HLA Ligands Research Articles

The antigenic landscape of multiple myeloma: mass spectrometry (re)defines targets for T-cell–based immunotherapy

AbstractDirect analysis of HLA-presented antigens by mass spectrometry provides a comprehensive view on the antigenic landscape of different tissues/malignancies and enables the identification of novel, pathophysiologically relevant T-cell epitopes. Here, we present a systematic and comparative study of the HLA class I and II presented, nonmutant antigenome of multiple myeloma (MM). Quantification of HLA surface expression revealed elevated HLA molecule counts on malignant plasma cells compared with normal B cells, excluding relevant HLA downregulation in MM. Analyzing the presentation of established myeloma-associated T-cell antigens on the HLA ligandome level, we found a substantial proportion of antigens to be only infrequently presented on primary myelomas or to display suboptimal degrees of myeloma specificity. However, unsupervised analysis of our extensive HLA ligand data set delineated a panel of 58 highly specific myeloma-associated antigens (including multiple myeloma SET domain containing protein) which are characterized by frequent and exclusive presentation on myeloma samples. Functional characterization of these target antigens revealed peptide-specific, preexisting CD8+ T-cell responses exclusively in myeloma patients, which is indicative of pathophysiological relevance. Furthermore, in vitro priming experiments revealed that peptide-specific T-cell responses can be induced in response-naive myeloma patients. Together, our results serve to guide antigen selection for T-cell–based immunotherapy of MM.

KIR–HLA genotypes suggestive of opposing natural killer cell responses are associated with tuberculosis and sarcoidosis, the granulomatous diseases with similar pathology

Aim The clinical, radiological and pathological similarities between pulmonary tuberculosis (PTB) and sarcoidosis (PS) can make disease differentiation challenging, particularly in countries with high burden of TB. Herein we analyzed KIR–HLA genotypes that might provide insight into immunological relationship between the diseases and allow us to distinguish between them. Methods We typed 375 PTB patients, 201 PS patients and 249 healthy controls collected from All India Institute of Medical Sciences, India for KIR and HLA class I genes using Luminex rSSO. All controls were +ve to PPD, but displayed normal chest radiograph and did not suffer from any disease. Results Differences between controls and patients in KIR genotypes, HLA ligands, and KIR–HLA pairs were tested by two-tailed Fisher’s exact probabilities (Table). Distribution of AA and Bx KIR genotype did not vary between study groups. However, B haplotype-specific KIRs of centromeric region (2DL2, 2DS3) were reduced significantly in PS. Moreover, 3DL1+Bw4 and 2DL2+C1 pairs decreased significantly in PS. These results suggest the possibility of generating functionally compromised NK cells due to missing 3DL1+Bw4 and/or 2DL2+C1-mediated licensing, which is consistent with known diminished cellular immunity in PS. Contrasting to PS, telomeric half of KIR complex was associated with PTB. Specifically, an increase of B-haplotype associated KIRs (3DS1, 2DS1) and a decrease of A-haplotype associated KIRs (3DL1, 2DS4) were observed in PTB. Moreover, a substantial increase of 2DL1+C2 (p = 0.0001) and a decrease of 3DL1+Bw4 (p = 0.023) were observed in PTB. These results suggest the possibility of generating more activating NK cells by 2DL1+C2-mediated licensing and expressing activating KIR 3DS1/2DS1 phenotypes, which is consistent with active immune response in PTB. Conclusions Association of distinct KIR-HLA genotypes predicts potentially distinct immune mechanisms for PTB and PS, and provides a more promising approach to differential disease diagnosis. Download : Download full-size image

HLA ligandomics identifies histone deacetylase 1 as target for ovarian cancer immunotherapy

abstractThe recent approval of clincially effective immune checkpoint inhibitors illustrates the potential of cancer immunotherapy. A challenging task remains the identification of specific targets guiding immunotherapy. Facilitated by technical advances, the direct identification of physiologically relevant targets is enabled by analyzing the HLA ligandome of cancer cells. Since recent publications demonstrate the immunogenicity of ovarian cancer (OvCa), immunotherapies, including peptide-based cancer vaccines, represent a promising treatment approach. To identify vaccine peptides, we employed a combined strategy of HLA ligandomics in high-grade serous OvCa samples and immunogenicity analysis. Only few proteins were naturally presented as HLA ligands on all samples analyzed, including histone deacetylase (HDAC) 1 and 2. In vitro priming of CD8+ T cells demonstrated that two HDAC1/2-derived HLA ligands can induce T-cell responses, capable of killing HLA-matched tumor cells. High HDAC1 expression shown by immunohistochemistry in 136 high-grade serous OvCa patients associated with significantly reduced overall survival (OS), whereas patients with high numbers of CD3+ tumor-infiltrating lymphocytes (TILs) in the tumor epithelium and CD8+ TILs in the tumor stroma showed improved OS. However, correlating HDAC1 expression with TILs, high levels of TILs abrogated the impact of HDAC1 on OS. This study strengthens the role of HDAC1/2 as an important tumor antigen in OvCa, demonstrating its impact on OS in a large cohort of OvCa patients. We further identified two immunogenic HDAC1-derived peptides, which frequently induce multi-functional T-cell responses in many donors, suitable for future multi-peptide vaccine trials in OvCa patients.

