scholarly journals Identification of Naturally Presented HLA Ligands of Enriched Leukemic Progenitor Cells for Peptide-Based Immunotherapy in Acute Myeloid Leukemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4046-4046 ◽  
Author(s):  
Annika Nelde ◽  
Heiko Schuster ◽  
Daniel J. Kowalewski ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Progenitor Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Isolation And Identification ◽  
Hla Ligands ◽  
Hla Ligandome

Abstract Several studies demonstrated that peptide-based cancer immunotherapy can induce specific immune responses and affect clinical outcome in a variety of different cancer entities. We recently conducted a study, which directly characterized the antigenic landscape of acute myeloid leukemia (AML) by mass spectrometric analysis of naturally presented HLA ligands and identified a panel of AML-specific CD4+ as well as CD8+ T-cell epitopes as suitable targets for T-cell based immunotherapy (Berlin et al. Leukemia 2015). One main reason for the high relapse rates in AML patients after standard polychemotherapy and allogeneic stem cell transplantation is the presence of minimal residual disease (MRD), which is associated with the persistence of leukemic stem cells (LSCs) in the bone marrow of patients. For clinically effective immunotherapy it is therefore indispensable to target the highly chemotherapy resistant LSCs. Here we present a mass spectrometry-based study, which for the first time analyzes the naturally presented HLA ligandome of stem cell enriched (LSCenr) fractions of primary AML samples to identify novel LSC-associated antigens using the approach of direct peptide isolation and identification. The enrichment of LSCs was performed using fluorescence-activated cell sorting of the originally described phenotype of lineage-negative CD34+CD38- cells of PBMCs from eight AML patients. The original stem cell containing population of 1-3% within the PBMCs of most patients was enriched to >90% purity with cell counts of 20-200x106 for the LSCenr fraction per sample. Consistent with our own previous results, all samples showed comparable expression levels of HLA class I molecules on primary leukemia blasts as well as for the LSCenr fractions, with HLA class I molecule counts ranging from 145,000 to 175,000 molecules/cell for the LSCenr fractions. To specifically identify leukemia-associated antigens on LSCenr cells, the HLA ligandome results obtained from the sorted LSCenrfractions were combined with data acquired from AML blasts of 20 AML patients (HLA class I n=19, HLA class II n=20) in previous studies as well as our normal tissue database that comprises 153 HLA class I and 82 HLA class II ligandomes of various healthy tissues (e.g. blood, bone marrow, spleen, kidney, liver, brain, skin, ovary, bowl). We identified more than 14,600 different naturally presented HLA class I ligands representing ̴6,500 source proteins on LSCenr fractions of primary AML samples (n=8) and their autologous blast cells by mass spectrometry. Overlap analysis of the HLA class I ligandomes of LSCenr fractions and autologous AML blasts with the benign peptidome revealed 45.4% (3,132/6,896) and 40.2% (4,922/12,244) of the LSCenr fraction and the autologous AML blast ligandomes to be represented in the benign-associated HLA ligandome, respectively. 79.1% (5,458/6,896) of the mapped LSCenr fraction ligandome was also presented on autologous AML blasts. 1,029 (14.9%) of these identified HLA class I ligands were presented exclusively on LSCenr fractions and not found on autologous AML blasts, previously analyzed AML blasts or any benign tissue. Furthermore, we were able to identify more than 8,000 different naturally presented HLA class II ligands representing ̴1,700 source proteins. Overlap of the HLA class II ligandomes revealed 45.0% (2,800/4,624) and 39.9% (2,706/6,790) of the LSCenr fraction and autologous AML blast ligandomes to be represented in the benign-associated HLA ligands, respectively. The HLA ligandomes of the LSCenr fraction and the autologous AML blasts showed an overlap of 69.7% (3,224/4,624). 941 (11.5%) HLA class II ligands showed exclusive representation in the LSCenr fraction ligandomes and were never identified on AML blast or benign tissue. These LSC-associated peptides represent highly interesting targets for immunotherapeutic approaches in AML patients and will be further evaluated for their potential to elicit a specific T-cell response. Taken together these preliminary results prove the feasibility of our approach to enrich leukemic progenitor cells of primary AML samples for the successful isolation and identification of HLA presented peptides associated with enriched leukemic progenitor cells. Disclosures Schuster: Immatics Biotechnologies GmbH: Employment. Kowalewski:Immatics Biotechnologies GmbH: Employment.

