Reference Citation Analysis: Find an Article, Find a Category, Find a Journal, Find a Scholar

For: Zhang W, Caspell R, Karulin AY, Ahmad M, Haicheur N, Abdelsalam A, Johannesen K, Vignard V, Dudzik P, Georgakopoulou K, Mihaylova A, Silina K, Aptsiauri N, Adams V, Lehmann PV, McArdle S. ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring--even in hands of ELISPOT-inexperienced investigators. J Immunotoxicol 2010;6:227-34. [PMID: 19908941 DOI: 10.3109/15476910903317546] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [What about the content of this article? (0)] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open

For:	Zhang W, Caspell R, Karulin AY, Ahmad M, Haicheur N, Abdelsalam A, Johannesen K, Vignard V, Dudzik P, Georgakopoulou K, Mihaylova A, Silina K, Aptsiauri N, Adams V, Lehmann PV, McArdle S. ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring--even in hands of ELISPOT-inexperienced investigators. J Immunotoxicol 2010;6:227-34. [PMID: 19908941 DOI: 10.3109/15476910903317546] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [What about the content of this article? (0)] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open

Number

Cited by Other Article(s)

Lehmann PV, Karulin AY, Becza N, Yao L, Liu Z, Chepke J, Maul-Pavicic A, Wolf C, Köppert S, Valente AV, Gorbachev AV, Tary-Lehmann M, Kirchenbaum GA. Theoretical and practical considerations for validating antigen-specific B cell ImmunoSpot assays. J Immunol Methods 2025;537:113817. [PMID: 39864733 DOI: 10.1016/j.jim.2025.113817] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2024] [Revised: 10/17/2024] [Accepted: 01/20/2025] [Indexed: 01/28/2025]

Carreto-Binaghi LE, Nieto-Ponce M, Palencia-Reyes A, Chávez-Domínguez RL, Blancas-Zaragoza J, Franco-Mendoza P, García-Ramos MA, Hernández-Lázaro CI, Torres M, Carranza C. Validation of the Enzyme-Linked ImmunoSpot Analytic Method for the Detection of Human IFN-γ from Peripheral Blood Mononuclear Cells in Response to the SARS-CoV-2 Spike Protein. Biomolecules 2024;14:1286. [PMID: 39456219 PMCID: PMC11506497 DOI: 10.3390/biom14101286] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Revised: 09/25/2024] [Accepted: 10/01/2024] [Indexed: 10/28/2024] Open

Affiliation(s)

Laura E. Carreto-Binaghi Laboratorio de Inmunobiología de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas, Mexico City 14080, Mexico; (L.E.C.-B.); (M.N.-P.); (A.P.-R.); (R.L.C.-D.); (J.B.-Z.); (P.F.-M.); (M.A.G.-R.)
Milton Nieto-Ponce Laboratorio de Inmunobiología de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas, Mexico City 14080, Mexico; (L.E.C.-B.); (M.N.-P.); (A.P.-R.); (R.L.C.-D.); (J.B.-Z.); (P.F.-M.); (M.A.G.-R.)
Andrea Palencia-Reyes Laboratorio de Inmunobiología de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas, Mexico City 14080, Mexico; (L.E.C.-B.); (M.N.-P.); (A.P.-R.); (R.L.C.-D.); (J.B.-Z.); (P.F.-M.); (M.A.G.-R.)
Rodolfo L. Chávez-Domínguez Laboratorio de Inmunobiología de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas, Mexico City 14080, Mexico; (L.E.C.-B.); (M.N.-P.); (A.P.-R.); (R.L.C.-D.); (J.B.-Z.); (P.F.-M.); (M.A.G.-R.)
Jessica Blancas-Zaragoza Laboratorio de Inmunobiología de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas, Mexico City 14080, Mexico; (L.E.C.-B.); (M.N.-P.); (A.P.-R.); (R.L.C.-D.); (J.B.-Z.); (P.F.-M.); (M.A.G.-R.)
Pablo Franco-Mendoza Laboratorio de Inmunobiología de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas, Mexico City 14080, Mexico; (L.E.C.-B.); (M.N.-P.); (A.P.-R.); (R.L.C.-D.); (J.B.-Z.); (P.F.-M.); (M.A.G.-R.)
Montserrat A. García-Ramos Laboratorio de Inmunobiología de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas, Mexico City 14080, Mexico; (L.E.C.-B.); (M.N.-P.); (A.P.-R.); (R.L.C.-D.); (J.B.-Z.); (P.F.-M.); (M.A.G.-R.)
Claudia I. Hernández-Lázaro Laboratorio Clinico, Instituto Nacional de Enfermedades Respiratorias (INER) “Ismael Cosío Villegas”, Mexico City 14080, Mexico;
Martha Torres Laboratorio de Inmunobiología de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas, Mexico City 14080, Mexico; (L.E.C.-B.); (M.N.-P.); (A.P.-R.); (R.L.C.-D.); (J.B.-Z.); (P.F.-M.); (M.A.G.-R.)
Claudia Carranza Laboratorio de Inmunobiología de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas, Mexico City 14080, Mexico; (L.E.C.-B.); (M.N.-P.); (A.P.-R.); (R.L.C.-D.); (J.B.-Z.); (P.F.-M.); (M.A.G.-R.)

