Tetramer-positive T-cell Research Articles

Combining CD34+ stem cell selection with prophylactic pathogen and leukemia directed T-cell immunotherapy to simultaneously reduce graft versus host disease, infection, and leukemia recurrence after allogeneic stem cell transplant.

We designed a trial to simultaneously address the problems of graft versus host disease (GVHD), infection, and recurrence of malignancy after allogeneic stem cell transplantation. CD34+ stem cell isolation was used to minimize the development of acute and chronic GVHD. Two prophylactic infusions, one combining donor-derived cytomegalovirus, Epstein-Barr virus, and Aspergillus fumigatus specific T-cells and the other comprising donor-derived CD19 directed chimeric antigen receptor (CAR) bearing T-cells, were given 21-28 days after transplant. Two patients were transplanted for acute lymphoblastic leukemia from HLA identical siblings using standard doses of cyclophosphamide and total body irradiation without antilymphocyte globulin. Patients received no post-transplant immune suppression and were given no pre-CAR T-cell lymphodepletion. Neutrophil and platelet engraftment was prompt. Following adoptive T-cell infusions, there was rapid appearance of antigen-experienced CD8+ and to a lesser extent CD4+ T-cells. Tetramer-positive T-cells targeting CMV and EBV appeared rapidly after T-cell infusion and persisted for at least 1 year. CAR T-cell expansion occurred and persisted for up to 3months. T-cell receptor tracking confirmed the presence of product-derived T-cell clones in blood targeting all three pathogens. Both patients are alive over 3 years post-transplant without evidence of GVHD or disease recurrence. Combining robust donor T-cell depletion with directed T-cell adoptive immunotherapy targeting infectious and malignant antigens permits independent modulation of GVHD, infection, and disease recurrence. The combination may separate GVHD from the graft versus tumor effect, accelerate immune reconstitution, and improve transplant tolerability.

Analysis of the Multiple Myeloma HLA Peptidome Identifies a Naturally Presented Bcma-Derived Peptide As an Immunogenic T-Cell Epitope for Immunotherapeutic Approaches

The B-cell maturation antigen (BCMA) is selectively expressed by cells of the B-lineage, including multiple myeloma (MM) cells, and constitutes a promising target for immunotherapeutic approaches. At present, BCMA is being evaluated as target for immunotherapeutic approaches, such as CAR T cells and bispecific antibodies, which have demonstrated promising results in phase I clinical trials. The utilization of cytotoxic T cells bearing T-cell receptors against BCMA constitutes an alternative promising approach to target MM cells. Therefore, the identification of BCMA-derived peptides that are naturally presented by human leukocyte antigens (HLA) and thus can serve as target structures for CD8+ T cells, is indispensable.In a previous study, we characterized the immunopeptidomic landscape of MM by mass spectrometry-based analysis of naturally presented HLA ligands from primary MM samples and MM cell lines (Walz et al., Blood, 2015). Comparative HLA peptidome profiling of the MM-derived HLA ligands versus the immunopeptidome of numerous benign samples from different tissues identified several strictly MM-associated antigens. Here, we evaluated this dataset for the presence of BCMA-derived MM-exclusive antigens and identified two HLA class I-restricted, BCMA-derived peptides in the immunopeptidome of our cohort comprising 15 primary MM samples and MM cell lines. Notably, one of these peptides showes strictly MM-associated presentation and was never detected on any benign tissues according to our extensive immunopeptidome database (135,354 HLA ligands originating from 16,626 source proteins detected in 337 samples from various benign tissues including blood, bone marrow, lung, kidney, liver, and spleen). This HLA-B*18-restricted ligand P(BCMA)B*18 is represented in 20% (3/15) of the analyzed MM immunopeptidomes.For immunological characterization of the P(BCMA)B*18 peptide, we performed in vitro artificial antigen-presenting cell-based priming experiments engaging naïve CD8+ T cells obtained from healthy volunteers (HV). Induction of tetramer-positive T-cell populations with frequencies ranging from 0.1-2.9% of viable CD8+ T cells was observed for all analyzed healthy whole blood donors, which demonstrates the immunogenicity of P(BCMA)B*18. Subsequently, we functionally characterized the induced P(BCMA)B*18-specific CD8+ T cells using intracellular cytokine staining. Upon stimulation with P(BCMA)B*18, we observed an increased IFNγ and TNF production specifically in the peptide-specific CD8+ T cells. In addition, the degranulation marker CD107a was found to be upregulated in the analyzed tetramer-positive T cells, confirming the activity of CD8+ T cells upon peptide-stimulation. Priming experiments using naïve CD8+ T cells obtained from MM patients as well as in vitro cytotoxicity assays with polyclonal peptide-specific effector T cells are presently ongoing in order to assess the capacity of P(BCMA)B*18-specific CD8+ T cells to induce antigen-specific cell lysis.Taken together, we identified a naturally presented and MM-associated, BCMA-derived peptide, which constitutes a promising target for tailored T cell-based immunotherapeutic approaches. DisclosuresSalih:Several patent applications: Patents & Royalties: e.g. EP3064507A1. Kowalewski:Immatics Biotechnologies GmbH: Employment. Weisel:Amgen, BMS, Celgene, Janssen, and Takeda: Honoraria; Amgen, BMS, Celgene, Janssen, Juno, Sanofi, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen, Celgene, Janssen, and Sanofi: Research Funding.