HLA Ligandome Analysis of Different Hematological Malignancies Identifies a Small Panel of "Pan-Leukemia"-Associated Antigens

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2169-2169
Author(s):  
Linus Backert ◽  
Daniel J. Kowalewski ◽  
Simon D. Walz ◽  
Heiko Schuster ◽  
Claudia Berlin ◽  
...  
Keyword(s):  
T Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Hematological Malignancies ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Hla Ligands ◽  
Hla Ligandome

Abstract Effective antigen-specific T-cell-based cancer immunotherapy requires exact knowledge of tumor-associated epitopes that can act as rejection antigens. While the current paradigm views mutation-derived neoantigens as the most promising targets, we have recently demonstrated that tumor-specific T-cell responses target panels of non-mutated tumor-associated self antigens in patients with hematological malignancies. Using the approach of direct HLA ligandome analysis by mass spectrometry, we were able to identify and characterize multiple immunogenic and naturally presented tumor-associated antigens for chronic lymphocytic leukemia (CLL, Kowalewski et. al., PNAS 2015), acute myeloid leukemia (AML, Berlin/Kowalewski et. al., Leukemia 2014), multiple myeloma (MM, Walz/Stickel et. al., Blood 2015) and chronic myeloid leukemia (CML, unpublished data). In this project we performed a comprehensive meta-analysis of our HLA ligandome data from different hematological malignancies (HM) to screen for the existence of "pan-leukemia" antigens for the broad application in T-cell based immunotherapy approaches in hematological malignancies. In a first step we performed unsupervised cluster analyses to identify similarities and differences in the HLA ligandome landscape of HM. To avoid skewed clustering due to HLA types of the samples, these analyses were performed specifically for the most common HLA allotypes in our datasets (A*02 (n=46 HM), A*03 (n=28 HM)). Distinct clustering was shown for the different entities (CLL, MM, CML, AML) as well as for the lymphoid versus myeloid malignancies on the HLA ligandome level. To identify leukemia-exclusive HLA ligands we compared the HLA ligandomes of CLL (HLA class I, n=35; HLA class II, n=30), AML (HLA class I, n=19; HLA class II, n=20), MM (HLA class I, n=15; HLA class II n=12) and CML (HLA class I, n=16; HLA class II n=15) with our normal tissue database including 153 HLA class I and 82 HLA class II ligandomes of various normal tissues (including normal blood, bone marrow and spleen). Cluster analysis of the leukemia-exclusive antigens showed identical clustering of the different entities and lymphoid/myeloid malignancies as shown before for the whole HLA ligandome and the respective source proteins. Overlap analysis revealed only 0.6% (16/2,716) and 0.3% (10/3,141) of the identified leukemia-exclusive HLA class I and class II antigens, respectively, to be represented across all analyzed hematological malignancies. These "pan-leukemia" antigens (n=26) include candidate antigens associated with T-cell activation (HSH2D), lymphoid development (IL2RF) and oncogenesis (LYN protooncogene, RAB5A). However, none of these "pan-leukemia" antigens shows frequent representation (>20%) across all 4 entities (CLL, AML, MM, CML). Furthermore, none of the "pan-leukemia" source proteins yielded corresponding peptides represented in all entities. To identify "pan-leukemia" HLA ligands, overlap analyses were performed in an allotype-specific fashion for the most frequent HLA allotypes (HLA-A*01, -A*02, -A*03, -A*24, -B*07, -B*08, -B*18) in our cohort. 0% (0/92) of HLA-A*01-, 1.6% (12/744) of HLA-A*02-, 1.4% (8/561) of HLA-A*03-, 0% (0/331) of HLA-A*24-, 0.1% (1/830) of HLA-B*07-, 0% (0/472) of HLA-B*08- and 0.8% (5/600) of the HLA-B*18-restricted peptides showed representation in all four entities. Out of these 26 "pan-leukemia" HLA ligands, only two (1 HLA-A*02-, 1 HLA-A*03-restricted peptide) showed frequent representation (>20%) in all entities. These peptides represent "pan-leukemia" targets that might be used for immunotherapeutic approaches in patients expressing the respective HLA allotype. Taken together, our approach of direct HLA ligandome analysis of hematological malignancies identified a small panel of "pan-leukemia"- proteins and peptides that show cancer-exclusive representation across all 4 included hematological malignancies. However, due to the low presentation frequencies of the candidate targets within the different entities, target discovery and compound development for the immunotherapy of HM may be more effectively achieved in an entity-specific or even patient-individualized manner. Disclosures Kowalewski: Immatics Biotechnologies GmbH: Employment. Schuster:Immatics Biotechnologies GmbH: Employment. Brümmendorf:Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Niederwieser:Novartis Oncology Europe: Research Funding, Speakers Bureau; Amgen: Speakers Bureau. Weisel:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Onyx: Consultancy; BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Novartis: Honoraria.

