Specific HLA Alleles Research Articles

P065 Updating the common and well documented allele list

Aim The goal of this study was to update the 2012 common and well documented (CWD) allele list using data from 3940 class I and 4124 class II assignments obtained by next generation sequencing (NGS) of two registry (USA and Argentina) populations and 360 donor-recipient pairs from the CIBMTR repository. Methods DNA was prepared from blood, buccal swabs, or cell lines. HLA-A, -B, -C alleles were amplified with primers annealing in the 5’ and 3’ UTRs. The sequences of exons and introns were determined by next generation sequencing (NGS) with an Illumina Miseq using an in-house protocol and Conexio Assign software analysis. Class II NGS sequencing focused on exons 2 and 3; however, some alleles had additional exons and introns characterized. Alleles that were on the well-documented list and alleles not considered CWD based on Mack et al. Tissue Antigens 2013 were identified. Results The majority of alleles detected had previously been designated as common. For example, from the Argentine registry samples, 92% of 2944 HLA-A alleles identified were common. In the total study population, 77 class I alleles previously designated as WD and 97 alleles not defined as C or WD were observed from 1 to >5 times. Thirty-seven of the 174 class I alleles occurred greater than 5 times, e.g., previously undefined A*02:17:02, B*44:02:01:03, and C*17:01:01:05. The majority (90%) of the 19 not CWD alleles were intron variants e.g., A*03:01:01:05, B*39:01:01:03, C*06:02:01:03. Forty-five class II alleles fell into the WD or not defined categories; 29% occurred >5 times. Class II alleles occurring >5 times included DRB1*04:06:02, DQB1*03:01:01:03 and DPB1*04:02:01:02. Conclusions The NGS data expand our knowledge of the frequency of specific full length HLA alleles. The majority of the uncommon alleles characterized in this study are synonymous variations of common alleles.

P227 Determination of HLA -A, -B and - DRB1 alleles and HLA-A -B haplotype frequencies in Egyptians based on family study

Knowledge of allele and haplotype frequencies in human populations guides the search for an HLA- matched unrelated hematopoietic stem cell donor. A new algorithm developed by the National Marrow Donor program uses this information to predict the likelihood that a volunteer of a particular ethnicity will carry specific HLA alleles when typed at high resolution. In the present study, we determined the HLA-A,-B and -DRB1 alleles and HLA-A-B haplotype frequencies in Egyptians and compared them to various populations. Specificities of HLA-A, -B and -DRB1 loci were determined in 286 matched related donors of allogeneic hematopoeitic stem cell transplantation (AHSCT). Typing was performed using serologic and low resolution PCR- reverse sequence-specific oligonucleotide (rSSO) techniques. HLA-A-B haplotypes were defined by segregation analysis. The most frequent alleles for HLA class I HLA-A were: HLA-A2 (19.2%), A1 (15.8%), A30 (9.2%) and A24 (8.8%), for HLA-B, B41 (10.9%), B35 (10.2%), B44 (6%) and B50 (5.4%) and for DRB1∗13 (18.7%), ∗04 (15%) ,∗11(14.7%) and ∗03 (13.2%). The most frequent HLA-A-B haplotypes were: A24-B35 (3.8%), A2-B41 (3.4%), A1- B35 (3.4%) and A33-B14 (3.4%). This study report HLA allele and HLA-A-B haplotype patterns in Egyptians. These patterns were close to Jordanians and Lebanese and different from Saudi Arabians, Europeans, Americans and Asians. These data explain the difficulty of finding donors in world registries for Egyptian patients in need of a stem-cell transplant and highlight the need to for planning an Egyptian national unrelated donor registry to meet the demands of these patients.

P160 Development of a multiplexed assay and software package for HLA disease association screening with NGS

HLA genes are the most polymorphic genes in the human genome. The encoded HLA molecules play a pivotal role in generating a target dependent immune response. Several HLA molecules are even associated with a specific disease or drug responsiveness. For example; HLA-B∗27 is associated with Ankylosing Spondilitis, HLA-B∗57:01 is associated with abacavir hypersensitivity, and the HLA-DQ2 and HLA-DQ8 haplotypes are associated with Celiac Disease. Prototypes of the assay and software were tested in an Early Access Program (EAP). We have developed a single tube multiplexed amplification assay for fast and cost-effective genotyping of HLA-related diseases based on exon 2 and exon 3 of HLA-B, HLA-DQA1 and HLA-DQB1 genes (NGSgo-HLA-LinkX). Amplicons were processed in our NGSgo workflow for Illumina, and the resulting library was sequenced on the Illumina MiSeq. Obtained sequencing data was analyzed in our newly developed software package LINKengine, which reports whether a sample is positive or negative for the specific HLA allele. Furthermore, LINKengine reports the reliability of its results. Here we present the first results of the Early Access Program, including findings on the amplification protocol as well as details of the analyzed data generated for HLA-B, HLA-DQA and HLA-DQB. The reagents in combination with the software provide an easy method to genotype disease-related HLA genes. As the workflow after amplification is identical to the workflow of other NGSgo amplified products, amplicons can be combined in a single sequencing experiment, enabling more efficient use of the capacity of the NGS sequencing equipment.