Abstract 2501: Vaccination using epitopes with higher MHC class I affinity elicits CD8+ T cells with increased PD-1 expression

Abstract We have been interested in DNA vaccines as treatments for human prostate cancer, and have demonstrated that this approach can elicit antigen-specific CD8+ T-cells in humans that can persist over years. However, the persistence of antigen-expressing tumors demonstrates that mechanisms of tumor escape are at play. Consequently, in preclinical studies we have focused on mechanisms of resistance and efforts to improve the immunological activity of DNA vaccines. As one model antigen, we have used the synovial sarcoma, X breakpoint 2 (SSX2) protein, a cancer-testis antigen expressed in metastatic prostate cancer. We have previously demonstrated that SSX2 encodes two HLA-A2-restricted epitopes, and we have utilized HHD-II mice, in which HLA-A2 and HLA-DR1 (but not murine MHC class I and II) are expressed, as a means to study CD8+ T cells generated by vaccination. We have shown in this system that immunization with a DNA plasmid encoding SSX2 elicits robust CD8+ cells, and anti-tumor responses to syngeneic tumors expressing SSX2. We have recently identified that a DNA vaccine altered to encode peptide epitopes with increased HLA-A2 binding elicited a greater frequency of CD8+ T cells specific for SSX2 when compared to the native vaccine, however these T cells were less effective against tumors due to higher expression of PD-1. In the current study, we investigated the mechanisms underlying this regulation of T-cells following vaccination. Altered peptide ligands (APL) specific for one HLA-A2-restricted epitope were modified at anchor residues to generate peptides of varying affinities for HLA-A2. Groups of HHD-II mice were then immunized with one of five SSX2 APLs with varying HLA-A2 affinity. After one week the resulting antigen-specific CD8+ T cells were assessed for regulatory ligand expression and cytokine expression as an indicator of function. We found that a single immunization with high affinity APLs resulted in an increased percentage of SSX2 tetramer positive T-cells that displayed persistent PD-1 expression compared with lower-affinity APL. Upon ex vivo re-encounter of the native SSX2 peptide, T-cells from mice immunized with higher affinity APLs showed an increased multifunctional Th1 response as evidenced by increased IFNγ, IL2, and TNFα expression when compared to mice immunized with native or low affinity SSX2 peptides, but also higher PD-1 expression. Ongoing studies are further exploring the kinetics and persistence of PD-1 expression following antigen presentation. Notwithstanding, our results have implications for the design of anti-tumor vaccines that seek to incorporate epitopes with altered HLA-A2-binding ability as these may affect the persistence of PD-1 expression on CD8+ T cells following immunization. Citation Format: Christopher D. Zahm, Douglas McNeel. Vaccination using epitopes with higher MHC class I affinity elicits CD8+ T cells with increased PD-1 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2501. doi:10.1158/1538-7445.AM2015-2501

Detection of different virus-specific CD8+ T cells after kidney transplantation.