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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4518-4518 ◽  
Author(s):  
Daniel J. Kowalewski ◽  
Mirle Schemionek ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
Tim H. Brümmendorf ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Myeloid Leukemia ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Peptide Vaccination ◽  
Hla Ligands ◽  
Hla Ligandome

Abstract 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. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mapping the HLA Ligandome Landscape of Chronic Myeloid Leukemia Identifies Novel CD8+ and CD4+ T Cell-Epitopes for Immunotherapeutic Approaches

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4232-4232
Author(s):  
Tatjana Bilich ◽  
Annika Nelde ◽  
Daniel J. Kowalewski ◽  
Janet Peper ◽  
Mirle Schemionek ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Hla Ligands ◽  
Tki Treatment ◽  
Positive Controls

Abstract While the discovery of BCR-ABL and the respective tyrosine kinase inhibitors (TKI) resulted in a significant prolongation of patient survival rates, there still is no curative treatment for chronic myeloid leukemia (CML) except for allogeneic stem cell transplantation. The concept of T cell-based immunotherapy is a promising opportunity to eliminate residual leukemic cells, which might promote disease relapse after TKI discontinuation. As effective antigen-specific immunotherapy requires exact knowledge of tumor-associated epitopes that can act as rejection antigens, we have developed a mass spectrometry-based approach, which allows for the direct identification of naturally presented tumor-associated HLA ligands in hematological malignancies. In this study we used this approach to identify HLA class I and II CML-associated peptides as targets for T cell-based immunotherapy. Analysis of HLA class I ligandomes of primary CML cells (n=16) identified 8,291 HLA ligands representing 4,337 source proteins. Comparative ligandome profiling using a benign HLA class I database, which includes various healthy tissues (n=188, 65,949 HLA ligands, 14,030 source proteins) originating from peripheral blood, bone marrow, kidney, lung, liver, colon, spleen and others, revealed 38 CML-exclusive HLA class I ligands with frequencies ≥ 25% of CML patients. Because of the important indirect and direct roles of CD4+ T cells in anti-cancer immune responses, an optimal immunotherapy approach requires the inclusion of HLA class II epitopes. Hence we also analyzed the HLA class II ligandomes of primary CML cells (n=15, 2,822 HLA ligands, 794 source proteins). Comparative ligandome analysis (benign tissue, n=114, 54,149 HLA ligands, 8,584 source proteins) identified 44 CML-associated HLA class II ligands showing CML-exclusive representation in > 25% of the analyzed CML samples. To validate the immunogenicity of our HLA class I and II CML-associated peptides, we performed IFNγ- ELISPOT assays after 12-days of in vitro peptide stimulation. For HLA class II antigens, a panel of 4 peptides was implemented for stimulation of PBMCs obtained from CML patients and healthy volunteers (HV). The ELISPOT assay revealed peptide-specific immune recognition of 4/4 (100%) CML-exclusive peptides in CML patients. The frequencies of the detected immune responses ranged from 17% (4/23 patients) to 4% (1/23 patients) within the tested CML samples. These immune responses were mediated by functional CML patient-derived CD4+ T cells and strictly CML-directed, as no immune response against CML-associated peptides could be detected in HV (0/8). For HLA class I antigens, ELISPOT assays were performed using a panel of 8 peptides. Immune responses were only detected for 1/8 (13%) peptides with a low frequency of 6% (1/18 patients) of tested CML patient samples. A possible explanation for the observed weak immune response to our HLA class I CML-associated peptides compared to the immune responses shown for HLA class II peptides and for HLA class I peptides in other hematological malignancies (e.g. CLL (Kowalewski et. al. PNAS 2015)) might be an inhibition of CD8+ T cell-responses, that reportedly occurs upon TKI treatment of CML patients. To prove this hypothesis in our CML patient cohort (all patients included were under TKI treatment at the time of sample collection), we compared the ELISPOT positive controls (stimulated with a set of 5 Epstein-Barr viral peptides) of all analyzed CML samples with positive controls derived from HV and CLL samples. We could show a highly significant mean spot count reduction (per 100,000 cells) in CML samples (mean 74±16 spots, n=19) compared to HV (mean 241±24 spots, n=42, p<0.001, two-tailed t-test) or CLL (mean 218±16 spots, n=125, p=0.008) samples, confirming the general debilitated CD8+ T cell-response in CML patients under TKI treatment. To prove the immunogenicity of our HLA class I CML-associated peptides, we performed in vitro artificial antigen-presenting cell (aAPC)-based priming experiments of HV CD8+ cells. For one HLA-A*03-restricted peptide we observed tetramer-positive CD8+ populations with frequencies ranging from 0.12% to 1.41% of viable cells in 2/2 HVs so far. Taken together, these results are a first step towards a successful validation of these newly defined HLA class I and II CML-associated antigens as prime targets for further T cell-based immunotherapy approaches in CML patients. Disclosures Kowalewski: Immatics Biotechnologies GmbH: Employment. Brümmendorf:Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau.