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Krivoshik SR, Dzielak L, Masters AR, Hall J, Johnson AJ. Development of an Enzyme-Linked Immunosorbent Spot Assay for the Assessment of Adeno-Associated Virus Peptides to Examine Immune Safety. Hum Gene Ther 2024;35:506-516. [PMID: 38264994 DOI: 10.1089/hum.2023.180] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2024] Open

Traska AK, Nowacki TM, Vollenberg R, Rennebaum F, Meier JA, Schomacher T, Reinartz Groba SN, Fischer J, Trebicka J, Tepasse PR. Immunomonitoring via ELISPOT Assay Reveals Attenuated T-Cell Immunity to CMV in Immunocompromised Liver-Transplant Patients. Cells 2024;13:741. [PMID: 38727277 PMCID: PMC11083338 DOI: 10.3390/cells13090741] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2024] [Revised: 04/12/2024] [Accepted: 04/21/2024] [Indexed: 05/13/2024] Open

Abstract

Assessing immune responses to cytomegalovirus (CMV) after liver transplant in patients on immunosuppressive therapy remains challenging. In this study, employing ELISPOT assays, 52 liver-transplant recipients were evaluated for antiviral T-cell activity in peripheral blood mononuclear cells (PBMCs), measuring interferon-γ (IFN-γ) secretion upon stimulation with CMV-specific peptides (CMV peptide pool, CMV IE-1, and pp65 antigens). Parameters such as stimulation index, mean spot size, and mean spot count were measured. The study found that heightened immunosuppression, especially with prednisolone in triple therapy, significantly dampened CMV-specific immune responses. This was demonstrated by decreased IFN-γ production by CMV-specific T-cells (CMV peptide pool: p = 0.036; OR = 0.065 [95% CI: 0.005-0.840], pp65 antigen: p = 0.026; OR = 0.048 [95% CI: 0.003-0.699]). Increased immunosuppression correlated with reduced IFN-γ secretion per cell, reflected in smaller mean spot sizes for the CMV peptide pool (p = 0.019). Notably, shorter post-transplant intervals correlated with diminished antiviral T-cell IFN-γ release at two years (CMV peptide pool: p = 0.019; IE antigen: p = 0.010) and five years (CMV peptide pool: p = 0.0001; IE antigen: p = 0.002; pp65 antigen: p = 0.047), as did advancing age (pp65 antigen: p = 0.016, OR = 0.932, 95% CI: 0.881-0.987). Patients with undetectable CMV antigens had a notably higher risk of CMV reactivation within six months from blood collection, closely linked with triple immunosuppression and prednisolone use. These findings highlight the intricate interplay between immunosuppression, immune response dynamics, and CMV reactivation risk, emphasizing the necessity for tailored immunosuppressive strategies to mitigate CMV reactivation in liver-transplant recipients. It can be concluded that, particularly in the early months post-transplantation, the use of prednisolone as a third immunosuppressant should be critically reconsidered. Additionally, the use of prophylactic antiviral therapy effective against CMV in this context holds significant importance.