The early diagnosis of viral reactivation after kidney transplantation (KTX) is an unsolved problem. Survey of virus-specific T-cell responses may identify patients at risk for viral reactivation. We therefore quantified virus-specific CD8+ T-cells to evaluate their potential predictive value for viral reactivation and infection in KTX patients. We quantified the virus-specific responses of CD8+ T-cells for CMV, EBV, HPV and HHV in 23 patients undergoing KTX for 6 mo after transplantation. We enumerated T-cells for 36 virus-specific binding peptides and five different human leukocyte antigen (HLA) alleles through the binding of Class I iTAg major histocompatibility complex (MHC) tetramers. The patients' pre-operative serologic status for CMV and CMV-specific CD8+ T-cell numbers were correlated with one another (p=0.0046). Three patients had clinical CMV disease and all three remained or became CMV-tetramer-positive for at least one HLA allele during follow-up. Three of the four patients with viral infections caused by or reactivations of viruses other than CMV were initially negative for CMV-specific CD8+ T-cells but became CMV-positive. Most of the patients who were initially CMV-tetramer positive also had tetramer-positive T-cells specific for Epstein-Barr virus (EBV); human papillomavirus (HPV)-6b, -11, -16, or -18; or human herpesvirus (HHV)-8. All of the patients who developed viral disease other than that caused by CMV remained or became positive for at least one binding peptide that was specific for a virus not directly related to the clinical features of a viral disease. Patients who were positive for any virus had a significantly greater risk of developing complications of viral disease during the 6-mo follow-up period in the study (p=0.026), suggesting a general susceptibility to viral reactivation. The evaluation of virus-specific CD8+ T-cells may prospectively help to identify patients at risk for viral reactivation after KTX.

Distinct activation phenotype of a highly conserved novel HLA‐B57‐restricted epitope during dengue virus infection

Variation in the sequence of T-cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T-cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS1(26-34) -specific CD8(+) T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T-cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57-NS1(26-34) -specific and other DENV epitope-specific CD8(+) T cells, as well as total CD8(+) T cells, expressed an activated phenotype (CD69(+) and/or CD38(+)) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8(+) T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T-cell activation.

Dynamics of the CD8 T‐cell response following yellow fever virus 17D immunization

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8(+) T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-gamma (IFN-gamma) responses of almost 1% of CD8(+) T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7-9 days, before detectable IFN-gamma-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA(+) CCR7(-) CD62L(-)) phenotype. The T-cell receptor Vbeta analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness.

Wilms Tumor gene WT1 peptide-based immunotherapy induced a minimal response in a patient with advanced therapy-resistant multiple myeloma

The product of the Wilms tumor gene, WT1, is a universal tumor antigen. We performed WT1 peptide-based immunotherapy for a patient with multiple myeloma (MM). This patient was a 57-year-old woman with chemotherapy-resistant MM (Bence Jones kappa type). The patient received weekly intradermal injections of an HLA-A*2402-restricted 9-mer WT1 peptide emulsified with Montanide ISA 51 adjuvant for 12 weeks and achieved a minimal response according to European Group for Blood and Marrow Transplantation criteria without experiencing systemic adverse effects. The proportion of myeloma cells in the bone marrow (BM) decreased from 85% to 25%, and the amount of M protein in the urine decreased from 3.6 to 0.6 g/day after WT1 vaccination. Furthermore, a bone scintigram showed an improvement after the vaccination. As for immunologic parameters, the frequency of WT1 tetramer-positive cells among CD8+ T-cells, which was higher than in healthy donors, temporarily decreased at weeks 4 and 8 but increased at week 12, whereas the frequency of WT1 peptide-responding CD107a/b+ cells among WT1 tetramer-positive T-cells increased from 27.0% to 38.6% after the vaccination. After WT1 vaccination, the frequency of CXCR4+ cells among WT1 tetramer-positive T-cells increased in the BM, where stromal cells expressed the ligand for CXCR4, stromal-derived factor 1 (SDF-1), but decreased in the peripheral blood (PB), implying that WT1-specific cytotoxic T-lymphocytes had migrated from the PB to the BM, a tumor site.