Impact of HLA High-Resolution Matching on Outcomes of Myeloablative Unrelated Donor Hematopoietic Stem Cell Transplantation in Chinese Population

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4361-4361
Author(s):  
He Huang ◽  
Yi Luo ◽  
Jimin Shi ◽  
Yamin Tan ◽  
Xiaoyan Han ◽  
...  
Keyword(s):  
Stem Cell ◽  
High Resolution ◽  
Chinese Population ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Unrelated Donor ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Unrelated donor hematopoietic stem cell transplantation (URD-HSCT) is more frequently associated with severe graft-versus-host disease (GVHD) or graft rejection, and the success of URD-HSCT is influenced by the degree of HLA compatibility between the donor and patient. However, HLA mismatched unrelated donors should to be considerable for patients awaiting allogeneic HSCT who lack a suitable related donor or matched unrelated donor. The purpose of the study was to observed the impact of HLA-A, -B, -DRB1/B3 high-resolution matching on outcomes of URD-HSCT in Chinese population. Patients and methods: 182 patients with hematological malignancies received URD-HSCT (bone marrow, n=130; peripheral blood stem cell, n=52) in our center between Nov. 1998 and May. 2008, and donors were from Chinese Marrow Donor Program (Chinese Mainland) and Tzu Chi Stem Cells Center (Chinese Taiwan), and the median age of all patients was 26 years (range 8–52 years). The selection of unrelated donor relied on donor-recipient HLA-A, -B, -DRB1/B3 matching by high-resolution molecular typing by PCR-SSP or PCR-SSO, with 121 cases of HLA 6/6 alleles matched, 51 cases of 1/6 allele mismatched and 10 cases of 2/6 alleles mismatched. The distribution of single HLA class I or class II mismatching was as follows: 37 HLA class I mismatching with 21 HLA-A and 16 HLA-B, and 14 HLA class II mismatching with 12 HLA-DRB1 and 2 HLA-DRB3. All of the patients were received Bu/Cy or Bu/Cy modified myeloablative conditionging regimen. MMF combined with CsA and short course MTX were performed as aGVHD prophylaxis, while other 18 patients received additional anti-CD25 monoclonal antibody to prevent severe aGVHD. Results: After a median follow-up of 14.9 months, 170 patients achieved sustained engraftment with the engraft failure of 6.6%, early treatment-related mortality (TRM) of all patients was 14.4% at 100 days after transplant, and clinical relapse was observed in 8 patients (16.5%). aGVHD developed in 106 (58.2%) patients of all with grade I–II 82 (45.1%) and grade III–IV 24 (13.1%). By Kaplan-Meier method, the accumulative probability of 5-year overall survival (OS) and disease free survival (DFS) of all patients was 51.65±4.15% and 47.38±4.05%, respectively. The incidences of aGVHD was a little higher in HLA 1–2 alleles mismatched group (n=61) compared to HLA matched group (n=121) (67.2% vs 53.7%, p&gt;0.05), and the incidences of grades I–II and III–IV aGVHD in HLA mismatched transplants were 45.9% and 21.3% respectively, while those in HLA matched transplants were 44.6% and 9.1% respectively. Comparing the outcomes between HLA 1–2 alleles mismatched and HLA matched transplants, the engraft failure were 9.8% and 5.0% (P&gt;0.05), and early TRM were 18.0% and 12.4% (P&gt;0.05), respectively. The Kaplan-Meier probability OS at 5 years were 44.31±6.86% and 55.66±5.11% in HLA mismatched and matched group (P&gt;0.05) respectively. In HLA 2 alleles mismatched URD-HSCT, the incidence of engraft failure and aGVHD were 30.0% and 80.0%, and the outcomes were really inferior to HLA matched transplants. The impact of single HLA class I (n=37) or HLA class II mismatched (n=14) on the results of URD-HSCT had been also studied, and incidences of aGVHD in HLA class I or class II mismatched transplants was not significantly different compared with HLA matched transplants. In HLA class I and class II mismatched URD-HSCT, the engraft failure were 5.4% and 7.1% (p&gt;0.05), and early TRM were 13.5% and 35.7% (p&gt;0.05), respectively. The probability OS at 5 years in single HLA class II mismatched transplants was significantly lower compared with HLA matched transplants (23.81±12.94% vs 55.66±5.11%, p&lt;0.01). Conclusion: URD-HSCT could be optimized by comprehensive and precise donor-recipient alleles matching, however, HLA mismatching was associated with the risk of URD-HSCT. Moreover, HLA 2 alleles mismatches of donor-recipient HLA-A, B, DRB high-resolution matching was correlated with an inferior clinical outcome. For patients with high-risk diseases without a suitable matched unrelated donor, alternative methods to URD-HSCT with a single HLA mismatch may permit early treatment before disease progression. In our study, it also demonstrated that HLA class I mismatching was correlated with a high incidence of aGVHD, and HLA class II mismatching was associated with an inferior overall survival in Chinese population, however, larger studies would have to dissect out the magnitude of the risk incurred with specific mismatches more clearly owing to small patient numbers in each group.

