Quantification of Tumor-Specific T Lymphocytes With the ELISPOT Assay

Schmittel, Alexander; Keilholz, Ulrich; Thiel, Eckhard; Scheibenbogen, Carmen

doi:10.1097/00002371-200005000-00001

Cited by 61 publications

(26 citation statements)

References 39 publications

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“…The ex vivo 18-hour Elispot assay did not require cell expansion as it detected memory effector cells (both CD4 + and CD8 + cytokine-producing). It is a sensitive method that has been used to monitor cellular immune responses in patients receiving immunotherapy (30,31). Purified protein derivative was used as a positive control antigen to validate the stored PBMC samples, and to which all patients were expected to respond.…”

Section: Resultsmentioning

confidence: 99%

Mannan-MUC1–Pulsed Dendritic Cell Immunotherapy: A Phase I Trial in Patients with Adenocarcinoma

Loveland

Zhao

White

et al. 2006

Clinical Cancer Research

150

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Purpose: Tumor antigen-loaded dendritic cells show promise for cancer immunotherapy.This phase I study evaluated immunization with autologous dendritic cells pulsed with mannan-MUC1 fusion protein (MFP) to treat patients with advanced malignancy. Experimental Design: Eligible patients had adenocarcinoma expressing MUC1, were of performance status 0 to 1, with no autoimmune disease. Patients underwent leukapheresis to generate dendritic cells by culture ex vivo with granulocyte macrophage colony-stimulating factor and interleukin 4 for 5 days. Dendritic cells were then pulsed overnight with MFP and harvested for reinjection. Patients underwent three cycles of leukapheresis and reinjection at monthly intervals. Patients with clinical benefit were able to continue with dendritic cell-MFP immunotherapy. Results: Ten patients with a range of tumor types were enrolled, with median age of 60 years (range, 33-70 years); eight patients were of performance status 0 and two of performance status 1. Dendritic cell-MFP therapy led to strong T-cell IFNg Elispot responses to the vaccine and delayed-type hypersensitivity responses at injection sites in nine patients who completed treatments. Immune responses were sustained at 1 year in monitored patients. Antibody responses were seen in three patients only and were of low titer. Side effects were grade 1 only. Two patients with clearly progressive disease (ovarian and renal carcinoma) at entry were stable after initial therapy and went on to further leukapheresis and dendritic cell-MFP immunotherapy. These two patients have now each completed over 3 years of treatment. Conclusions: Immunization produced T-cell responses in all patients with evidence of tumor stabilization in 2 of the 10 advanced cancer patients treated. These data support further clinical evaluation of this dendritic cell-MFP immunotherapy.

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Section: Resultsmentioning

confidence: 99%

Mannan-MUC1–Pulsed Dendritic Cell Immunotherapy: A Phase I Trial in Patients with Adenocarcinoma

Loveland

Zhao

White

et al. 2006

Clinical Cancer Research

150

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“…For this reason, besides this method, other two assays are recommended as first-line monitoring tools in clinical vaccination studies: IFN-γ ELISPOT assays and intracellular cytokine staining (ICS) in flow cytometry [136]. IFN-γ ELISPOT owns the lowest detection limit for specific T cell responses at the single cell levels [137], while ICS allows the simultaneous analysis of multiple cytokines and phenotypic markers of T-cell activation, thus identifying multifunctional T cells [109,138]. These 3 assays, strongly suggested for the immune-monitoring of vaccination trials, require the early identification of the target of interest, and thus may benefit from previously identified epitopes.…”