Abstract 4978: Adoptively transferred CMV-specific T-cells recognizing dominant and sub-dominant pp65 epitopes demonstrate improved in vivo inhibition of tumor xenografts in combination with PD-1 inhibition

Abstract Adoptive transfer of transplant donor or third party donor derived CMV-specific T cells (CMV-CTL) can effectively treat CMV infections in HSCT recipients. In clinical trials, infusion of partially matched third party CMV-CTLs, has demonstrated high response rates against persistent CMV infection. T-cells (TC) generated in vitro or directly selected in vivo demonstrate a striking preponderance of specificity for 1-2 immunodominant (ID) epitopes presented by specific HLA alleles. ID epitopes elicit higher TC functional activity in vivo, compared to sub-dominant (SD) epitopes. The relative clinical efficacy of TC directed against ID versus SD epitopes in vivo remains undefined. Agents augmenting activity of TC responsive to SD epitopes are unexplored. When these alleles are co-inherited in humans, epitopes of CMVpp65 presented by HLA A*02:01 are ID over HLA A*24:02 presented epitopes. We describe an in vivo model to assess efficacy of CMV-CTLs using colon carcinoma cells (coca)transduced to express CMVpp65, as a surrogate system. HLA A*02:01 + and A*24:02 + human coca cells were transduced to express CMVpp65 and GFP-firefly luciferase (cocapp65). CMV-CTLs responding to either the A*0201 presented ID NLV epitope (A2-NLV) or the A*24:02 presented SD QYD epitope (A24-QYD) were generated from donors co-inheriting HLA A*02:01 and A*24:02 by in vitro stimulation using NIH 3T3 artificial antigen presenting cells, expressing HLA A*02:01 or A*24:02, B7.1, LFA-3, and ICAM1. Tumor cells (105 cells) were injected subcutaneously into groups of 5-6 NSG mice on the R flank, and 105 cells from a pp65 expressing melanoma cell line (melpp65), lacking expression of HLA A*02:01 or A*24:02 were injected on the L shoulder as control. 2 Groups each received 106 of tetramer+ A2-NLV or A24-QYD CMV-CTLs i.v per mouse; one of each CMV-CTL treated group also received 2 i.v doses ( 200µg /dose) of anti-PD1 antibody (Nivolumab-BMS) at day 2 and 7 post CTL infusion. Control groups received IL-2, with or without anti-PD1, or HLA mismatched CMV-CTLs. Tumor growth was monitored by bioluminescent imaging. CMV-CTLs responsive to SD A24-QYD epitope induced significant cocapp65 growth suppression compared to controls, but did not eradicate tumors in any animal. Combined treatment of A24-QYD CMV-CTLs with anti-PD-1 Ab induced complete cocapp65 eradication in 2 of 5 mice, with minute residual tumors in 3 mice. Treatment with ID A2-NLV CMV-CTLs induced complete cocapp65 eradication in 2 of 5 mice, and smaller residual tumors compared to SD A24-QYD CTL treatment. Combined treatment with anti-PD-1 and A2-NLV CMV-CTLs led to complete cocapp65 eradication in 3 of 5 mice, with minute tumors in 2 mice. Taken together, these data provide evidence that blocking the PD-1/PD-L1 interaction may significantly augment the antiviral activity of both ID and SD CMV-CTLs. Citation Format: Aisha N. Hasan, Annamalai Selvakumar, Tzu-Yun Kuo, Richard J. O'Reilly. Adoptively transferred CMV-specific T-cells recognizing dominant and sub-dominant pp65 epitopes demonstrate improved in vivo inhibition of tumor xenografts in combination with PD-1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4978. doi:10.1158/1538-7445.AM2017-4978

Molecular dynamics approach to probe the antigenicity of PagN – an outer membrane protein of Salmonella Typhi

PagN is a highly immunogenic 27-kDa outer membrane adhesin present in Salmonella Typhi. It plays a major role in the pathogenesis of typhoid fever and has emerged as a strong vaccine candidate. In this report, we predict the three-dimensional structure of PagN and describe the conformational dynamics associated with its four extracellular loops based on two 100-ns molecular dynamics simulations at 300 and 310 K. The formation and deformation of the secondary structures on these loops were also investigated during the simulations which revealed loops L1 and L2 to be highly flexible, whereas the relative flexibility of loops L3 and L4 was minimal. Essential dynamics and principal component analysis deciphered more realistic dynamic behaviours of the loops, particularly at 310 K. Moreover, our epitope predictions suggest that the antigenic peptides for B-cell recognition are located within the loops L1 and L2, while those for T-cell recognition are located within the loops L3 and L4. The binding specificities of the antigenic peptides towards specific human MHC-I and MHC-II HLA alleles closely resembled the stability of the loops L3 and L4 inferred from the simulations. Finally, we identified potential antigenic peptides in the flexible (L1 and L2) as well as stable (L3 and L4) regions of PagN for both B- and T-cell recognitions, which can help in developing effective sub-unit vaccines.