Identification of Novel Tumor-Associated Antigens for Chronic Lymphocytic Leukemia (CLL) Based On HLA Ligandome Analysis – New Targets for Peptide Based Immunotherapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4119-4119
Author(s):  
Daniel Johannes Kowalewski ◽  
Heiko Schuster ◽  
Claudia Berlin ◽  
Lothar Kanz ◽  
Helmut R Salih ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
B Cells ◽  
Affinity Chromatography ◽  
Hla Class I ◽  
T Test ◽  
Class I ◽  
Isolation And Identification ◽  
Tumor Associated Antigens ◽  
Hla Ligands ◽  
Hla Ligandome

Abstract Abstract 4119 Recurrent disease, selection for chemo resistant clones and inhibition of immune effector functions are limitations of chemotherapeutic treatment of cancer including CLL. This is even more since graft-versus-leukemia effects and remissions after donor lymphocyte infusions have been correlated to long-term CLL free survival. Clonotype analysis of such cases suggested clonally expanded CD8+ T cells that recognize tumor associated antigens (TAAs) presented on HLA (human leukocyte antigen) as mediators of the observed effects, thus making CLL an attractive target for peptide vaccine based immunotherapy. For this goal we established the approach of direct isolation and identification of naturally processed and presented HLA ligands from tissues of interest by affinity chromatography and mass spectrometry. Comparative, semi-quantitative analysis of the HLA ligandomes of malignant and benign samples provided the rationale for selection of potential targets. 42 CLL patients (ages 48–85; median 70 years) of different HLA types and disease stages (Binet A, 24 patients; Binet B, 11 patients; Binet C, 7 patients) were enrolled in this study. Furthermore we collected blood samples from 50 healthy volunteers. HLA class I ligands were isolated from CLL cells as well as benign B cells from healthy donors and unsorted healthy PBMC using a standard affinity chromatography protocol implementing the pan-HLA class I specific antibody W6/32. Liquid chromatography coupled mass spectrometry (LC-MS/MS) based peptide analysis was performed on an LTQ Orbitrap hybrid mass spectrometer followed by annotation of fragment spectra using the MASCOT search algorithm against the human proteome comprised in the SwissProt database. Spectral counting based analysis provided semi-quantitative information regarding the abundance of HLA ligands and their source proteins in the respective ligandomes. In addition, HLA quantification experiments on the cell surface of CLL cells and autologous healthy B cells were performed using a flow cytometric indirect immunofluorescence assay. For this interim analysis we completely analyzed the HLA ligandomes of 7 CLL patients and 10 healthy controls. In total, we identified more than 15.000 different HLA ligands representing more than 7300 different proteins. This comprised more than 6,500 different ligands from CLL cells representing a total of 4,149 source proteins. Comparative analysis of representation in the HLA ligandomes of CLL cells, healthy B cells and healthy PBMC identified 1741 different source proteins as being exclusively expressed in CLL. Semi-quantitative evaluation revealed 138 of these proteins as being highly expressed on CLL (e.g. SET proto-oncogene, Pim-1 Oncogene, Mucin 1). Flow cytomerty based quantification of surface HLA expression revealed similar amounts of HLA class I (p=0.23, unpaired t-test) and II (p=0.33, unpaired t-test) molecules on CLL cells and autologous benign B lymphocytes, with a trend to higher HLA expression on CLL cells. Taken together, the presented strategy enabled the identification of a vast array of both known and novel TAAs and their corresponding naturally processed and presented HLA ligands in CLL. It pinpointed highly overrepresented TAAs for further analysis and immunogenicity testing. Expansion of the dataset after analysis of all enrolled patients will provide an unprecedented in-depth characterization of the HLA ligandome of CLL for future immunotherapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

HLA Class I Ligandome Analysis In Acute Myeloid Leukemia – Novel T-Cell Epitopes For Peptide-Based Immunotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5431-5431
Author(s):  
Stickel S. Juliane ◽  
Claudia Berlin ◽  
Daniel J. Kowalewski ◽  
Heiko Schuster ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
T Cell ◽  
Conflicts Of Interest ◽  
Peptide Vaccine ◽  
T Cell Responses ◽  
Hla Class I ◽  
Class I ◽  
Isolation And Identification ◽  
Cell Responses ◽  
Healthy Donors ◽  
Hla Ligands