Section: Discussionmentioning

confidence: 99%

1086 IGHV1-69 as a Promising Candidate for the Development of a Shared Immunotherapy to B-cell Lymphomas

Muraro¹,

Martorelli²,

Bomben³

et al. 2012

European Journal of Cancer

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B-cell Non-Hodgkin Lymphomas (B-NHL) are a heterogeneous group of cancers, broadly diffused worldwide and often relapsing after standard treatment and rituximab. Therapeutic vaccines targeting B-NHL idiotype (Id) represent a promising approach to maintain the complete response induced by standard treatments. However, customized idiotypic vaccination still remains a non-approved, experimental therapeutic option, mostly due to the personalized use and penalized by the lack of reliable clinical or biological markers of patient eligibility and responsiveness. Nevertheless, the molecular characterization of different lymphoid tumor histotypes revealed a set of stereotyped immunoglobulins among distinct B-cell lymphoma types. On this ground, we focused our attention on the IGHV1-69 protein, frequently expressed in HCV-associated lymphomas, chronic lymphocytic leukaemia, and auto-immunity related lymphoproliferations, and we characterized the ex vivo immunogenicity of this protein.Seventy IGHV1-69 sequences obtained from patients affected by different B-NHLs or pre-malignant lymphoproliferations were compared to design an optimized sequence characterized by the highest degree of similarity among studied cancers, and thus CDR3 hypervariable region free. Within this "immunogenically" optimized sequence, we identified 13 potential HLA class-I cytotoxic T lymphocyte (CTL) epitopes and synthesized the corresponding pentamers (Pent). We assessed by flow cytometry the presence and extent of epitope-specific T-cell responses in peripheral blood of patients with IGHV1-69 + B-cell lymphoproliferative disorders and healthy donors, and validated these data in IFN-γ ELISPOT (Enzyme-linked immunosorbent spot) assays. Finally, we boosted in vitro IGHV1-69-specific responses by stimulating peripheral blood lymphocytes (PBL) from donors and patients with different protocols for the generation of epitope-specific CTL cultures.Interestingly, the IGHV1-69 Pent + population observed in patients' samples was generally larger than in donors, supporting the existence of spontaneous memory T-cell responses against IGHV1-69, at least for some HLA-restrictions. Surprisingly, in patients' samples, IGHV1-69-recognizing T cells displayed higher IFN-γ release in ELISPOT assays compared to viral-specific T cells. Moreover, we obtained peptide-specific CTL lines, which showed a weak but specific lysis against peptide-pulsed targets, especially when derived from patients' PBLs. In addition, we were able to generate IGHV1-69-epitope specific CTL clones from healthy donors CD8 + T cells, employing synthetic artificial APC, developed to elicit and expand low-avidity tumor-directed human CTL lines. Finally, IGHV1-69-induced CTL lines showed specific lysis also towards an IGHV1-69 naturally expressing cell line, suggesting that IGHV1-69 memory T-cell responses could be boosted for therapeutic purposes.These results show that IGHV1-69 constitutes a potential target for the development of a subset-specific Id vaccine. Furthermore, multimer (tetramers...

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“…However, the ELISPOT assay also allows for the stimulation of T cells with complex antigens such as whole proteins, cell lysates, or whole or apoptotic cells. In the case of complex antigens, these would need to be pre-incubated with antigen-presenting cells (APC) (Schmittel et al 2000). In this case, antigens would be processed by the APC and appropriately presented to CD4+ and CD8+ T cells, with co-stimulatory signals, according to each cell's specific requirements.…”

Section: Discussionmentioning

confidence: 99%

Dual-Color ELISPOT Assay for the Simultaneous Detection of IL-2 and/or IFN-γ Secreting T Cells

Boulet

Ndongala

Bernard

2010

Cold Spring Harb Protoc

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INTRODUCTIONThe enzyme-linked immunospot (ELISPOT) assay measures the secretion intensity of effector molecules released by immune cells in response to ex vivo antigenic stimulation, as well as the frequency of these responding cells. This assay is highly sensitive, quantitative, easy to use, and amenable to high-throughput screening. For these reasons, the ELISPOT assay is considered by many as a gold standard for monitoring cellular immune responses. Until recently, ELISPOT assays using chromophores to detect the T cell secretion of cytokines were limited to the characterization of a single effector molecule. Notably, studies evaluating the immune response to chronic viral infections often measured IFN-γ secretion by ELISPOT because of the known antiviral effects of this cytokine as well as its correlation to the cytotoxic capacity of T cells. However, maintenance of both IFN-γ and IL-2 secretion by pathogen-specific T cells has been linked to a more favorable clinical outcome in human immunodeficiency virus (HIV) and Leishmania infections. Therefore, an ELISPOT assay able to simultaneously characterize T cell responses in terms of IL-2 and IFN-γ secretion is potentially relevant for the monitoring of immune responses to certain infectious agents. In this protocol, we describe an ELISPOT assay for the simultaneous detection of IL-2 and IFN-γ upon stimulation with viral peptides.

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Quantification of Tumor-Specific T Lymphocytes With the ELISPOT Assay

Cited by 61 publications

References 39 publications

Mannan-MUC1–Pulsed Dendritic Cell Immunotherapy: A Phase I Trial in Patients with Adenocarcinoma

Mannan-MUC1–Pulsed Dendritic Cell Immunotherapy: A Phase I Trial in Patients with Adenocarcinoma

1086 IGHV1-69 as a Promising Candidate for the Development of a Shared Immunotherapy to B-cell Lymphomas

Dual-Color ELISPOT Assay for the Simultaneous Detection of IL-2 and/or IFN-γ Secreting T Cells

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