Severe Delayed Cutaneous and Systemic Reactions to Drugs: A Global Perspective on the Science and Art of Current Practice

Most immune-mediated adverse drug reactions (IM-ADRs) involve the skin, and many have additional systemic features. Severe cutaneous adverse drug reactions (SCARs) are an uncommon, potentially life-threatening, and challenging subgroup of IM-ADRs with diverse clinical phenotypes, mechanisms, and offending drugs. T-cell-mediated immunopathology is central to these severe delayed reactions, but effector cells and cytokines differ by clinical phenotype. Strong HLA-gene associations have been elucidated for specific drug-SCAR IM-ADRs such as Stevens-Johnson syndrome/toxic epidermal necrolysis, although the mechanisms by which carriage of a specific HLA allele is necessary but not sufficient for the development of many IM-ADRs is still being defined. SCAR management is complicated by substantial short- and long-term morbidity/mortality and the potential need to treat ongoing comorbid disease with related medications. Multidisciplinary specialist teams at experienced units should care for patients. In the setting of SCAR, patient outcomes as well as preventive, diagnostic, treatment, and management approaches are often not generalizable, but rather context specific, driven by population HLA-genetics, the pharmacology and genetic risk factors of the implicated drug, severity of underlying comorbid disease necessitating ongoing treatments, and cost considerations. In this review, we update the basic and clinical science of SCAR diagnosis and management.

Impact of genetic variations and transcriptional alterations of HLA class I genes on cervical cancer pathogenesis.

In a novel attempt to understand the variations in DNA sequences underlying HLA class I alleles associated with HPV16-related CaCx, we determined the alleles by reconstructing SNP-based haplotypes from resequencing of the most polymorphic exons 2 and 3 of HLA-A, HLA-B and HLA-C. We also determined the impact of SNPs and transcriptional alterations of the genes on CaCx. A high density of SNPs was identified from resequencing. HLA expression was determined by real-time PCR. We identified that even a single associated HLA allele had many underlying SNP-based haplotypes. Out of the most frequent (≥5%) HLA class I alleles, HLA-B*40:06 and HLA-B*15:02 respectively imparted significant risk towards and protection from CaCx as well as HPV16 infection. Employing median-joining networks to detect clusters of sequence-variations for specific HLA alleles, we found the protective SNP-based signature, GAATTTA, in all SNP-based haplotypes of HLA-B*15:02 allele. The signature was derived from seven SNPs within HLA-B which were newly associated with the disease. Contrarily, similarly derived risk-signature, TTGCGCC, mapped only to 52% of SNP-based haplotypes of HLA-B*40:06 allele. This indicated that all SNP-based haplotypes underlying a particular associated HLA allele might or might not have a single signature of risk/protection. HLA-A, HLA-B and HLA-C expressions were downregulated among CaCx cases compared to asymptomatic infections and HPV-negative controls. HLA-A and HLA-B were repressed in both cases harbouring episomal and integrated HPV16, whereas HLA-C in only the latter. Novel genetic variations and differential downregulation-patterns of HLA class I have a significant bearing on HPV16-related CaCx pathogenesis.

Abstract B12: Independent comparison of the state of the art in-silico HLA typing software

Abstract To accurately characterize the neo-antigen repertoire in tumors, identifying the HLA alleles of the patient is paramount. The binding of the neo-antigens to the patient's specific HLA alleles HLA-restriction has to be determined to identify neo-antigens that might play a role in stimulating an immune response in immunotherapy. The information on the patient's HLA alleles is captured in the next-generation sequencing (NGS) data and has to be accurately teased out using in-silico HLA typing software. However, to our knowledge, until now an independent study of HLA typing software has not been carried out. In this study, five publicly available HLA typing software packages were compared using 106 whole exome sequencing samples from the 1000 Genomes Project and a published dataset of 56 Asian nasopharyngeal tumor-normal pairs with experimentally determined HLA types. Whole exome sequencing samples of varying depth and coverage were chosen to simulate real-world data and test the robustness of the HLA typing software. Citation Format: Alvin Ng, Steve Rozen. Independent comparison of the state of the art in-silico HLA typing software. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B12.

A Cross-Validation Design Using Age and HLA Alleles for the Assessment of Clinical Schizophrenia Risk.

Schizophrenia (SCH) is a highly heritable disease that occurs mostly in young adults. Genetic factors usually play important roles in the onset of SCH. Human leukocyte antigen complex (HLA) genes are considered to be the important genetic predisposition factor and the genetic markers in SCH and other diseases. To screen SCH-associated HLA alleles, alleles for HLA-DRB1 and HLA-DQB1 loci were determined using the PCR-SSP typing kit. Statistically significant differences between observed and expected frequencies of allele combinations were identified. To further determine allele combinations related to SCH, an analysis of the age of onset of SCH in the patient who carried an allele or specific allele combination was conducted. Finally, a crossvalidation of the two sets of analytical results was carried out. Statistical differences in frequency values between the SCH group and the control population were observed for five HLA alleles. The results of cross-validation showed that there was a significant difference for a combination of DRB1*07 and DQB1*02, whereas no significant differences were observed for the other four alleles. We suggested that the combination of DRB1*07-DQB1*02 may be related to SCH susceptibility. The cross-validation design using age and HLA alleles could enable additional discoveries pertaining to the relationship between specific HLA allele combinations at different loci and the onset of complex diseases.