Abstract Data regarding the graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion strongly suggest that T lymphocytes play a major role in the rejection of leukemic cells. Immunotherapy directed against leukemia- associated antigens might elicit specific immune responses that may serve to eliminate minimal residual disease after chemotherapy, or enhance the GVL effect after SCT. To achieve this goal there is need to identify appropriate leukemia associated HLA ligands, which are able to induce specific T cell responses. We here aimed to characterize the HLA class I ligandome in AML patients to provide novel tumor associated antigens (TAA) for peptide-based immunotherapy employing our recently implemented approach of direct isolation and identification of naturally presented HLA ligands by affinity chromatography and mass spectrometry (LC-MS/MS) in AML (Stickel et.al., abstract in Blood 2012). Absolute HLA surface expression on AML cells and autologous monocytes and granulocytes was quantified by flow cytometry. HLA class I ligands were isolated from AML cells as well as bone marrow and peripheral blood mononuclear cell (BMNCs/PBMCs) of healthy donors. LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived TAA (LiTAA) and to select peptide vaccine candidates. The most abundantly detected peptide candidates were checked for immunogenicity by ELISpot and confirmed by intracellular interferon-g staining of CD8+ T-cells. Meanwhile 15 AML patients (8 FLT3-ITD mutant) and 35 healthy donors were analyzed. We observed overexpression of HLA class I and II on AML cells as compared to autologous monocytes and granulocytes, with the level of significance reached for HLA class II (p=0,04). A total of more than 12,000 AML derived HLA ligands representing >6,000 different source proteins were identified; of which 2,220 were exclusively represented in AML, but not in healthy PBMC/BMNC. Data mining for broadly represented LiTAA pinpointed 98 TAA as most promising targets. HLA ligands derived from these TAA were presented exclusively on more than 33% of all analyzed AML samples, amongst them already described TAA (e.g. JUP, FAF1) as well as several new leukemia-associated proteins (e.g. MTCH2, METTL7A). Subset analysis of the FLT3-ITD positive AML cohort revealed 21 LiTAA presented exclusively on more than 50% of FLT3-ITD positive AML cases. Additional screening for HLA ligands derived from described leukemia associated antigens revealed overrepresentation for e.g. FLT3, NUSAP, RHAMM and RGS5. Specific CD8+ T cell responses were detected against two A*03 epitope pools (pool 1: APLP2, DKGZ, FAF1, MTCH2; pool 2: KLF2, METTL7A, VCIP1, WIPI1) in AML patients. Notably, the chosen A*03 epitope pools did not elicit specific responses of CTL from healthy donors. Taken together, our HLA class I ligandome analysis in AML for the first time identified naturally presented HLA ligands from patients including a vast array of new leukemia associated antigens representing promising targets for a multipeptide-based immunotherapy approach in AML. Disclosures: No relevant conflicts of interest to declare.

Characterization Of The HLA Class II Ligandome In Acute Myeloid Leukemia (AML) Reveals Novel Candidates For Peptide-Based Immunotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5012-5012 ◽  
Author(s):  
Juliane S. Stickel ◽  
Claudia Berlin ◽  
Daniel J. Kowalewski ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Response ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
Class I ◽  
Tumor Associated Antigens ◽  
Healthy Donors

Abstract CD4+ T cells are crucial for the induction and maintenance of cytotoxic T cell responses, but can also mediate direct tumor rejection. The therapeutic efficacy of peptide-based cancer vaccines may thus be improved by including HLA class II epitopes to stimulate T helper cells. In contrast to HLA class I ligands, only a small number of class II ligands of TAA has been described so far. We recently reported on the overexpression of HLA class II in AML cells as compared to autologous monocytes and granulocytes as well as on the first HLA class I leukemia associated antigens identified directly on the cell surface of primary AML cells (Stickel et. al. abstract in Blood 2012). In this study we characterized the HLA class II ligandome in AML to identify additional ligands for a peptide-based immunotherapy approach. HLA class II ligands from primary AML cells as well as bone marrow and peripheral blood mononuclear cell (BMNCs/PBMCs) of healthy donors were analyzed using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry (LC-MS/MS). LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived tumor associated antigens (LiTAAs) and to select peptide vaccine candidates. Most abundantly detected peptides were functionally characterized with regard to their ability to induce a specific CD4+ T-cell response in healthy donors and in tumor patients using ELISpot. Samples from 10 AML patients (5 FLT3-ITD mutated) and 18 healthy donors were analyzed. We identified more than 2,100 AML-derived HLA class II ligands representing >1,000 different source proteins, of which 315 were exclusively represented in AML, but not in healthy PBMC/BMNC. Data mining for broadly represented LiTAAs pinpointed 26 HLA class II ligands from 8 source proteins that were presented exclusively on more than 40% of all analyzed AML samples as most promising targets. Amongst them were already described TAAs (e.g., RAB5A) as well as several so far understated proteins (e.g. calsyntenin 1, glycophorin A, mannose-binding lectin 2). Subset analysis revealed 58 LiTAAs presented exclusively on FLT3-ITD mutated AML cells. Additional screening for HLA class II ligands from described leukemia associated antigens showed positive results for NPM1 (1 peptide sequence) and MPO (13 peptide sequences). Peptides from calsyntenin 1 and RAB5A were able to elicit CD4+-T-cell response in 25% of tested AML patients (n=16). Thus, our study identified, for the first time, HLA class II tumor associated antigens directly obtained from the HLA ligandomes of AML patients and thereby represents a further step to our goal of developing a multipeptide vaccine for immunotherapy of AML. Disclosures: No relevant conflicts of interest to declare.