Susceptibility to IBD is associated with with specific KIR and HLA ligand genotypes (BA4P.142)

Abstract Adaptive and innate host immune responses to intestinal microbes have been implicated in the pathogenesis of Ulcerative Colitis and Crohn’s Disease, the subtypes of Inflammatory Bowel Disease (IBD). Polymorphisms of the Killer Ig-like Receptors (KIR) genes, which encode receptors on natural killer cells, influence immune mediated diseases. Here we study the genetic contribution of KIR and their HLA ligands in UC and CD. HLA genotyping was performed by next generation sequencing and KIR genotyping with our MALDI-TOF assay. The KIR HLA ligands, Bw4/6 and C1/C2 were assigned using HLA-B and HLA-C residue positions 77-83. The case-control analysis consisted of 1232 Caucasian IBD patients (461 with UC, 771 with CD) and 203 Caucasian controls. The homozygous genotypic combination of KIR2DL3, KIR3DL1 (homozygous KIR A/A haplotype) and Bw6 (absence of Bw4) with a heterozygous C1/C2 genotype was protective (cf, C1/C1 and C2/C2) against ulcerative colitis (OR = 0.29, p < 0.001). No significant association was found with KIR loci and their HLA-ligands with CD. In a C1/C2 heterozygote, signaling would be expected via both the KIR2DL3 receptor and the KIR2DL1 receptor. These data reveal a central role for a specific combination of KIRs and HLA ligands (i.e., homozygous KIR A haplotype, absence of Bw4 and C1/C2 heterozygosity) in protecting against UC disease and underscore the importance of analyzing KIR genotypes in the context of their HLA ligands in disease association studies.

The role of KIR and HLA polymorphism in HIV acquisition: association and in vitro functional studies (VIR1P.1159)

Abstract Polymorphism in the genes that encode the HLA antigens and the KIR receptors on NK cells influence both adaptive and innate immunity. We analyzed the role of HLA and KIR polymorphisms in HIV acquisition by comparing 405 HIV+ and 523 HIV- men in the Chicago MACS cohort. We found no significant association with any HLA allele, in contrast to the well established strongly protective effect of HLA-B*57:01 for HIV disease progression. The inhibitory KIR ligands are HLA class I epitopes and fall into the groups: HLA-C1,C2, HLA-Bw4, and HLA-A3/A11. Association analyses of individual KIR genes in the presence/absence of their cognate HLA ligand revealed that one KIR/HLA ligand genotype combination was protective for HIV acquisition. The HLA ligand genotype C1/C1 was protective compared to C1/C2 and C2/C2 but only with genotype KIR2DL3+/KIR2DL2- (OR = 1.76, p = 0.007). This KIR genotype marks homozygosity for the centromeric KIR A haplotype, which is more inhibitory, has fewer genes, and less diverse than KIR B haplotypes. Since C2 is the ligand for the KIR2DL1 receptor, and C1 is the ligand for the KIR2DL3 receptor, the protective association observed with C1/C1 homozygotes suggests that a lack of signaling via the strongly inhibitory KIR2DL1 receptor in addition to signaling via the KIR2DL3 receptor is protective. In vitro assays of NK cells from KIR2DL3 homozygous individuals demonstrate that C1/C1 vs C1/C2 and C2/C2 (C2+) genotypes are more active in terms of cytokine release.

P0463 : Influence of killer cell immunoglobulin-like receptors (KIRs) and their HLA ligands on vertical transmission and chronicity of hepatitis C virus in children

P0463 : Influence of killer cell immunoglobulin-like receptors (KIRs) and their HLA ligands on vertical transmission and chronicity of hepatitis C virus in children

Mycosis fungoides: association of KIR ligands and HLA-DQB1*05 with bad prognosis of the disease.

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma. We previously reported that the prognosis of MF patients is not only related on clinical variables but it is also associated with peculiar HLA alleles. Until today, the association of HLA ligands for KIR with the prognosis of the disease has not yet been analysed. We investigated the frequency of HLA ligands for killer cell Immunoglobulin-like receptors (KIRs) in MF patients, evaluating if the presence of particular HLA alleles that are ligands for KIR may have prognostic value. The study includes 46 Caucasian MF patients that, between 1993 and 1997, underwent HLA genomic typing. All patients were diagnosed and followed up from 1977 to 2012 (mean follow-up of 11 years). MF patients have been divided into two groups (long survivors and dead patients). We noticed that the HLA-Bw6/Bw6 specificity increased among the group of seven dead patients compared to the group of 39 long survivors (71.4% vs. 41.0%, P = ns, OR = 3.59), while in the long survivors group the HLA- Bw4/Bw4 specificity increased when compared to dead patients (23.0% vs. 0%, P = ns). Moreover, we observed that six of the seven dead patients had HLA-DQB1*05; the phenotypic frequency of this HLA allele, in dead and long survivors patients, was 85.7% and 23.0% respectively (P = 0.004; OR = 20). Our observations suggest that the presence of the HLA-DQB1*05 alleles characterizes the patients with the poorest prognosis in MF. In addition, absence of the KIR-ligand epitope HLA-B Bw4 showed a trend of being more prominent in MF patients with the poorest prognosis.

