Peptide-specific IFNγ Production Research Articles

Enzyme-linked ImmunoSpot (ELISpot) assay to quantify peptide-specific IFN-γ production by splenocytes in a mouse tumor model after radiation therapy.

To develop new effective therapeutic strategies for cancer patients, there is a need for extensive and precise insights into the mechanisms involved in the immune response to anti-cancer treatments. The enzyme-linked immunospot (ELISpot) assay is a rapid and reproducible technique that allows the detection of cytokine-producing antigen-specific T cells at the single cell level. This protocol describes an interferon gamma (IFN-γ) ELISpot method for measuring antigen-specific murine CD8+ T cells that produce IFN-γ, a marker of their activation and cytotoxicity. Splenocytes from tumor-bearing mice treated with radiation therapy were used as source of CD8+ T cells and were stimulated with a tumor-derived peptide. This method was facilitated by a ready-to-use assay kit and provides a tool to analyze the specificity, intensity, and kinetics of specific CD8+ T cells.

Human rhinovirus-specific CD8 T cell responses target conserved and unusual epitopes.

Human Rhinovirus (HRV) is a major cause of common cold, bronchiolitis, and exacerbations of chronic pulmonary diseases such as asthma. CD8 T cell responses likely play an important role in the control of HRV infection but, surprisingly, HRV‐specific CD8 T cell epitopes remain yet to be identified. Here, we approached the discovery and characterization of conserved HRV‐specific CD8 T cell epitopes from species A (HRV A) and C (HRV C), the most frequent subtypes in the clinics of various pulmonary diseases. We found IFNγ‐ELISPOT positive responses to 23 conserved HRV‐specific peptides on peripheral blood mononuclear cells (PBMCs) from 14 HLA I typed subjects. Peptide‐specific IFNγ production by CD8 T cells and binding to the relevant HLA I were confirmed for six HRV A‐specific and three HRV C‐specific CD8 T cell epitopes. In addition, we validated A*02:01‐restricted epitopes by DimerX staining and found out that these peptides mediated cytotoxicity. All these A*02:01‐restricted epitopes were 9‐mers but, interestingly, we also identified and validated an unusually long 16‐mer epitope peptide restricted by A*02:01, HRVC1791‐1806 (GLEPLDLNTSAGFPYV). HRV‐specific CD8 T cell epitopes describe here are expected to elicit CD8 T cell responses in up to 87% of the population and could be key for developing an HRV vaccine.

Abstract 4575: Efficacy proof of concept studies with universal mRNA vaccines in various mouse tumor models

Abstract Introduction: Cancer immunotherapies have rapidly advanced in recent years and are revolutionizing cancer care. Among them, cancer vaccines are an attractive treatment approach because they may represent a specific, safe, and well tolerated therapy as well as the potential to overcome drug resistance and achieve durable response. Currently, personalized mRNA cancer vaccines are being actively pursued in clinical trials. However, only a few universal mRNA cancer vaccines have been investigated. This study assessed the antitumor efficacy of universal mRNA cancer vaccines targeting three tumor-associated antigens (TAAs) in CT26.WT-CEA syngeneic tumors, as well as KPC (KrasG12D; Trp53KO; Pdx1-cre) MuPrime™ tumors. Methods: We have designed 27 epitopes against 3 TAAs, cloned into 4 DNA constructs in different combinations for mRNA synthesis by in vitro transcription (IVT). Prophylactic and therapeutic efficacy studies were carried out using CT26.WT-CEA tumor bearing mice with vaccine monotherapy or its combination with an anti-PD-1 antibody. At the end of the efficacy phase, mouse splenocytes were collected from half of the treated animals to measure peptide-specific IFN-γ production by ELISpot assay. In the remaining tumor-free mice, we re-grafted CT26.WT-CEA and parental CT26.WT (1st re-challenge) or a different syngeneic line H22 (2nd re-challenge), which shares two common antigens with CT26.WT. Finally, we selected the most efficacious mRNA vaccine to assess its efficacy in treating pancreatic KPC tumors. Results: While anti-PD-1 monotherapy led to ~80% TGI, its combination with mRNA vaccines resulted in complete eradication of CT26.WT-CEA tumors in both prevention and intervention settings. In the ELISpot assay, splenocytes from vaccine treated mice showed significantly increased IFN-γ secreting cells after co-culture with specific peptides, in comparison to splenocytes from treatment naïve mice indicating existence of antigen specific CD8+ T cells. In the serial re-challenge studies, the vaccine treated, tumor-free mice showed no tumor growth in 2 rounds of re-challenge, either with the same tumor line (CT26.WT-CEA), its parental line (CT26.WT), or a different syngeneic line (H22), suggesting durable tumor growth inhibition by memory T cells after vaccine therapy. This universal mRNA cancer vaccine also demonstrated robust TGI against KPC tumors. Conclusions: We demonstrated the effectiveness of universal mRNA cancer vaccines targeting different epitopes of multiple common tumor antigens in various mouse tumor models, including hard-to-treat KPC tumors. We are now performing toxicology studies using rats and NHPs to move the universal mRNA cancer vaccines toward IND filing. Citation Format: Chenyan Wu, Tina Tingting Zhang, Xuefei Yan, Ying Jin, Henry Q. X. Li, Davy Xuesong Ouyang. Efficacy proof of concept studies with universal mRNA vaccines in various mouse tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4575.