Identification of Four New HLA Class II Restricted Minor Histocompatibility Antigens Contributing to Graft Versus Leukemia Reactivity.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3247-3247
Author(s):  
Anita N. Stumpf ◽  
Edith D. van der Meijden ◽  
Cornelis A.M. van Bergen ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Class I ◽  
T Cell Clones ◽  
Cell Clones

Abstract Patients with relapsed hematological malignancies after HLA-matched hematopoietic stem cell transplantation (HSCT) can be effectively treated with donor lymphocyte infusion (DLI). Donor-derived T cells mediate beneficial graft-versus-leukemia (GvL) effect but may also induce detrimental graft-versus-host disease (GvHD). These T cell responses are directed against polymorphic peptides which differ between patient and donor due to single nucleotide polymorphisms (SNPs). These so called minor histocompatibility antigens (mHag) are presented by HLA class I or II, thereby activating CD8+ and CD4+ T cells, respectively. Although a broad range of different HLA class I restricted mHags have been identified, we only recently characterized the first autosomal HLA class II restricted mHag phosphatidylinositol 4-kinase type 2 beta (LB-PI4K2B-1S; PNAS, 2008, 105 (10), p.3837). As HLA class II is predominantly expressed on hematopoietic cells, CD4+ T cells may selectively confer GvL effect without GvHD. Here, we present the molecular identification of four new autosomal HLA class II restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLAmatched HSCT who experienced long-term complete remission after DLI with only mild GvHD of the skin. By sorting activated CD4+ T cells from bone marrow mononuclear cells obtained 5 weeks after DLI, 17 highly reactive mHag specific CD4+ T cell clones were isolated. Nine of these T cell clones recognized the previously described HLADQ restricted mHag LB-PI4K2B-1S. The eight remaining T cell clones were shown to exhibit five different new specificities. To determine the recognized T cell epitopes, we used our recently described recombinant bacteria cDNA library. This method proved to be extremely efficient, since four out of five different specificities could be identified as new HLA-class II restricted autosomal mHags. The newly identified mHags were restricted by different HLA-DR molecules of the patient. Two mHags were restricted by HLA-DRB1 and were found to be encoded by the methylene-tetrahydrofolate dehydrogenase 1 (LBMTHFD1- 1Q; DRB1*0301) and lymphocyte antigen 75 (LB-LY75-1K; DRB1*1301) genes. An HLA-DRB3*0101 restricted mHag was identified as LB-PTK2B-1T, which is encoded by the protein tyrosine kinase 2 beta gene. The fourth mHag LB-MR1-1R was restricted by HLA-DRB3*0202 and encoded by the major histocompatibility complex, class I related gene. All newly identified HLA class II restricted mHags exhibit high population frequencies of 25% (LB-MR1-1R), 33% (LB-LY75-1K), 68% (LB-MTHFD1- 1Q), and 70% (LB-PTK2B-1T) and the genes encoding these mHags show selective (LY- 75) or predominant (MR1, MTHFD1, PTK2B) expression in cells of hematopoietic origin as determined by public microarray databases. All T cell clones directed against the newly identified mHags recognized high HLA class II-expressing B-cells, mature dendritic cells (DC) and in vitro cultured leukemic cells with antigen-presenting phenotype. The clone recognizing LB-MTHFD1-1Q also showed direct recognition of CD34+ CML precursor cells from the patient. In conclusion, we molecularly characterized the specificity of the CD4+ T cell response in a patient with CML after HLA-matched HSCT who went into long-term complete remission after DLI. By screening a recombinant bacteria cDNA library, four new different CD4+ T cell specificities were characterized. Our screening method and results open the possibility to identify the role of CD4+ T cells in human GvL and GvHD, and to explore the use of hematopoiesis- and HLA class II-restricted mHag specific T cells in the treatment of hematological malignancies.

