscape of natural HLA class I ligand peptides of cancer cells

Abstract HLA class I ligand repertoire on cancer cells directly influences immune surveillance system against cancer. We performed large-scale mass spectrometric profiling of the natural HLA-A*2402 ligand peptides and unveiled the landscape of natural HLA class I ligand peptides derived from various cancer cells including colon cancer cells (SW480, Colo320, HCT15) and lung cancer cells (LHK2, Sq-1). Known A24-binding anchors (Y/F at P2 and F/L/I at P9) were largely conserved among the ligands; however, a subset of peptides had an unusual anchor (K/R at the C-terminal P9 or P10), suggesting diverse usage of anchors in certain types of cancer cells. Moreover, a certain amino acid residue had significantly low frequency in the N-terminal flanking sequence, suggesting that it might inhibit processing of the peptides. Profiling of the natural HLA ligands of cancer cells revealed "neoantigen" peptides derived from mutated genome DNA and "aberrant antigen" peptides derived from long noncoding RNAs as well as "cancer-germ cell antigen" peptides. Among the natural HLA ligands derived from cancer cells, immunogenicity of the "neoantigen" peptide was the highest. The landscape clarified immunogenic peptide repertoire that might be suitable for cancer vaccine as well as as-yet-unknown rules for antigen processing. Citation Format: Yasuhiro Kikuchi, Takayuki Kanaseki, Ayumi Hongo, Serina Tokita, Toshihiko Torigoe, Kochin Vitaly. Landscape of natural HLA class I ligand peptides of cancer cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B018.

Abstract B089: Application of precision cancer immunotherapy design tools to bladder cancer: Non-self-like neoepitopes as a prognostic biomarker

Abstract Precision cancer immunotherapy targeting mutations expressed by cancer cells has proven to effectively control the tumor of patients in multiple clinical trials (Sahin et al., Nature 2017; Ott et al., Nature 2017). However, the selection of immunogenic T-cell neo-epitopes remains challenging and many epitopes selected using traditional methodologies fail to induce effector T-cell responses. Poor performance may partially be due to inclusion of mutated epitopes cross-conserved with self-epitopes recognized by regulatory (Treg), anergic, or deleted T-cells. Vaccination with self-epitopes can lead to weak effector responses, active immune suppression, and toxicity due to immune-mediated adverse effects. In addition, most cancer vaccine studies focus on the selection of CD8 T-cell neo-epitopes due to an apparent lack of robust and accurate CD4 T-cell epitope prediction tools. We have developed Ancer, an integrated and streamlined neo-epitope selection pipeline, that accelerates the selection of both CD4 and CD8 T-cell neo-epitopes from next-generation sequencing (NGS) data. Ancer leverages EpiMatrix and JanusMatrix, predictive algorithms that have been extensively validated in prospective vaccine studies for infectious diseases (Moise et al., Hum Vaccines Immunother 2015; Wada et al., Sci Rep 2017). Distinctive features of Ancer are its ability to accurately predict Class II HLA ligands, or CD4 epitopes, with EpiMatrix, and to identify tolerated or Treg epitopes with JanusMatrix. In addition, screening candidate sequences with JanusMatrix enables to the removal of neo-epitopes that may trigger off-target events, which have in some cases abruptly halted the development of promising cancer therapies. Ancer was applied to NGS data derived from the BLCA bladder cancer cohort from The Cancer Genome Atlas (TCGA) database. On average, 55 out of 204 missense mutations in bladder cancer patients’ tumors met Ancer’s quality control standards, in an initial analysis carried out for a representative set of 11 patients. This subset of high-quality missense variants was then screened using Ancer settings defined by the unique HLA of each patient, to derive the best vaccine candidate sequences encompassing these mutations. A median number of 24 (interquartile range: 15-64) candidate sequences were generated for each patient under study. The time required to select sequences for all of the patients in this study was less than two days. This initial analysis of eleven BLCA bladder cancer cohort patients demonstrates the capacity of Ancer to define a sufficient number of candidate sequences for vaccinating bladder cancer patients in a precision immunotherapy setting. We also assessed Ancer’s ability to predict patient outcomes on a larger subset of 58 individuals. While the disease-free status of BLCA patients could not be explained by their tumor mutational burden (AUC = 0.55, p-value = 0.1328), nor by their load of missense mutations (AUC = 0.54, p-value = 0.1740), the number of neoepitopes highly different from self, as defined by Ancer, significantly segregated disease-free patients from patients who recurred or progressed (AUC = 0.68, p-value = 0.0214). These results suggest that defining the number of true neoepitopes using Ancer may represent a novel biomarker for more robust antitumor immune response and higher likelihood of disease-free survival.Our analysis of the BLCA cohort from the TCGA database showcases the value of Ancer in clinical settings. Ancer can be used to identify high-value candidate sequences for inclusion in personalized therapies while removing potentially tolerated or tolerogenic self-epitopes from consideration. Our next step will be to investigate whether Ancer-defined neoepitope load will serve as a biomarker for prognosis and response to therapy in the full BLCA cohort. Citation Format: Guilhem Richard, Randy F. Sweis, Leonard Moise, Matthew Ardito, William A. Martin, Gad Berdugo, Gary D. Steinberg, Anne S. De Groot. Application of precision cancer immunotherapy design tools to bladder cancer: Non-self-like neoepitopes as a prognostic biomarker [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B089.

Differential contribution of education through KIR2DL1, KIR2DL3, and KIR3DL1 to antibody-dependent (AD) NK cell activation and ADCC.