Analysis of hepatitis C virus-specific CD8+ T-cells with HLA-A*24 tetramers during phlebotomy and interferon therapy for chronic hepatitis C

Hepatitis C virus (HCV)-specific, HLA class I-restricted, CD8-positive (CD8+) T lymphocytes are thought to contribute to viral clearance as well as liver disease in chronic hepatitis C. For the patients who do not respond to interferon (IFN) therapy, phlebotomy can be used as a tool to reduce inflammation and lower transaminase levels; however, the immunological aspects have not been clearly defined. In this study, we evaluated the HCV-specific CD8+ T-cell responses during phlebotomy and IFN therapy using HLA-A*24 tetramers in 6 Japanese patients with chronic hepatitis C. During phlebotomy, 4 of the 6 cases achieved a biochemical response, but there was no clear correlation between its efficacy and HCV viral loads or changes in frequencies or activation status of tetramer-positive T-cells. In contrast, the frequencies of tetramer-positive cells and the proportions of T-cells expressing activation marker HLA-DR were higher in sustained viral responders than in transient responders to IFN therapy. Furthermore, expression of the activation marker was enhanced in the initial period of IFN therapy. The results suggest that the immunological aspects of phlebotomy obviously differ from those of IFN therapy and these differences may provide clues as to a therapeutic strategy of their combination for patients who do not respond to IFN monotherapy.

NY-ESO-1 Specific T-Cells Are Spontaneously Present in High-Risk Myeloma and Kill Primary Myeloma Cells.

We have previously reported that the Cancer/Testis antigen NY-ESO-1 is frequently expressed in patients with high-risk myeloma or advanced myeloma characterized by cytogenetic abnormalities. We tested 3 HLA-A*0201 positive MM patients with abnormal cytogenetics for the spontaneous presence of NY-ESO-1 specific CD8+ T-cells in the peripheral blood. We were able to detect 0.1% and 0.6% NY-ESO-1 specific CD8+ T-cells respectively in 2 patients using HLA-A*0201/NY-ESO-1157-165 specific tetramers prior to any re-stimulation with peptide. The 3rd patient did not have NY-ESO-1 specific T-cells. This last patient had been very heavily pre-treated and in contrast to the other patients, influenza matrix peptide58–66 specific T-cells failed to expand indicating that this patient's immune system may have been exhausted. CD3+/8+ T-cells were expanded in multiple experiments by re-stimulation with autologous monocyte derived dendritic cells (Mo-DCs) pulsed with NY-ESO-1157-C165V analogue peptide. Substitution of cysteine (C) for valine (V) at position 165 improves binding affinity for HLA-A*0201 and antigenicity by eliminating dimerization of the carboxy-terminus cysteine residue. The lines contained 45–79% tetramer positive T-cells after stimulation. The CTLs were highly specific and lysed NY-ESO-1 positive/ HLA-A*0201 positive primary myeloma cells (50%), the NY-ESO-1 positive/ HLA-*0201 positive myeloma cell line U266 (55%) and autologous PHA-blasts pulsed with NY-ESO-1157–165 wild-type peptide (75%), but not K562 or patient PHA-blasts pulsed with the HLA-A*0201 binding MAGE-3112–120 peptide at E:T ratios of 10:1. The CTLs were of Tc1 type (g-IFN positive and IL4 negative) and stained positive for granzymes suggesting killing via the perforin/granzyme pathway. Immunophenotyping showed that the CTLs expressed the IL2 receptor and the integrin VLA-4, which is associated with the ability to transgress endothelial barriers. Further, the CTLs predominantly belonged to the terminal effector compartment (CD45RO+/CCR7−). We were unable to detect or expand NY-ESO-1 specific T-cells in HLA-A*0201 positive normal donors or NY-ESO-1 negative MM patients suggesting that pre-existent T-cell immunity to NY-ESO-1 is a prerequisite for the expansion of NY-ESO-1 specific CTLs. It also ruled out the possibility that the use of autologous APC's induced de novo NY-ESO-1 specific CTL. In conclusion, these data suggest that NY-ESO-1 positive myeloma tumors are able to elicit spontaneous T-cell immunity and that expanded CTLs from these patients can kill primary myeloma cells. These CTLs recognize on the cell surface of myeloma cells naturally processed and presented NY-ESO-1, which is displayed in the context of HLA-A*0201. These results suggest that patients with high-risk NY-ESO-1 positive myeloma have a pool of memory CTLs, which can be exploited for vaccine or other immuno-therapeutic approaches.