Identification of a novel HLA-A24-restricted cytotoxic T lymphocyte epitope peptide derived from mesothelin in pancreatic cancer.

Pancreatic cancer involves highly malignant tumors, and the development of new therapeutic strategies is critical. Mesothelin is overexpressed in infiltrating pancreatic cancer cells and plays an important role in the invasion and migration processes. In this study, we focused on mesothelin as a tumor-specific antigen target for a pancreatic cancer vaccine. We first investigated the mesothelin-derived epitope peptide restricted to HLA-A*2402. A total of 19 candidate peptides were synthesized, and we then determined their potential to induce peptide-specific cytotoxic T lymphocytes (CTLs). Peptide-specific CTLs were induced by five peptides derived from mesothelin, and these CTLs successfully exhibited peptide-specific IFN-γ production. After the expansion of each CTL, two CTL lines were established, which were induced by mesothelin-10-5 peptide (AFYPGYLCSL). These CTL lines exhibited peptide-specific cytotoxicity and IFN-γ production. Moreover, we were able to generate mesothelin-10-5 peptide-specific CTL clones. These CTL clones also had specific cytotoxic activity against HLA-A*2402-positive pancreatic cancer cells that endogenously expressed mesothelin. These results indicate that the mesothelin-10-5 peptide is a novel HLA-A*2402 restricted CTL epitope and that it is a promising candidate target for antigen-specific immunotherapy against pancreatic cancers.

Personalised peptide vaccination combined with radiotherapy for patients with advanced hepatocellular carcinoma: a phase 1 study