The engagement of activating NK receptors (aNKR) stimulates NK cell activity, provided that interactions between inhibitory NK receptors (iNKR) with their HLA ligands do not override them. Abs bound to target cells can also activate NK cells by engaging the CD16 aNKR. NK cell education status is an important factor for Ab‐dependent NK cell activation (ADNKA) of some NK cell subsets. However, whether NK cell education also influences Ab‐dependent cellular cytotoxicity (ADCC) levels is not fully known. ADCC‐GranToxiLux (GTL) assays measured ADCC activity as the frequency of granzyme B positive (%GzB+) target cells. Target cells were anti‐HIV Immunoglobulin G (HIVIG)‐opsonized CEM‐NKr.CCR5 (CEM) cells. Lymphocytes and sorted single positive (SP) NKG2A+, KIR2DL1+, KIR2DL3+, and KIR3DL1+ NK cells, to self‐ and nonself HLA, were used as effectors in ADCC‐GTL assays to examine how education status influenced ADCC activity. ADNKA activity was assessed by stimulating lymphocytes with HIVIG‐opsonized CEMs and measuring the frequency of NK cell populations defined by their expression of iNKRs, along with IFN‐γ, CCL4, and CD107a functions. ADCC: the %GzB+ CEM cells generated by self‐ versus nonself HLA‐specific SPiNKR did not differ. ADNKA: More NK cells educated through KIR2DL1 and KIR3DL1, but not KIR2DL3, responded to ADNKA than their uneducated counterparts. CD16 engagement induced ADCC and ADNKA activity. With the proviso that groups’ sizes were small, our results support the notion that NK cell education does not influence ADCC levels but does contribute to ADNKA activity.

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

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

Long non-coding RNA expression analysis during mouse testis development using the FANTOM5 data

Specific killer cell immunoglobulin-like receptor (KIR) in women with recurrent pregnancy loss (RPL) and HLA ligands in couples invoke a susceptibility to RPL. However, the relationship between KIR2DL2 and its cognate ligand HLA-C1 has not been explored. In this prospective cohort study, 160 Caucasian women with RPL and 99 partners were included. KIR/HLA-C typing, NK assay, Th1/Th2 intracellular cytokine ratios, 25-(OH)-vitamin D level, and the presence of autoantibodies were analyzed. KIR2DL2 positive women (P = 0.023) and their partners (P = 0.017) had lower allele frequencies of HLA-C1 than those of KIR2DL2 negative women. KIR2DL2 positive women had significantly lower genotype frequency of HLA-C1C1 as compared to the North American Caucasian population controls (P < 0.05). In the partners of KIR2DL2 positive women, there was a substantially higher frequency of HLA-C2C2 than controls (P = 0.016). Besides, KIR2DL2 negative women had a higher prevalence of anti-ssDNA antibody as compared with that of KIR2DL2 positive women (P = 0.043). There were no differences in the distribution of HLA-C genotypes based on KIR2DL2, regardless of pregnancy outcome in women with RPL and their partners while on immunomodulation treatment. In conclusion, decreased ligands for inhibitory KIRs (inhKIR) could lead to insufficient inhibition of maternal uterine NK cells toward the trophoblast, thereby contributing to the pathogenesis of RPL. Specific KIR and HLA-C genotyping may predict the reproductive outcome of women with RPL.

P006 NK KIR receptor genotypes are associated with relapse in mexican HLA matched sibling patients with acute lymphoblastic leukemia (ALL)

Aim The outcome of Hematopoietic Stem Cell Transplantation (HSCT) is strongly associated with the HLA identity of donor/recipient, and GVHD remains the most frequent associated factor with mortality and impact on the quality of life. A successful HSCT depends also on T-cell mediated graft vs. leukemia (GvL) effect. Relapse is a major important challenge. NK cells, KIR and their HLA ligands are involved with an increase in overall survival, as well as better engraftment and reduced incidence of GVHD in patients with AML. The aim of the study was to determine the KIR gene content, their HLA ligands and their association on the outcome of ALL patients transplanted with HLA-identical sibling donors. Methods Twenty-seven Mexican patients with their HLA-identical siblings were included. Of these, 20 had ALL and 7 AML. KIR gene content was determined with the LinkSēq™ KIR kit and was run on an ABI 7500 Real-Time PCR instrument. Analysis was done with SureTyper 5. HLA intermediate resolution typing was performed by SSOP on a Luminex. HLA alleles were analyzed with the Match it DNA 1.2.0 software. KIR haplotypes and HLA ligands were assessed using https://www.ebi.ac.uk/ipd/kir/ligand.html . Allele Frequency (AF) and multiple parameter comparison analysis was performed using the SPSS17 software. Results We looked if activating or inhibitory KIR genes had any effect on relapse, long term disease free survival (DFS), GVHD or mortality. An association with relapse was observed in ALL patients, when the matched sibling donor carried the 3DL1 -Bw4 ligand (50% of ALL patients were carriers); (Chi2 = 5.93; p = 0.03) with no differences in Bw4 80I and 80T ligand. The AA and Bx haplotypes did not have any association with mortality, GVHD, relapse or DFS. Conclusions The results in ALL regarding relapse show that the presence of 3DL1 -Bw4, conveys higher susceptibility for relapse, as shown for AML. Contrary to AML, no effect was detected with 80I or 80T with intermediate relapse and mortality, implying a non benefit of education and sensitivity to inhibition as reported for AML. More cases of ALL are being analyzed to confirm if HLA and KIR allele typing in donor selection, enables the antileukemic benefits of NK alloreactivity in HSCT, in ALL, as it has been pointed out in AML, since the affected cell population in ALL, is totally different of the latter.