Are Polymorphisms in Genes Relevant to Drug Disposition Predictors of Susceptibility to Drug-Induced Liver Injury?

Despite considerable progress in identifying specific HLA alleles as genetic risk factors for some forms of drug-induced liver injury, progress in understanding whether genetic polymorphisms relevant to drug disposition also contribute to risk for developing this serious toxicity has been more limited. Evidence from both candidate-gene case control studies and genome-wide association studies is now discussed. In the case of genes relevant to drug metabolism, polymorphisms in cytochromes P450, UDP-glucuronosyltransferases, N-acetyltransferases and glutathione S-transferases as risk factors for DILI are assessed. The relevance of ABC transporters to drug-induced liver injury is also considered, together with data showing associations of particular ABCB11, ABCB1 and ABCC2 polymorphisms with some forms of drug-induced liver injury. Very few of the associations with genes relevant to drug disposition that have been reported have been well replicated. Even apparently well-studied associations such as that between isoniazid liver injury and N-acetyltransferase 2 slow acetylators remain problematic, though it seems likely that polymorphisms in drug metabolism genes do contribute to risk for some specific drugs. A better understanding of genetic risk factors for drug-induced liver injury will require further genome-wide association studies with larger numbers of cases, especially for forms of drug-induced liver injury where HLA genotype does not appear to be a risk factor.

Correlation Between Somatic Mutation Burden, Neoantigen Load and Progression Free Survival in Multiple Myeloma: Analysis of MMRF CoMMpass Study

IntroductionTumor specific somatic mutations can potentially influence patient response to targeted immunotherapies. Since somatic mutations generate neoantigens, novel tumor-specific peptides can be processed, loaded into MHC class I molecules, and presented to T-cells as potentially immunogenic targets. Indeed, both somatic mutation burden and neoantigen burden have been correlated with responsiveness to checkpoint inhibitor blockade in highly mutagenic advanced melanoma and lung cancer. However, correlation of response with mutation and neoantigen burden has not been reported for cancers of lower characteristic mutational burden such as multiple myeloma (MM). Indeed early results of checkpoint inhibitors in MM have been underwhelming. We therefore wished to better understand the mutation and neoantigen landscape of MM patients as it relates to responsiveness to current and future therapies.Materials/MethodsSomatic mutation burden, neoantigen load, and response to therapy was obtained using interim data from theCoMMpassstudy (NCT01454297), a study by the Multiple Myeloma Research Foundation that collects longitudinal clinical and molecular data on 1,000 newly diagnosed MM patients from baseline through treatment and disease progression. Next-generation exome sequencing data was used to call somatic mutations andbioinformaticallydetermine patient specific HLA type. Neoantigen binding affinity was predicted for all unique patient specific somatic missense mutation and HLA allele combinations using a well validated neural network and machine learning based algorithm. Binding affinity of the corresponding non-mutated peptide was also predicted to identify mutated neoantigens witha high binding affinity while the wild type counterpart has low predicted binding affinity.ResultsFor 627 patients with available sequencing and clinical data, mutation and neoantigen profiles were created. Within this population, the mean overall canonical somatic mutation load was 405.84 (s=608.55) mutations per patient. These mutations included 38,222 unique missense mutations in 12,994 genes. The mean somatic missense mutation load was 64.51(s=95.29) mutations per patient including immunoglobulin genes (52.70, s=94.85 excluding immunoglobulins). There was a positive linear correlation between mutation burden and neoantigen burden. The mean neoantigen load using thresholds of mutant peptide binding affinity (half maximal inhibitory concentration, IC50) less than 500nM was 69.57 (s=124.76) neoantigens (58.53, s=123.58 excluding immunoglobulins). Limiting binding affinity of wild type peptide sequence to greater than 500nM decreased the mean neoantigen load to 23.84 (s=53.55) neoantigens (20.74, s=53.28 excluding immunoglobulins).Progression free survival (PFS) following induction therapy was used to determine correlates of mutation burden excluding immunoglobulin mutations with response. Kaplan-Meier survival analysis revealed significantly shorter PFS time in patients with greater than average tumor somatic missense mutation load (N=126) relative to those with less than average mutation load (49.1% vs. 73.0% PFS at 2yrs, p=0.0055, Figure 1) Similarly, patients with greater than average neoantigen load (N=164) had significantly shorter PFS relative to those with less than average neoantigen load (48.0% vs. 74.2% PFS at 2yrs, p=0.00259, Figure 2). Moreover, patients with both high mutation and neoantigen load (N=85) had significantly shorter PFS relative to those with less than average mutation and neoantigen load (45.3% vs. 72% PFS at 2yrs, p= 0.0168).ConclusionWhile mutation and neoantigen load has been correlated with response to targeted immunotherapies in highly mutated solid tumors, we report here a negative correlation between mutation, neoantigen load and MM patient survival. This indicates that a high mutation andneoantigen load negatively impacts survival of myeloma patients under current standard of care. It is hypothesized that this is because current treatments fail to recruit T cell immunity necessary to exploit tumor neoantigens fundamental to immune-mediated killing of cancer cells. This presents an encouraging basis for pursuing targeted and personalized immunotherapies for multiple myeloma, especially for patients with a higher mutational burden. [Display omitted] [Display omitted] DisclosuresLonial:BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Novartis: Consultancy; BMS: Consultancy; Millenium: Consultancy; Merck: Consultancy.