BackgroundWe aimed to investigate a new treatment method for the treatment of advanced hepatocellular carcinoma. The method was a cellular immune therapy based on personalised peptide vaccination (PPV-DC-CTL) combined with radiotherapy. MethodsWe did a phase 1 study in which patients with advanced hepatocellular carcinoma who were not eligible for surgery, as confirmed by multidisciplinary consultation, were treated with precise radiotherapy combined with PPV-DC-CTL in the Comprehensive Cancer Centre of Nanjing Drum Tower Hospital, Nanjing, China. To personalise the vaccine, the peptide candidate library, including mutated peptides and highly expressed peptides, was established according to the gene mutation and expression spectra of hepatocellular carcinoma and previous studies about PPV. Peptides for vaccination of every patient were selected from the peptide candidate library, with the consideration of the pre-existing immunity of the host before vaccination. The assay of peptide-specific IFN-γ production was used to define pre-existing immunity. Side-effects were measured with a haemogram. This study is registered with the Chinese Clinical Trial Registry, number ChiCTR-OIC-16010025. FindingsA total of nine patients with advanced hepatocellular carcinoma were admitted. Four patients had multiple liver metastases (liver lesions more than three pieces), one patient had liver metastasis and portal vein tumour thrombosis, one patient had lung and bone metastases, two patients had liver and lung metastases, and one patient had liver metastasis and peritoneal metastasis. Following radiotherapy and 1–3 cycles of PPV-DC-CTL treatment, AFP concentrations were significantly reduced in six patients compared with baseline concentrations, and imaging assessment of the lesions showed a partial response in three of the patients and stable disease in the other three patients. An objective response was achieved in 33% and disease control in 66%. This regimen was found to be safe and well tolerated because none of the patients developed liver or kidney side-effects. Only one patient developed grade 2 bone marrow suppression, and the other patients had no significant side-effects. InterpretationRadiotherapy combined with PPV-DC-CTL provides a new therapeutic strategy for patients with advanced hepatocellular carcinoma, which is well tolerated, safe, feasible, and effective. FundingNational Natural Science Foundation of China, Jiangsu Provincial Medical Youth Talent, and the Key Medical Science and Technology Development Project of Nanjing.

Identification of novel Lck-derived T helper epitope long peptides applicable for HLA-A2(+) cancer patients as cancer vaccine.

The present study attempted to identify T helper epitope long peptides capable of inducing cytotoxic T lymphocytes (CTL) from Lck antigen (p56(Lck) ), the src family tyrosine kinase, which is known to be aberrantly expressed in metastatic cancers cells, in order to develop a long peptide-based cancer vaccine for HLA-A2(+) cancer patients. Based on the biding motif to the HLA-DR and HLA-A2 alleles, 94 peptides were prepared from the Lck antigen. These peptides were screened for their reactivity to immunoglobulin G (IgG) from plasma of cancer patients, followed by testing of their ability to induce both CD4(+) and CD8(+) T lymphocytes showing not only peptide-specific IFN-γ production but cytotoxicity against HLA-A2(+) cancer cells from peripheral blood mononuclear cells (PBMC) of HLA-A2(+) cancer patients. Among 94 peptides tested, the three T helper epitope long peptides and their inner CTL epitope short peptides with HLA-A2 binding motifs were frequently recognized by IgG of cancer patients, and efficiently induced both CD4(+) IFN-γ(+) and CD8(+) IFN-γ(+) T lymphocytes. Patients' PBMC stimulated with these long peptides showed cytotoxicity against HLA-A2(+) Lck(+) cancer cells in HLA-class I and HLA-class II dependent manners. These three peptides might be useful for long peptide-based vaccines for HLA-A2(+) cancer patients with Lck(+) tumor cells.

Dendritic cell-mediated, DNA-based vaccination against hepatitis C induces the multi-epitope-specific response of humanized, HLA transgenic mice.

Hepatitis C virus (HCV) is the etiologic agent of chronic liver disease, hepatitis C. Spontaneous resolution of viral infection is associated with vigorous HLA class I- and class II-restricted T cell responses to multiple viral epitopes. Unfortunately, only 20% of patients clear infection spontaneously, most develop chronic disease and require therapy. The response to chemotherapy varies, however; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success. Vector-mediated vaccination with multi-epitope-expressing DNA constructs alone or in combination with chemotherapy offers an additional treatment approach. Gene sequences encoding validated HLA-A2- and HLA-DRB1-restricted epitopes were synthesized and cloned into an expression vector. Dendritic cells (DCs) derived from humanized, HLA-A2/DRB1 transgenic (donor) mice were transfected with these multi-epitope-expressing DNA constructs. Recipient HLA-A2/DRB1 mice were vaccinated s.c. with transfected DCs; control mice received non-transfected DCs. Peptide-specific IFN-γ production by splenic T cells obtained at 5 weeks post-immunization was quantified by ELISpot assay; additionally, the production of IL-4, IL-10 and TNF-α were quantified by cytokine bead array. Splenocytes derived from vaccinated HLA-A2/DRB1 transgenic mice exhibited peptide-specific cytokine production to the vast majority of the vaccine-encoded HLA class I- and class II-restricted T cell epitopes. A multi-epitope-based HCV vaccine that targets DCs offers an effective approach to inducing a broad immune response and viral clearance in chronic, HCV-infected patients.