The HLA Ligand Atlas: A novel immuno-oncology resource for T-cell antigen discovery.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3128-3128
Author(s):  
Ana Marcu ◽  
Leon Bichmann ◽  
Leon Kuchenbecker ◽  
Linus Backert ◽  
Daniel J Kowalewski ◽  
...  
Keyword(s):  
T Cell ◽  
Hla Class I ◽  
Class Ii ◽  
Tumor Rejection ◽  
Class I ◽  
Data Set ◽  
Essential Information ◽  
Data Resource ◽  
Hla Ligand ◽  
Hla Ligands

3128 Background: The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system, which is the basis for T cell-dependent tumor rejection. Therefore, a comprehensive map of the entirety of both HLA class I- and class II-presented peptides from various benign tissues is a highly sought after resource, as it enables the definition of tumor-association on the immunologically pivotal level of the HLA ligandome. Methods: Human tissue samples were snap frozen post mortem during autopsy. The study was approved by the local IRB. HLA ligands were immunopurified and characterized using an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC Nano UHPLC System. Data acquisition was performed as technical triplicates in data-dependent mode, and data were analyzed using the containerized, computational pipeline MHCquant. Results: In this work, we describe the HLA Ligand Atlas, a comprehensive collection of matched HLA class I and class II ligandomes from 29 non-malignant tissues and 13 human subjects (208 samples in total), covering 38 HLA class I, and 17 HLA*DRB alleles and comprising 48,381 HLA class I and 16,146 HLA class II peptides. Nearly 50% of HLA ligands have not been previously described. The HLA Ligand Atlas is publicly available as a raw data resource, but also in the form of a user-friendly web interface that allows users to quickly formulate complex queries against the data set. Both downloadable data and the query interface are available at www.hla-ligand-atlas.org. Conclusions: This data set provides a valuable tool for research in diverse fields such as systems biology, general immunology, autoimmune disease and organ transplantation. Most importantly, the HLA Ligand Atlas provides essential information for translational applications in immuno-oncology. The knowledge of HLA ligands from benign tissues strongly supports the informed design of proteogenomic HLA-dependent target discovery approaches.

scholarly journals Characterization of cells emerging at the time of graft failure after bone marrow transplantation from an unrelated marrow donor

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 1023-1029 ◽  
Author(s):  
J Donohue ◽  
M Homge ◽  
NA Kernan
Keyword(s):  
T Cell ◽  
Mononuclear Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Unrelated Donor ◽  
Peripheral Blood Mononuclear ◽  
Hla Dr

Abstract To help elucidate the mechanism responsible for graft failure (GF) following a T-cell depleted bone marrow transplant (BMT) from an unrelated donor, five patients (2 chronic myelogenous leukemia, 1 acute undifferentiated leukemia, 2 myelodysplastic syndrome) who experienced this complication were studied. All patients were HLA class I identical with their donors as determined by serology and one-dimensional isoelectric focusing (IEF); two were serologically matched with their donors for HLA class II antigens, whereas three donor-recipient pairs were serologically mismatched for one HLA-DR antigen. All patients received total body irradiation (fractionated, 1,500 rads), VP-16 (750 mg/m2), and cyclophosphamide (120 mg/kg) pre-BMT and antithymocyte globulin (15 mg/kg every other day) and methylprednisolone (2 mg/kg) post-BMT. Three patients experienced primary nonengraftment and two experienced secondary GF. Peripheral blood mononuclear cells obtained from the patients at the time of GF were studied to examine their functional and phenotypic characteristics. Emerging cells were of host origin and were found to be specifically cytotoxic to donor target cells and suppressive to the in vitro growth of donor BM, especially in the cases of primary nonengraftment. Peripheral blood mononuclear cells from these patients were expanded to form T-cell lines (TcLs). The cytotoxic activities of TcLs were tested in the presence of blocking MoAbs directed against various HLA determinants in an attempt to determine if HLA antigens expressed on donor cells were the target for cytotoxicity. The observed cytotoxic activity was blocked by antibodies to HLA-B, -C (1 patient), HLA-DR (1 patient), and HLA-DQ (1 patient). In two cases, antidonor cytotoxicity could not be blocked by MoAb directed against HLA-A, -B, -C, or -DR. Phenotypic characterization of four successfully maintained TcLs showed 100% CD3+ cells with 100% CD4+ (3 patients) or 50% CD4+/50% CD8+ (1 patient). In two of the three patients with 100% CD4+ cells, antidonor cytotoxicity was blocked by an anti-HLA class II MoAb. In contrast to our previous findings in cases of GF following T-cell-depleted HLA nonidentical family member BMT in which host T cells were CD8+ and cytotoxicity was directed against HLA class I antigens, our present study indicates host T cells emerging at the time of GF following BMT from an HLA class I IEF-identical unrelated donor can be of the CD4+ subset and seem to be capable of recognizing antigenic disparities in the HLA class II region.

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