Comparison of the KIR3DS1/Bw4 distribution in Chinese healthy and acute myeloid leukemia individuals

Killer cell immunoglobulin like receptor (KIR) 3DS1 is one of the most important activating receptors and some studies revealed that KIR3DS1 combined with HLA ligand was not related to acute myeloid leukemia (AML), but rare data was reported in Chinese population. In this study, KIR3DS1 gene polymorphisms and HLA-Bw4 were investigated in 189 Chinese AML patients compared with 166 healthy individuals. The results showed that the distributions of KIR3DS1, Bw4, 3DS1/Bw4 and 3DS1/Bw4-80I were insignificantly different between AML and healthy individuals. This study suggests that the presence of 3DS1 and HLA-Bw4 ligands have no effect on AML disease.

Role of Donor Activating KIR–HLA Ligand–Mediated NK Cell Education Status in Control of Malignancy in Hematopoietic Cell Transplant Recipients

Some cancers treated with allogeneic hematopoietic stem cell transplantation (HSCT) are sensitive to natural killer cell (NK) reactivity. NK function depends on activating and inhibitory receptors and is modified by NK education/licensing effect and mediated by coexpression of inhibitory killer-cell immunoglobulin-like receptor (KIR) and its corresponding HLA I ligand. We assessed activating KIR (aKIR)-based HLA I–dependent education capacity in donor NKs in 285 patients with hematological malignancies after HSCT from unrelated donors. We found significantly adverse progression-free survival (PFS) and time to progression (TTP) in patients who received transplant from donors with NKs educated by C1:KIR2DS2/3, C2:KIR2DS1, or Bw4:KIR3DS1 pairs (for PFS: hazard ratio [HR], 1.70; P = .0020, Pcorr = .0039; HR, 1.54; P = .020, Pcorr = .039; HR, 1.51; P = .020, Pcorr = .040; and for TTP: HR, 1.82; P = .049, Pcorr = .096; HR, 1.72; P = .096, Pcorr = .18; and HR, 1.65; P = .11, Pcorr = .20, respectively). Reduced PFS and TTP were significantly dependent on the number of aKIR-based education systems in donors (HR, 1.36; P = .00031, Pcorr = .00062; and HR, 1.43; P = .019, Pcorr = .038). Furthermore, the PFS and TTP were strongly adverse in patients with missing HLA ligand cognate with educating aKIR-HLA pair in donor (HR, 3.25; P = .00022, Pcorr = .00045; and HR, 3.82; P = .027, Pcorr = .054). Together, these data suggest important qualitative and quantitative role of donor NK education via aKIR-cognate HLA ligand pairs in the outcome of HSCT. Avoiding the selection of transplant donors with high numbers of aKIR-HLA-based education systems, especially for recipients with missing cognate ligand, is advisable.

Impact of killer immunoglobulin-like receptor-human leukocyte antigens ligand incompatibility among renal transplantation

Killer immunoglobulin-like receptor (KIR) gene shows a high degree of polymorphism. Natural killer cell receptor gets activated once they bind to self-human leukocyte antigens (HLAs) with specific ligand. KIR gene and HLA ligand incompatibility due to the presence/absence of KIR in the recipient and the corresponding HLA ligand in the allograft may impact graft survival in solid organ transplantation. This study evaluates the effect of matches between KIR genes and known HLA ligands. KIR genotypes were determined using sequence specific primer polymerase chain reaction. Presence of certain KIR in a recipient, where the donor lacked the corresponding HLA ligand was considered a mismatch. The allograft was considered matched when both KIR receptor and HLA alloantigen reveald compatibility among recipient and donor. The data revealed better survival among individuals with matched inhibitory KIR receptors and their corresponding HLA ligands (KIR2DL2/DL3-HLAC2, KIR3DL1-HLABw4). On the contrary, no adverse effect was seen for matched activating KIR receptors and their corresponding HLA ligands. One of the activating gene KIR2DS4 showed risk (P = 0.0413, odds ratio = 1.91, 95% confidence interval = 1.02-3.57) association with renal allograft rejection. We conclude that the presence of inhibitory KIR gene leads to better survival; whereas activating motifs show no significant role in renal allograft survival.