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Affiliation(s)

Ann-Kristin Traska Department of Medicine B for Gastroenterology, Hepatology, Endocrinology and Clinical Infectiology, University Hospital Münster, 48149 Münster, Germany; (A.-K.T.); (R.V.); (F.R.); (J.A.M.); (T.S.); (S.N.R.G.); (J.F.); (J.T.)
Tobias Max Nowacki Department of Internal Medicine and Gastroenterology, Marienhospital Steinfurt, 48565 Steinfurt, Germany;
Richard Vollenberg Department of Medicine B for Gastroenterology, Hepatology, Endocrinology and Clinical Infectiology, University Hospital Münster, 48149 Münster, Germany; (A.-K.T.); (R.V.); (F.R.); (J.A.M.); (T.S.); (S.N.R.G.); (J.F.); (J.T.)
Florian Rennebaum Department of Medicine B for Gastroenterology, Hepatology, Endocrinology and Clinical Infectiology, University Hospital Münster, 48149 Münster, Germany; (A.-K.T.); (R.V.); (F.R.); (J.A.M.); (T.S.); (S.N.R.G.); (J.F.); (J.T.)
Jörn Arne Meier Department of Medicine B for Gastroenterology, Hepatology, Endocrinology and Clinical Infectiology, University Hospital Münster, 48149 Münster, Germany; (A.-K.T.); (R.V.); (F.R.); (J.A.M.); (T.S.); (S.N.R.G.); (J.F.); (J.T.)
Tina Schomacher Department of Medicine B for Gastroenterology, Hepatology, Endocrinology and Clinical Infectiology, University Hospital Münster, 48149 Münster, Germany; (A.-K.T.); (R.V.); (F.R.); (J.A.M.); (T.S.); (S.N.R.G.); (J.F.); (J.T.)
Sara Noemi Reinartz Groba Department of Medicine B for Gastroenterology, Hepatology, Endocrinology and Clinical Infectiology, University Hospital Münster, 48149 Münster, Germany; (A.-K.T.); (R.V.); (F.R.); (J.A.M.); (T.S.); (S.N.R.G.); (J.F.); (J.T.)
Julia Fischer Department of Medicine B for Gastroenterology, Hepatology, Endocrinology and Clinical Infectiology, University Hospital Münster, 48149 Münster, Germany; (A.-K.T.); (R.V.); (F.R.); (J.A.M.); (T.S.); (S.N.R.G.); (J.F.); (J.T.)
Jonel Trebicka Department of Medicine B for Gastroenterology, Hepatology, Endocrinology and Clinical Infectiology, University Hospital Münster, 48149 Münster, Germany; (A.-K.T.); (R.V.); (F.R.); (J.A.M.); (T.S.); (S.N.R.G.); (J.F.); (J.T.)
Phil-Robin Tepasse Department of Medicine B for Gastroenterology, Hepatology, Endocrinology and Clinical Infectiology, University Hospital Münster, 48149 Münster, Germany; (A.-K.T.); (R.V.); (F.R.); (J.A.M.); (T.S.); (S.N.R.G.); (J.F.); (J.T.)

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Lehmann AA, Roen DR, Megyesi Z, Lehmann PV. Reagent Tracker^™ Platform Verifies and Provides Audit Trails for the Error-Free Implementation of T-Cell ImmunoSpot^® Assays. Methods Mol Biol 2024;2768:105-115. [PMID: 38502390 DOI: 10.1007/978-1-0716-3690-9_7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/21/2024]

Waerlop G, Leroux-Roels G, Pagnon A, Begue S, Salaun B, Janssens M, Medaglini D, Pettini E, Montomoli E, Gianchecchi E, Lambe T, Godfrey L, Bull M, Bellamy D, Amdam H, Bredholt G, Cox RJ, Clement F. Proficiency tests to evaluate the impact on assay outcomes of harmonized influenza-specific Intracellular Cytokine Staining (ICS) and IFN-ɣ Enzyme-Linked ImmunoSpot (ELISpot) protocols. J Immunol Methods 2023;523:113584. [PMID: 37918618 DOI: 10.1016/j.jim.2023.113584] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2023] [Revised: 09/30/2023] [Accepted: 10/28/2023] [Indexed: 11/04/2023]