T cells engineered with a T cell receptor against the prostate antigen TARP specifically kill HLA-A2 + prostate and breast cancer cells

To produce genetically engineered T cells directed against prostate and breast cancer cells, we have cloned the T-cell receptor recognizing the HLA-A2-restricted T-cell receptor γ-chain alternate reading-frame protein (TARP)(4-13) epitope. TARP is a protein exclusively expressed in normal prostate epithelium and in adenocarcinomas of the prostate and breast. Peripheral blood T cells transduced with a lentiviral vector encoding the TARP-TCR proliferated well when exposed to peptide-specific stimuli. These cells exerted peptide-specific IFN-γ production and cytotoxic activity. Importantly, HLA-A2(+) prostate and breast cancer cells expressing TARP were also killed, demonstrating that the TARP(4-13) epitope is a physiologically relevant target for T-cell therapy of prostate and breast cancer. In conclusion, we present the cloning of a T cell receptor (TCR) directed against a physiologically relevant HLA-A2 epitope of TARP. To our knowledge this report on engineering of T cells with a TCR directed against an antigen specifically expressed by prostate cells is unique.

Bystander suppression of experimental arthritis by nasal administration of a heat shock protein peptide

ObjectivesMucosal immune therapy with disease-inducing antigens is an effective way to prevent experimental arthritis, but in humans these antigens are unknown. In juvenile idiopathic arthritis, however, T cell recognition of...

T cell responses to bluetongue virus are directed against multiple and identical CD4 + and CD8 + T cell epitopes from the VP7 core protein in mouse and sheep

Bluetongue virus (BTV), an economically important orbivirus of the Reoviridae family, is a non-enveloped, dsRNA virus that causes a haemorrhagic disease mainly in sheep, but little is known of the cellular immunity elicited against BTV. We observed that vaccination of interferon type I-deficient mice (IFNAR (−/−)), or inoculation of the wild type C57BL/6 strain with BTV-8, induced a strong T cell response. Therefore, we proceeded to identify some of the T cell epitopes targeted by the immune system. We selected, using H-2 b-binding predictive algorithms, 3 major histocompatibility complex (MHC)-class II-binding peptides and 7 MHC-class I binding peptides from the BTV-8 core protein VP7, as potential T cell epitopes. Peptide binding assays confirmed that all 7 MHC-class I predicted peptides bound MHC-class I molecules. Three MHC-class I and 2 MHC-class II binding peptide consistently elicited peptide-specific IFN-γ production (as measured by ELISPOT assays) in splenocytes from C57BL/6 BTV-8-inoculated mice and IFNAR (−/−)-vaccinated mice. The functionality of these T cells was confirmed by proliferation and cytotoxicity assays. Flow cytometry analysis demonstrated that CD8 + T cells responded to MHC-class I binding peptides and CD4 + T cells to MHC-class II binding peptides. Importantly, these 5 epitopes were also able to induced IFN-γ production in sheep inoculated with BTV-8. Taken together, these data demonstrate the activation of BTV-specific T cells during infection and vaccination. The characterisation of these novel T cell epitopes may also provide an opportunity to develop DIVA-compliant vaccination approach to BTV encompassing a broad-spectrum of serotypes.

Phenotypic and functional characteristics of HIV-specific CD8 T cells and gag sequence variability after autologous dendritic cells based therapeutic vaccine

A decrease in HIV-1 specific CD8 T-cell responses associated with a partial control of viral replication occurred in 12 HIV-1-infected patients during autologous dendritic cells vaccination. HIV CD8 T cells were detected in 6/10 patients during immunizations, increasing after HAART discontinuation in 3 of them. Tet+ CD8 cells mainly had an effector phenotype (CD45RA−/+ CCR7− and CD28− and Perf+/−) and maintained IFN-γ release throughout follow-up. By contrast, patients with CD45RA−/+ CCR7+ Perf+ HIV-specific cells showed a decrease in peptide-specific IFN-γ production during vaccinations while levels were recovered when off HAART. No major mutations in either Gag p24 and p17 immunodominant epitopes were observed that might have explained the impaired CD8+ T-cell responses. Taken together, heterogeneity in the maturation status of HIV-specific CD8 T cells may be partially involved in the drop of peptide-specific IFN-γ production during immunizations.