Genes of killer cell immunoglobulin-like receptors and their HLA ligands after allogeneic hematopoietic stem cell transplantation in myeloid leukemia patients

To study the impact of the genes of donor killer cell immunoglobulin-like receptors (KIR) and HLA-KIR ligands on overall (OS) and event-free survival (EFS) rates in patients with myeloid leukemia after transplantation with allogeneic hematopoietic stem cells (allo-HSCT) from HLA-identical related and HLA-compatible unrelated donors. The investigation enrolled 29 patients who had undergone allo-HSCT from KIR-genotyped donors at the Department of Bone Marrow Transplantation, Hematology Research Center (see symbol) in 2010-2013. OS and EFS rates after allo-HSCT were calculated using the Kaplan-Meier method. The main predictor of recurrence and survival in patients after allo-HSCT was a recurrence-risk group the patient belonged to before transplantation. The standard-risk group patients whose donors had telomeric gene-content motifs of KIR-B haplotypes had higher EFS rates than those whose donors lacked these genes. The standard-risk patients homozygous for HLA-1 alleles (i.e. without HLA-C2 ligand) tended to have higher EFS rates, so did the patients without HLA-Bw4 ligand. The donors having telomeric gene-content motifs of KIR-B haplotypes are more preferred for allo-HSCT for patients with myeloid leukemia as the presence of donor telomeric KIR-B genes increases EFS rates in standard-risk patients.

NK cells with KIR2DS2 immunogenotype have a functional activation advantage to efficiently kill glioblastoma and prolong animal survival.

Glioblastomas (GBMs) are lethal brain cancers that are resistant to current therapies. We investigated the cytotoxicity of human allogeneic NK cells against patient-derived GBM in vitro and in vivo, as well as mechanisms mediating their efficacy. We demonstrate that KIR2DS2 immunogenotype NK cells were more potent killers, notwithstanding the absence of inhibitory killer Ig-like receptor (KIR)-HLA ligand mismatch. FACS-sorted and enriched KIR2DS2(+) NK cell subpopulations retained significantly high levels of CD69 and CD16 when in contact with GBM cells at a 1:1 ratio and highly expressed CD107a and secreted more soluble CD137 and granzyme A. In contrast, KIR2DS2(-) immunogenotype donor NK cells were less cytotoxic against GBM and K562, and, similar to FACS-sorted or gated KIR2DS2(-) NK cells, significantly diminished CD16, CD107a, granzyme A, and CD69 when in contact with GBM cells. Furthermore, NK cell-mediated GBM killing in vitro depended upon the expression of ligands for the activating receptor NKG2D and was partially abrogated by Ab blockade. Treatment of GBM xenografts in NOD/SCID mice with NK cells from a KIR2DS2(+) donor lacking inhibitory KIR-HLA ligand mismatch significantly prolonged the median survival to 163 d compared with vehicle controls (log-rank test, p = 0.0001), in contrast to 117.5 d (log-rank test, p = 0.0005) for NK cells with several inhibitory KIR-HLA ligand mismatches but lacking KIR2DS2 genotype. Significantly more CD56(+)CD16(+) NK cells from a KIR2DS2(+) donor survived in nontumor-bearing brains 3 wk after infusion compared with KIR2DS2(-) NK cells, independent of their proliferative capacity. In conclusion, KIR2DS2 identifies potent alloreactive NK cells against GBM that are mediated by commensurate, but dominant, activating signals.

The Impact of HLA and KIR Ligand Mismatching on Unrelated Allogeneic Hematopoietic Stem Cell Transplantation in Korean Adult Patients

BackgroundThe impact of HLA and KIR ligand mismatching on the outcome of hematopoietic stem cell transplantation (HSCT) remains unclear. Previous reports have identified considerable ethnic differences in the impact of HLA and KIR ligand mismatches, as well as KIR ligand status, on HSCT; however, to date, no data has been acquired in Korean adult patients.MethodsWe investigated the association of high-resolution HLA matching on five loci (HLA-A, -B, -C, -DRB1, and -DQB1), KIR ligand mismatching, and KIR ligand status on the outcome of allogeneic HSCT from unrelated donors in 154 Korean adult patients treated at Seoul National University Hospital.ResultsIn a multivariate analysis, less than 9/10 allelic matches in five HLA loci was an independent risk factor for acute graft-versus-host disease (GVHD) (grade II to IV) (P=0.019, odds ratio [OR]=2.7). In addition, HLA-A allele mismatching was increasingly prevalent in patients with acute GVHD compared to patients without (61.9% vs. 34.5%, P=0.06). For KIR ligand status, the patient and donor combination of both C1/C1 ligands showed better event-free and overall survival than combinations with C2 ligand patients or donors (P=0.048, P=0.034, respectively) by log-rank test.ConclusionsKorean adult transplant patients with less than 9 of 10 HLA allele matches in the HLA-A, -B, -C, -DRB1, and DQB1 loci have a higher likelihood of developing acute GVHD (grade II to IV). Impact of KIR ligand status on clinical outcome should be further studied in a larger patient population.