Brief Report: HLA-DRB1, DQA1, and DQB1 in Juvenile-Onset Systemic Sclerosis.

Systemic sclerosis (SSc) is a rare disease that is particularly uncommon in children. Specific HLA alleles have been associated with SSc in adults. This study was undertaken to investigate HLA class II alleles in juvenile-onset SSc. DRB1, DQA1, and DQB1 alleles were determined by DNA-based HLA typing. Analyses were conducted comparing Caucasian patients with juvenile-onset SSc (n = 76) to healthy Caucasian controls (n = 581). Initial analyses focused on HLA class II associations previously reported in adult Caucasian patients with SSc. The frequency of DRB1*11 was not significantly increased in juvenile-onset SSc (22.4% of patients with juvenile-onset SSc versus 17.6% of controls; odds ratio [OR] 1.35, P = 0.34), nor were the specific DRB1*11:01 or *11:04 alleles. DQA1*05, a risk factor previously identified in adult men with SSc, was increased in patients with juvenile-onset SSc versus controls (57.9% versus 44.1%; OR 1.76, P = 0.027), as was DRB1*03 (34.2% versus 22.5%; OR 1.79, P = 0.031). Secondary analyses of all DRB1 allele groups revealed an association with DRB1*10 (10.5% of patients with juvenile-onset SSc versus 1.5% of controls; OR 7.48, P = 0.0002). As this is a new observation, correction was made for multiple comparisons of 13 different DRB1 allele groups; results nevertheless remained significant (P = 0.003). Also, a lower frequency of DRB1*01 was observed in patients with juvenile-onset SSc who were younger at disease onset (OR 0.06, P = 0.01) and in those with antibodies to topoisomerase (OR 0.14, P = 0.024). Associations of HLA alleles with juvenile-onset SSc differed from associations with SSc in women, but were similar to associations with SSc in men. Additionally, a novel association with DRB1*10 was observed in children. The greatest proportion of genetic risk of SSc is contributed by the HLA complex, and the current study reveals the importance of the association of HLA class II genes in juvenile-onset SSc.

In silico proposition to predict cluster of B- and T-cell epitopes for the usefulness of vaccine design from invasive, virulent and membrane associated proteins of C. jejuni.

PurposeCampylobacter jejuni is the one of the leading causes of bacterial diarrheal illness worldwide. This study aims to design specific epitopes for the utility of designing peptide vaccine(s) against C. jejuni by targeting invasive, virulent and membrane associated proteins like FlaA, Cia, CadF, PEB1, PEB3 and MOMP.MethodsIn the present study, various immunoinformatics approaches have been applied to design a potential epitope based vaccine against C. jejuni. The tools include Bepipred, ABCpred, Immune Epitope databse (IEDB) resource portal, Autodock vina etc.ResultsPeptides “EINKN”, “TGSRLN”, “KSNPDI”, “LDENGCE” respectively from FlaA, MOMP, PEB3, CadF proteins were found to be the most potential B cell epitopes while peptides “FRINTNVAA”, “NYFEGNLDM”, “YKYSPKLNF”, “YQDAIGLLV”, “FRNNIVAFV” and “LIMPVFHEL” respectively from Fla, CadF, MOMP, PEB1A, PEB3 and Cia might elicit cell mediated immunity and “IFYTTGSRL” from MOMP protein might elicit both humoral and cell-mediated immunity. All these potential peptidic epitopes showed almost 80–100 % conservancy in different strains of C jejuni with varying proportions of population coverage ranging from 22–60 %. Further authentication of these peptide epitopes as probable vaccine candidate was mediated by their binding to specific HLA alleles using in silico docking technique.ConclusionBased on the present study, it could be concluded that these predicted epitopes might be used to design a vaccine against C. jejuni bacteria and thus, could be validated in model hosts to verify their efficacy as vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s40203-016-0020-y) contains supplementary material, which is available to authorized users.

Immune DNA signature of T-cell infiltration in breast tumor exomes.