Vpr is preferentially targeted by CTL during HIV-1 infection.

The HIV-1 accessory proteins Vpr, Vpu, and Vif are essential for viral replication, and their cytoplasmic production suggests that they should be processed for recognition by CTLs. However, the extent to which these proteins are targeted in natural infection, as well as precise CTL epitopes within them, remains to be defined. In this study, CTL responses against HIV-1 Vpr, Vpu, and Vif were analyzed in 60 HIV-1-infected individuals and 10 HIV-1-negative controls using overlapping peptides spanning the entire proteins. Peptide-specific IFN-gamma production was measured by ELISPOT assay and flow-based intracellular cytokine quantification. HLA class I restriction and cytotoxic activity were confirmed after isolation of peptide-specific CD8(+) T cell lines. CD8(+) T cell responses against Vpr, Vpu, and Vif were found in 45%, 2%, and 33% of HIV-1-infected individuals, respectively. Multiple CTL epitopes were identified in functionally important regions of HIV-1 Vpr and Vif. Moreover, in infected individuals in whom the breadth of HIV-1-specific responses was assessed comprehensively, Vpr and p17 were the most preferentially targeted proteins per unit length by CD8(+) T cells. These data indicate that despite the small size of these proteins Vif and Vpr are frequently targeted by CTL in natural HIV-1 infection and contribute importantly to the total HIV-1-specific CD8(+) T cell responses. These findings will be important in evaluating the specificity and breadth of immune responses during acute and chronic infection, and in the design and testing of candidate HIV vaccines.

Novel approach to the characterization of melanoma associated-peptide-specific CTL lines from Japanese metastatic melanoma patients

Melanoma-associated antigens, MART-1, tyrosinase, gp100 and MAGEs, are typical melanoma-specific tumor antigens which can potently induce immune responses in metastatic melanoma patients treated with peptide vaccines. In the present study, we established a dendritic cell (DC)-based HLA-A2 melanoma-associated peptide (MART-1 or gp100)-specific CTL induction method and characterized the CTLs using HLA-A2 tetramer staining in 6 cases of HLA-A2+ melanoma treated with DC vaccines. Peripheral blood mononuclear cells (PBMC) from patients were stimulated twice with MART-1 A2 peptide-pulsed DCs in the presence of a low dose of IL-2. To boost CTL populations, CTL lines were further stimulated twice with MART-1 A2 peptide-pulsed T2 cells. The frequency of MART-1 A2 tetramer-positive CTLs increased from 0.16% (prior to stimulation) to 2.15% (after DC stimulation), and reached 46.5% on average (after additional T2 stimulation) in 4 cases which showed a successful expansion. The absolute numbers of MART-1 A2 tetramer-positive CTLs increased from 187- to 619-fold (average, 415-fold) compared to prior to DC stimulation. CTL assays using MART-1-specific CTL lines demonstrated potent killing activity against MART-1 peptide-pulsed T2 cells or HLA-A2+ melanoma cell lines in accordance with the frequency of tetramer-positive CTLs. Finally, we were successful in identifying melanoma peptide-specific T-cell receptor (TCR) cDNAs in 2 cases for MART-1 and 1 case for gp100 using the anti-TCR MoAb-based sorting as a novel approach instead of a conventional cell cloning, and confirmed peptide-specific IFN-gamma production in TCR cDNA-transduced naïve T cells. The results showed that cloned TCR cDNAs were efficient in reconstituting tumor-specific cytotoxicity and good candidates for novel immunotherapy.