HLA Ligandome Analysis in Primary Polycythemia Vera (PV) Samples Shows No Tumor-Association of Naturally Presented JAK2 Ligands but Reveals a Vast Array of Novel PV-Associated HLA Ligands

Polycythemia vera (PV) is a myeloproliferative disorder mainly driven by the JAK2V617F mutant kinase. Except allogeneic stem cell transplantation, the current standard therapies, including novel therapies aiming for specific inhibition of JAK2 that are presently under investigation, do not offer a chance for cure. Of note, clear clinical benefits of unspecific immunotherapy with pegylated IFN-α suggest that PV might also be targeted by specific peptide based vaccination. Here we aimed to identify tumor associated HLA ligands of PV with special focus on peptides derived from JAK2. Therefore we analyzed HLA class I and II ligands of primary PV samples using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry.We analyzed the HLA class I ligandomes of 7 PV patients with the JAK2V617F mutation as well as of 4 patients with secondary (JAK2V617F negative) polycythemia. As controls, we profiled the ligandomes of 40 healthy volunteers (30 blood and 10 bone marrow samples). We identified more than 7,000 unique HLA class I ligands representing 4,435 proteins on primary PV samples. Moreover, 3 different HLA ligands derived from JAK2 on PV (KYLINLETL, HLA-A*24; KIGDFGLTK, HLA-A*03; GQGTFTKIFK, HLA-A*11) were identified. None of these ligands was found to be PV-exclusive, with JAK2 being represented on 3/4 (75%) of PV samples (JAK2V617F positive) and 18/30 (60%) healthy controls. No JAK2-derived peptides were identified on JAK2V617F negative patients, and no peptides covering the JAK2V617F mutation were identified. Since our previous work strongly suggests that tumor exclusive representation of an antigen is a prerequisite for the immunogenicity of its corresponding T cell epitopes, JAK2-derived wild-type peptides are unlikely to constitute optimal vaccines in PV. On the other hand, comparative profiling of the HLA class I ligandomes identified 935 antigens to be exclusively presented in JAK2V617F positive PV with 12 ligandome-derived tumor-associated antigens (LiTAAs) representing 26 different HLA ligands with exclusive representation in ≥30% of PV patients. Overlap analysis of PV patients with and without JAK2V617F mutation revealed 92% of the exclusive antigens to be represented on both groups, while only 8% and 0% showed exclusive representation in JAK2V617F positive and negative PV patients, respectively.As CD4+ T cells play important indirect and direct roles in anti-tumor immunity, we further applied our approach to HLA class II ligandomes of 8 PV patients (6 with, 2 without JAK2V617F mutation) and 18 HV (13 blood and 5 bone marrow samples) Using this approach, we identified more than 7,700 different naturally presented HLA class II ligands (1,700 source proteins). Notably, no JAK2-derived HLA class II peptides were identified in PV patients and HV ligandomes. Applying the same antigen-ranking strategy as described for HLA class I, we identified 5 additional HLA class II LiTAAs (exclusive in >30% of PV ligandomes) represented by 19 corresponding LiTAPs (ligandome-derived tumor-associated peptides). Overlap analysis of PV-exclusive source proteins revealed 2 proteins (POLA1, polymerase (DNA directed), alpha 1 and RHOQ, ras homolog family member Q) to be exclusively represented both in HLA class I and II ligandomes of PV patients.The novel HLA class I and II ligandome-derived tumor associated antigens identified in this study may constitute prime candidates for peptide-based immunotherapy of PV. DisclosuresNo relevant conflicts of interest to declare.

HLA Ligandome Profiling Identifies a Novel Category of Pathophysiologically Relevant CLL Associated Antigens