Tumor infiltrating lymphocytes (TILs) have been associated with favorable prognosis in multiple tumor types. The Cancer Genome Atlas (TCGA) represents the largest collection of cancer molecular data, but lacks detailed information about the immune environment. Here, we show that exome reads mapping to the complementarity-determining-region 3 (CDR3) of mature T-cell receptor beta (TCRB) can be used as an immune DNA (iDNA) signature. Specifically, we propose a method to identify CDR3 reads in a breast tumor exome and validate it using deep TCRB sequencing. In 1,078 TCGA breast cancer exomes, the fraction of CDR3 reads was associated with TILs fraction, tumor purity, adaptive immunity gene expression signatures and improved survival in Her2+ patients. Only 2/839 TCRB clonotypes were shared between patients and none associated with a specific HLA allele or somatic driver mutations. The iDNA biomarker enriches the comprehensive dataset collected through TCGA, revealing associations with other molecular features and clinical outcomes.

High-risk HLA alleles for severe acute graft-versus-host disease and mortality in unrelated donor bone marrow transplantation.

HLA molecules play an important role for immunoreactivity in allogeneic hematopoietic stem cell transplantation. To elucidate the effect of specific HLA alleles on acute graft-versus-host disease, we conducted a retrospective analysis using 6967 Japanese patients transplanted with T-cell-replete marrow from an unrelated donor. Using unbiased searches of patient and donor HLA alleles, patient and/or donor HLA-B*51:01 (patient: HR, 1.37,P<0.001; donor: HR, 1.35,P<0.001) and patient HLA-C*14:02 (HR, 1.35,P<0.001) were significantly associated with an increased risk of severe acute graft-versus-host disease. The finding that donor HLA-C*14:02 was not associated with severe acute graft-versus-host disease prompted us to elucidate the relation of these high-risk HLA alleles with patient and donor HLA-C allele mismatches. In comparison to HLA-C allele match, patient mismatched HLA-C*14:02 showed the highest risk of severe acute graft-versus-host disease (HR, 3.61,P<0.001) and transplant-related mortality (HR, 2.53,P<0.001) among all patient mismatched HLA-C alleles. Although patient HLA-C*14:02 and donor HLA-C*15:02 mismatch was usually KIR2DL-ligand mismatch in the graft-versus-host direction, the risk of patient mismatched HLA-C*14:02 for severe acute graft-versus-host disease was obvious regardless of KIR2DL-ligand matching. The effect of patient and/or donor HLA-B*51:01 on acute graft-versus-host disease was attributed not only to strong linkage disequilibrium of HLA-C*14:02 and -B*51:01, but also to the effect of HLA-B*51:01 itself. With regard to clinical implications, patient mismatched HLA-C*14:02 proved to be a potent risk factor for severe acute graft-versus-host disease and mortality, and should be considered a non-permissive HLA-C mismatch in donor selection for unrelated donor hematopoietic stem cell transplantation.

CD4+ T Cells Targeting Dominant and Cryptic Epitopes from Bacillus anthracis Lethal Factor.

Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called ‘cryptic’ or ‘subdominant’ epitopes. We analyzed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISpot assays we characterized epitopes that elicited a response following immunization with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, as a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 transgenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were influenced by the specific HLA alleles presenting the peptide, and imply that construction of future epitope string vaccines which are immunogenic across a wide range of HLA alleles could benefit from a combination of both cryptic and immunodominant anthrax epitopes.

HLA genes in Chimila Amerindians (Colombia), the Peopling of America and Medical implications

Our aim is to study the HLA-A, -B, -C, -DRB1 and -DQB1 gene frequencies in the Chimila Amerindian (Colombia) ethnic group. Results are compared with other World populations in order to obtain information about Chimila and Amerindian Health promotion, Amerindian origins and America peopling. Written consent was obtained from Chimila subjects to be included in this study. Peripheral blood was drawn and HLA DNA genotyping was carried out by standard methods. Analyses of Chimila relatedness with other Amerindians and worldwide populations was performed with a standard NJ dendrogram and correspondence analyses methodology. Chimila HLA gene profile showed to be related to that of other Amerindians groups. New complete HLA extended haplotypes were obtained. Some of them are described for the first time. Also, specific genealogical comparisons were done between Chimila Amerindians and Pacific Islanders by using specific HLA alleles. Our conclusions are: 1) These new data and HLA extended haplotypes are useful for present and future Chimila Preventive Medicine (HLA linked diseases), HLA Pharmacogenomics and transplantation regional programs, 2) Classical accepted origin of America peopling should be revised: Pacific (Asian and Austronesian) and Atlantic (European) populations gene exchange may have occurred before 1492 AD. This is confirmed by our present studies using HLA autosomic genetic markers. 3) Genetic HLA Amerindian profile is separated from that of other World populations. Keywords : America peopling, Amerindians, Anthropology, Chimila, Epidemiology, HLA and Transplantation.