The breakthrough clinical success of immune checkpoint inhibition with PD-1 and CTLA-4 antibodies illustrates the potential of T cell based immunotherapy to effectively treat malignancies. However, a remaining major challenge is to increase the specificity and frequency of anti-cancer immune responses. A rational approach to achieve this goal is therapeutic vaccination, as highlighted by a recent phase II vaccination study using naturally presented HLA ligands, which induced vaccine-specific immune responses that were associated with improved clinical outcome (Walter et al., Nat. Med. 2012). This underscores the potential of identifying patho-physiologically relevant targets by direct differential analysis of the entire landscape of HLA-presented peptides, termed the HLA ligandome.Here we applied this approach to chronic lymphocytic leukemia (CLL) with the aim of developing a CLL-specific multi-peptide vaccine. Using cohorts of 30 patients and 30 healthy volunteers, we comparatively and extensively mapped the HLA ligandome landscape of CLL and identified more than 20,000 different HLA class I and II ligands representing 7,377 source proteins, attaining >95% of the maximum attainable coverage (as extrapolated from saturation analysis). A set of 49 HLA ligand source proteins (225 different HLA ligands) showed CLL-exclusive representation in >20% of CLL patients, thereby constituting the novel category of ligandome-derived tumor associated antigens (LiTAAs). These LiTAAs were validated to be broadly and frequently represented across different stages and mutational subtypes of CLL and found to be robustly represented in HLA ligandomes of patients undergoing standard chemo-/immunotherapy. Functional characterization by IFNγ-ELISPOT revealed peptide-specific immune recognition of 14/15 (93.3%) corresponding HLA ligands (LiTAPs) exclusively in CLL patients. These immune responses were strictly CLL (LiTAP-) directed and mediated by functional patient-derived CD8+ T cells. Notably, for immunoreactive LITAPs, a linear correlation between frequencies of representation in the CLL ligandomes and immune recognition by CLL patient PBMC was observed (R²=0.59), suggesting that LiTAP presentation on cancer cells is a direct prerequisite for the presence of anti-LiTAP immune responses. Moreover, we investigated the prognostic relevance of LiTAP-specific immune responses: Retrospective survival analysis of 45 CLL patients analyzed by IFNγ-ELISPOT assays comparing cases with T cell responses specific for 0-1 LiTAPs (n=32) versus >1 LiTAPs (n=13) suggests a prolonged overall survival in the multiple-response cohort as compared to the single/non-responders (P=0.0695, log-rank test, Fig. 1). Taken together these data provide clear evidence for tumor-dependent priming of LiTAP-specific T cells in CLL patients and indicate their involvement in tumor control in vivo. This directly implies these non-mutant self-peptides as pathophysiologically relevant tumor antigens in CLL thereby validating our target identification approach and encouraging future evaluation in clinical vaccination trials. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mapping the HLA Ligandome Landscape of Chronic Myeloid Leukemia (CML)—Towards Peptide Based Immunotherapy

Despite the success of targeted therapy with tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable. Immunotherapy, and in particular multi-peptide vaccination, may be a promising approach to eliminate residual CML cells. As of now, a multitude of potential vaccine targets have been proposed based on reverse immunology and functional genomic approaches focusing either on BCR-ABL junction peptides, which represent CML-specific neo-antigens, or on aberrantly expressed self-proteins such as WT-1, PR and hTERT. However, the results of clinical studies employing such antigens have so far not been encouraging. This might in part be due to the inherent limitations of the above mentioned approaches: evidence of natural presentation of the predicted epitopes is lacking and the correlation of transcript abundance and HLA restricted presentation of the corresponding gene product has been shown to be skewed. Modern mass spectrometry, on the other hand, enables the comprehensive analysis of the entirety of naturally presented HLA ligands on tissues of interest, termed the HLA ligandome.Here we implemented this direct approach and comparatively mapped the HLA ligandome landscape of 16 primary CML samples and 40 healthy volunteer (HV) controls (30 blood and 10 bone marrow samples). We identified more than 30,000 different naturally presented HLA class I ligands representing ~10,000 source proteins. Regression analysis suggests source protein identifications on CML (4,337 different proteins) to attain >95% of maximum achievable coverage with the implemented analytical setup. Based on this extensive dataset, we investigated the HLA restricted presentation of established CML-associated/specific antigens and applied a novel approach defining tumor-associated antigens strictly based on exclusive and frequent representation in CML ligandomes. Strikingly, we found the vast majority of previously described antigens including wild-type BCR protein (6% CML, 5% HV), Myeloperoxidase (56% CML, 15% HV) and Proteinase 3 (38% CML, 11% HV) to be (also) represented on normal PBMC or BMNC. No evidence of naturally presented BCR-ABL junction peptides was found. However, we identified a panel of 7 LiTAAs (ligandome-derived tumor-associated antigens) represented by 16 different HLA ligands, showing CML-exclusive representation in ≥25% of CML patient ligandomes.As CD4+ T cells mediate important indirect and direct effects in anti-tumor immunity, we further applied our approach to HLA class II ligandomes of 15 CML patients and 18 HV (13 blood and 5 bone marrow samples), identifying more than 9,000 different naturally presented HLA class II ligands (1,900 source proteins). Applying the same antigen-ranking strategy as described for HLA class I, we identified 7 additional HLA class II LiTAAs represented by 50 corresponding LiTAPs (ligandome-derived tumor-associated peptides). Overlap analysis of CML-exclusive source proteins revealed 6 proteins to be represented both in HLA class I and II ligandomes. Notably, for Galectin-1, which shows CML-exclusive representation in 19% of HLA class I and 13% of HLA class II ligandomes, one of the HLA class II ligands was found to contain a complete, embedded HLA class I peptide. Such naturally presented embedded HLA ligands might present optimal vaccine candidates that are recognized by both, CD4+ and CD8+ T cells.Functional analysis of the here defined HLA class I and II LiTAPs with regard to induction of T cell responses is presently ongoing and serves to validate them as prime targets for the development of an off-the-shelf peptide vaccination in CML patients. DisclosuresNo relevant conflicts of interest to declare.

Donor KIR Tel-B Haplotypes Beneficially Affect Outcome after Allogeneic Stem Cell Transplantation in Patients with Myeloid Malignancies

Introduction. Leukemia relapse after allogeneic hematopoietic stem-cell transplantation (allo-HSCT) remains a major clinical issue. Myelogenous leukemias are the most sensitive to natural killer (NK)-cell reactivity among blood malignancies treated with allo-HSCT. NK-cell function is controlled by an array of inhibitory and activating receptors, including killer cell immunoglobulin-like receptors (KIRs), whose genes vary in number and content. Inhibitory KIRs recognize the HLA class I ligands and mediate tolerance to self when they encounter self-HLA molecules on putative target cells. HLA-C allotypes with asparagine at position 80 (C1 ligands) are recognized by KIR2DL2/3, HLA-C allotypes with lysine at position 80 (C2 ligands) are recognized by KIR2DL1. HLA-A and –B allotypes with Bw4 motif are recognized by KIR3DL1. The ligands and function of activating KIRs are not well identified with the exception of KIR2DS1which recognizes HLA-C2 ligand group. KIR A haplotypes have simple, fixed gene content; B haplotypes have variable gene content and one or more of the B-specific genes: KIR2DS1, 2, 3, 5 KIR2DL2, KIR2DL5.The KIR locus divides into centromeric (Cen) and telomeric(Tel) regions. In both regions there are two distinctive types of variable KIR gene content motif - Cen-A/Cen-B and Tel-A/Tel-B. The aim of our prospective study was to evaluate the influence of immunogenetic factors (KIRs and their HLA ligands) on the outcome after allo-HSCT in patients with myeloid malignancies.Methods. 29 patients with different clonal myeloid disorders who underwent allo-HSCT in Research Center for Hematology (Moscow, Russian Federation) in 2011-2013 from HLA-identical related (n=19) and HLA-matched unrelated (n=10) donors were included in the study. The pre-transplantation risk category included standard or high risk. Acute myelogenous leukemia (AML) in complete remission 1 (CR1, n=15), chronic myelogenous leukemia (chronic phase 1, n=4), and myelodysplastic syndromes (n=2) classified as refractory anemia/refractory anemia with ringed sideroblasts were categorized as standard risk (n=21), whereas AML > CR1were considered as high risk (n=8). KIR genotyping was performed before allo-HSCT simultaneously with HLA-typing using a PCR-SSP kit (Invitrogen, WI, USA). Overall survival (OS) and event-free survival (EFS) were calculated by the Kaplan-Meier method, and compared with the log-rank test. OS was defined as survival without lethal event from any cause, EFS was defined as survival in complete remission without lethal event from any cause.Results. We found that the 3-year estimated OS and EFS in all patients were 59.7 and 50.0%, respectively. Conditioning regimen and graft source (related or unrelated) had no significant effect on transplant outcome. The pre-transplantation risk category was the main factor influencing survival. The probability of the 3-year OS and EFS for standard-risk patients was 85.7 and 69.0%, respectively; all high-risk patients died during 20 months after allo-HSCT. For standard-risk patients there was better EFS in patients whose donors had KIR Tel-B haplotypes in comparison with patients whose donors had no KIR Tel-B haplotypes (87.5 vs. 58.3%, p=0.08). Standard-risk recipients with HLA-C1/C1 ligands (i.e., C1 epitope present on both HLA-C alleles) had a tendency to an improved EFS compared with patients having either C1/C2 or C2/C2 ligands (88.9 vs. 55.6%, p=0.15). Also there was a tendency to better EFS in patients lacking HLA-Bw4 ligands compared with patients having those ligands (88.9 vs. 55.6%, p=0.15).Conclusions. Our small one single-center prospective study supports the hypothesis about the specific, genetically determined role of NK cell in the graft versus leukemia effect in myeloid malignancies. We suggest that donors having KIR Tel-B haplotypes are preferable for patients with myelogenous leukemias, and selection of the donors with KIR Tel-B haplotypes can improve outcome after allo-HSCT for myelogenous leukemias. DisclosuresNo relevant conflicts of interest to declare.

Correlation of Killer Immunoglobulin like Receptor Genes with the Rate of Cytomegalovirus Infection in Renal Transplantation Cases