Adoptively Transferred CMV-Specific T-Cells Responsive to Immunodominant CMVpp65 Epitopes Extend Durable in-Vivo Suppression of Clonogenic CMVpp65 [+] Human Colon Carcinoma Cells in Comparison to T-Cells Responsive to Subdominant Epitopes

Adoptively transferred transplant donor or third party donor derived CMV-specific T-cells (CMV-CTLs) can effectively prevent and treat CMV disease in HSCT recipients. T-cells (TC) respond to specific viral epitopes when presented by HLA class-I and class-II alleles on infected cells. We have infused third party CTLs from HLA partially matched donors that were chosen based on in-vitro CTL activity against epitopes presented by HLA alleles shared by the recipient, and this approach has afforded a 60% response rate among patients treated for CMV viremia and/or disease. A recurring feature of TC generated in-vitro or directly selected in-vivo is the striking preponderance of TC specific for 1-2 immunodominant epitopes presented by specific HLA alleles. However, the functional activity of such immunodominant CMV-CTLs as compared to CTLs directed against subdominant CMV epitopes has not been evaluated. To directly compare the activity of CMV-CTLs directed against immunodominant and subdominant epitopes, we developed an in-vivo model using human colon carcinoma cells transduced with CMVpp65 as a surrogate system. Human colon carcinoma cells co-expressing HLA A0201 and A2402 (cocapp65), and a melanoma cell line lacking expression of these HLA alleles (melpp65) were each transduced to also express CMVpp65 and a GFP-firefly luciferase transgene. Subcutaneous (s.c.) inoculation of 1 x 105 tumor cellsinto NOD/Scid-IL2Rgc-KO/J mice (NSG) provided consistent engraftment. CMV-CTLs responsive to either the immunodominant NLV epitope presented by HLA A0201 (A2-NLV) or the subdominant QYD epitope presented by HLA A2402 (A24-QYD) were generated from 2 donors, each co-inheriting HLA A0201 and A2402, using NIH 3T3 artificial antigen presenting cells, each expressing a single HLA class-I allele (A0201 or A2402), B7.1, LFA-3 and ICAM1.Groups of 5-6 NSG mice, each bearing 2 established tumors: cocapp65 and melpp65 (control), were intravenously injected with 2-4 x 106 of tetramer+ A2-NLV or A24-QYD specific CMV-CTLs per mouse. Control animals either did not receive any TC, or received HLA B0801- LTM specific CMV-CTLs, and IL-2 was given 2 x/week to all groups. Tumor growth was monitored for 6-8 weeks by bioluminescent imaging.Both A2-NLV and A24-QYD CMV-CTLs significantly suppressed growth of cocapp65 tumors in all treated animals with infusion of equivalent doses of epitope specific tet [+] T-cells (p = ns). No tumor suppression was observed in control animals, and there was no evidence of GvHD in CTL treated animals. A2-NLV CTLs eradicated cocapp65 tumor in 4/10 treated animals, while A24-QYD CTLs did not completely eradicate the tumor in any treated animal. In all A2-NLV treated animals, the cocapp65 remained suppressed until end of study (6 wks), while the melpp65 continued to grow. Treatment with A24-QYD CTLs induced tumor suppression after a time lag, as evidenced by initial cocapp65 growth for 10-14 days followed by suppression for 4-5 weeks and then stabilization of tumor size. In subsequent studies with animals bearing single cocapp65 xenografts, and infused with either A2-NLV or A24-QYD CTLs, the tumors remained suppressed for upto 8 weeks after A2-NLV CTL infusion, while recurrent tumor growth was observed 5 weeks after A24-QYD CTL infusion. Studies detailing the relative accumulation of CTLs within tumor tissue, as well as engraftment of CTLs in these animals are in progress. Phenotypic analysis of the infused epitope specific TC demonstrated 91-95% TEM with 5-9% TCM, with a highly restricted oligoclonal TCRVβ repertoire.This model provides a platform for direct comparative evaluation of the in-vivo cytotoxic activity of epitope specific TC. These studies demonstrate that CMV-CTLs responsive to both immunodominant as well as subdominant epitopes, that are generated using AAPC from the same donor co-inheriting the presenting HLA alleles, can suppress the growth of clonogenic human carcinoma cells co-expressing an immunogenic viral antigen in-vivo at equivalent doses of antigen specific TC. However, less robust antigen specific cytotoxic activity was demonstrated by the subdominant A24-QYD CMV CTLs, which maybe reflective of reduced in-vivo proliferation, effector function or persistence of such TC. Experiments of define these variables contributing to disparities in the in-vivo CTL activity are in progress. DisclosuresHasan:Atara Biotherapeutics: Research Funding. O'Reilly:Atara Biotherapeutics: Research Funding.