Aim: Immune mechanisms of Cytomegalovirus (CMV) infection suggest a possible relationship between CMV with development of acute graft rejection. Current immune suppression impairs antiviral specific T-cell immunity in solid organ transplantation. Inhibitory/ activating NK receptor bindings activated by self HLA antigens confront allogeneic cells that lack a ligand for specific receptor. KIR ligand incompatibility caused due to presence/absence of KIR receptor in recipient and corresponding HLA ligand by the allograft which is recognized by KIR, may have potential impact on chance of CMV infection and graft survival in renal transplantation recipient. We hypothesized that predominance of activating KIR genes may downplay the rate of CMV infection among kidney transplant recipients. Methods: We have evaluated matches/mismatches between KIR genes and known HLA ligands among CMV disease (n=27) and primary CMV infected (n=259) conditions among North Indian renal transplant cases. Sequence specific primed polymerase chain reaction method was used for KIR genotyping. Results: Survival analysis revealed increased CMV risk for individuals carrying inhibitory KIR genes 2DL1 (OR=3.45, p-value=0.013) and 3DL1(OR=3.23, p-value=0.032), while protective association was revealed for activating KIR2DS1 (OR=0.32, p-value=0.005). Compatible KIR2DL2-HLA-C1 combination showed protective association (OR=0.23, p=0.031) with its ligand HLA-Bw4. KIR-HLA ligand match-mismatch also revealed protective association (OR=0.95, p-value=0.014) in the absence of KIR3DS1-HLA-Bw4 combination. Conclusion: Graft outcome after renal transplantation revealed prolonged survival in the presence of certain KIR/HLA class I ligand combinations among CMV diseased and primary CMV infected cases.

18.: Killer immunoglobulin-like receptors and their HLA ligands in Guillain-Barré syndrome

Guillain-Barre syndrome (GBS) is an inflammatory neuropathy that occurs in some individuals after exposure to an infectious illness. We investigated the role of killer-immunoglobulin-like receptors (KIR) and their HLA ligands as potential genetic factors in the pathogenesis of GBS. These receptors are involved in the innate immune response to infections. Polymerase chain reaction of KIR and their HLA ligands using DNA obtained from subjects with GBS and healthy controls was carried out. Whilst no significant differences in the frequencies of KIR genes were found, HLA-C2 and HLA-B Bw4-T were more frequent in subjects with GBS than controls (p

Abstract 2807: Activated NK cells efficiently kill Glioblastoma through activating receptor-ligand interactions

Abstract Glioblastoma (GBM) is the most malignant brain tumor, where the patients' survival remains dismally low despite multimodal therapies. Due to the immune suppressed status of GBM patients, adoptive cellular immunotherapy might represent an attractive therapeutic strategy. We hypothesized that allogeneic NK cells may be potent effectors against GBM due to their direct cytotoxicity and immunomodulatory properties through cytokine production. Nevertheless, it is not known how concurrent inhibitory and activating signals coordinate to regulate NK effector function against solid tumors. In vitro cytotoxicity assays using n=3 patient-derived GBM and K562 cells co-cultured with NK cells obtained from n=9 healthy donors revealed that activated NK cells efficiently killed GBM cells in a dose- and donor-dependent manner. The GBM cells variably expressed MHC class I, but highly expressed ligands for NKG2D receptor: MICA, ULBPs 1,2,3,5,6, and to a less extent MICB by flow cytometric phenotyping. Activated NK cells highly expressed NKp46, NKG2D, and NKG2A on the protein level. Analysis of high resolution genotyping of NK cell Killer Immunoglobulin-like Receptors (KIR) and HLA ligands expressed by GBM revealed that NK cell cytotoxic potency was associated with activating KIRs and NKG2D, and was partially independent of inhibitory KIRs repertoire in vitro. Blocking NKG2D receptor with specific antibody attenuated tumor lysis providing proof of concept for the contribution of this receptor. Experiments with antibodies blocking KIR2DS2 and KIR2DS4 receptors are in progress. Efficacy of NK cells obtained from donors with different KIR repertoires and infused intratumorally into human GBM-bearing NOD-SCID mice was investigated. The primary end points were animals' survival and mechanisms of therapeutic efficacy. Donor A lacked activating KIR2DS2 and KIR2DS4 and had inhibitory KIR-HLA ligands mismatched in the human GBM xenografts. In contrast, Donor B possessed activating KIR2DS2 and partial deletion KIR2DS4, as well as, a full repertoire of inhibitory receptors. NK cells from both donors significantly prolonged animal survival after single dose treatment compared to vehicle controls (p=0.0005 and p=0.0001; donor A and B respectively). Despite diminished inhibitory KIR-HLA ligand mismatch, donor B's NK cells extended the median survival to 163 days compared to 117.5 days of donor A´s NK cells. Furthermore, donor B´s NK cells significantly increased tumor apoptosis compared to control (p=0.04) and tumors treated with donor A´s NK cells (P=0.0004). Donor B´s NK cells significantly diminished tumor proliferation (p=0.02) and angiogenesis (p=0.04) compared to controls. In conclusion, allogeneic NK cells may be potent effectors against GBM mediated by activating signals through receptor-ligand interactions. However, further studies are required to evaluate their toxicity and applicability to GBM management. Citation Format: Justyna Kmiecik, Andrea Gras Navarro, Per Øyvind Enger, Lina Leiss, Jacques Zimmer, Martha Chekenya. Activated NK cells efficiently kill Glioblastoma through activating receptor-ligand interactions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2807. doi:10.1158/1538-7445.AM2014-2807