T Regulatory Cells Exhibit Surface Expression of FoxP3 Derived Peptides Presented within Class I MHC

Background:FOXP3 is a transcription factor of central importance to the inhibitory function of CD4+, CD25+ FOXP3+ T regulatory cells (Tregs). Tregs function as critical inhibitory immunoregulatory cells and have been reported to be increased in tumor-bearing individuals, inhibiting optimal T effector mediated anti-tumor cytotoxicity. Treg depletion targeting CD25, a surface marker of Tregs as well as activated T cells, has been validated in pre-clinical models as a promising adjunctive therapeutic approach to improving T cell mediated therapy and is currently being explored in clinical trials. However, because CD25 expression is not limited to Tregs, CD25-directed therapies may also result in the undesired depletion of effector T cells. Although FOXP3 expression is more restricted to Tregs as compared to CD25, its intranuclear localization has made it challenging to target therapeutically. Vaccination of mice against FOXP3 using FOXP3 mRNA-transfected dendritic cells has been explored and can elicit FOXP3 specific CTL mediated cytotoxicity. This work implies that FOXP3 mRNA-transfected dendritic cells process FOXP3 and present FOXP3 derived peptides on the cell surface. Direct expression of FOXP3 derived peptides on the surface of Tregs has however, not been previously demonstrated.Methods:We utilized mass spectrometry to investigate if FOXP3 derived peptides presented within MHC class I complex are expressed on the surface of Tregs. CD4+, CD25+ T cells were isolated from healthy donors using magnetic bead selection to enrich for Tregs. Surface class I MHC was immunoprecipitated using a pan-MHC class I anti- HLA-A, HLA-B and HLA-C antibody and associated peptides were eluted from their MHC complex using acid elution. Peptide and fragment masses were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the spectra of resulting epitopes were matched to known protein sequences by SEQUEST.Results:Four unique FOXP3 derived peptides were identified from the primary donor samples enriched for FOXP3+ cells as described. Predictive HLA binding algorithms demonstrate that the peptides identified are predicted binders to the specific HLA alleles of the original donors. No FOXP3 derived peptides were identified from the CD4+, CD25- T cell fraction analyzed to date.Conclusions:This work directly demonstrates for the first time to our knowledge that, although FOXP3 is an intra-nuclear transcription factor, FOXP3 derived peptides are presented within class I MHC on the surface of Tregs, representing a targetable Treg associated surface protein complex. DisclosuresLevy:Bullet Biotechnology, Inc.: Consultancy.

Interplay Between the Immune System and Colorectal Carcinoma - Towards Tumor-Specific Peptide-Based Vaccination for Any HLA-Type

Introduction: Peptide-basedcancer vaccines targeting tumor-associated antigens comprised of synthetic peptides have shown promising clinical results and have progressed to Phase III clinical testing in several tumor entities, including colorectal carcinoma (CRC). As of yet, these vaccines have typically been limited to HLA-A*02 positive patients, which means that more than half of the patient pool will not be eligible for those treatment strategies. The development of novel strategies for vaccine design, which are applicable to a larger fraction of potential patients, is therefore required. CRC seems to be particularly suitable for antigen-specific immunotherapy as it is among those cancers with highest rates of genetic mutations leading to alterations in protein metabolism. In this study, we aimed to identify HLA class I and II tumor-associated antigens from CRC covering the most frequent HLA-allotypes, in order to develop a peptide warehouse containing HLA-specific peptide panels that can be compiled in a patient-individualized manner, according to the respective patient's HLA-typing.Methods: The HLA presented immunopeptidome of 30 primary CRC samples and matched autologous non-malignant colon tissue was analyzed after HLA-immunoprecipitation by uHPLC tandem mass spectrometry. Identified source proteins and peptides were cross-evaluated with an in-house database of ligandome data derived from different benign tissues to preclude off-tumor presentation of HLA-ligands in order to to prevent any possible induction of autoimmunity by antigen-specific T cells against these ligands. Furthermore, we evaluated pre-existing antigen-specific T cell responses against these novel CRC-associated peptides in a cohort of 25 CRC patients.Results: About 17,000 MHC class I presented peptides could be identified on CRC and were cross-evaluated with our in-house database containing 39,000 MHC class I peptides presented on non-malignant tissue. The ligands identified were derived from 7,500 unique tumor-associated proteins and from 11,500 tumor-exclusive source proteins, respectively. Moreover, about 2,000 different MHC class II presented peptides from more than 1,600 source proteins were identified on CRC tissue. Interestingly, MHC class II peptides could contain shorter amino-acid sequences which - after further proteasomal degradation - might also bind to MHC class I molecules. For clinical applicability, we prioritized the identified ligands according to their frequencies of presentation and detectability on CRC. For representation in the HLA-specific peptide sets, a cutoff expression on at least 25% of allotype-matched tumors was required. This guided the assembly of a peptide vaccine warehouse consisting of 40 highly specific CRC-associated HLA ligands. Therefore, this selected set of HLA-specific peptides allows vaccination against CRC for about 95% of Caucasian patients. So far, we detected specific T cell reactivity against two peptides (detection of 10-fold and 17-fold increase of IFNγ-producing T cells representing spots as compared to background in enzyme linked immunospot assays - ELISPOT) in CRC patients.Conclusions: We here provide for the first time a comprehensive evaluation of HLA-ligandomes in CRC across the most frequent Caucasian HLA-alleles. We developed a HLA-allotype specific warehouse of newly identified CRC associated HLA-ligands, which can be individually assembled according to patient specific HLA-alleles. This approach expands the applicability of peptide-based cancer vaccines to literally every CRC patient. DisclosuresNo relevant conflicts of interest to declare.