Abstract

The magnitude and quality of cell-mediated immune responses elicited by natural infection or vaccination are commonly measured by Interferon-ɣ (IFN-ɣ) Enzyme-Linked ImmunoSpot (ELISpot) and Intracellular Cytokine Staining (ICS). To date, laboratories apply a variety of in-house procedures which leads to diverging results, complicates interlaboratory comparisons and hampers vaccine evaluations. During the FLUCOP project, efforts have been made to develop harmonized Standard Operating Procedures (SOPs) for influenza-specific IFN-ɣ ELISpot and ICS assays. Exploratory pilot studies provided information about the interlaboratory variation before harmonization efforts were initiated. Here we report the results of two proficiency tests organized to evaluate the impact of the harmonization effort on assay results and the performance of participating FLUCOP partners. The introduction of the IFN-ɣ ELISpot SOP reduced variation of both background and stimulated responses. Post-harmonization background responses were all lower than an arbitrary threshold of 50 SFU/million cells. When stimulated with A/California and B/Phuket, a statistically significant reduction in variation (p < 0.0001) was observed and CV values were strongly reduced, from 148% to 77% for A/California and from 126% to 73% for B/Phuket. The harmonizing effect of applying an ICS SOP was also confirmed by an increased homogeneity of data obtained by the individual labs. The application of acceptance criteria on cell viability and background responses further enhanced the data homogeneity. Finally, as the same set of samples was analyzed by both the IFN-ɣ ELISpot and the ICS assays, a method comparison was performed. A clear correlation between the two methods was observed, but they cannot be considered interchangeable. In conclusion, proficiency tests show that a limited harmonization effort consisting of the introduction of SOPs and the use of the same in vitro stimulating antigens leads to a reduction of the interlaboratory variation of IFN-ɣ ELISpot data and demonstrate that substantial improvements for the ICS assay are achieved as comparable laboratory datasets could be generated. Additional steps to further reduce the interlaboratory variation of ICS data can consist of standardized gating templates and detailed data reporting instructions as well as further efforts to harmonize reagent and instrument use.

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Affiliation(s)

Gwenn Waerlop Center for Vaccinology (CEVAC), Ghent University and University Hospital, Ghent, Belgium.
Geert Leroux-Roels Center for Vaccinology (CEVAC), Ghent University and University Hospital, Ghent, Belgium
Anke Pagnon Sanofi, Research Global Immunology, Marcy l'Etoile, France
Sarah Begue Sanofi, Research Global Immunology, Marcy l'Etoile, France
Bruno Salaun GSK, Rixensart, Belgium
Michel Janssens GSK, Rixensart, Belgium
Donata Medaglini Laboratory of Molecular Microbiology and Biotechnology, Department of Medical Biotechnologies, University of Siena, Siena, Italy
Elena Pettini Laboratory of Molecular Microbiology and Biotechnology, Department of Medical Biotechnologies, University of Siena, Siena, Italy
Emanuele Montomoli Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy; VisMederi srl, 53100 Siena, Italy
Elena Gianchecchi VisMederi srl, 53100 Siena, Italy
Teresa Lambe Oxford Vaccine Group, Department of Paediatrics, Medical Sciences Division, University of Oxford, UK; Chinese Academy of Medical Science (CAMS) Oxford Institute (COI), University of Oxford, United Kingdom
Leila Godfrey Oxford Vaccine Group, Department of Paediatrics, Medical Sciences Division, University of Oxford, UK
Maireid Bull Oxford Vaccine Group, Department of Paediatrics, Medical Sciences Division, University of Oxford, UK; Chinese Academy of Medical Science (CAMS) Oxford Institute (COI), University of Oxford, United Kingdom
Duncan Bellamy The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
Håkon Amdam Influenza Centre, Department of Clinical Science, University of Bergen, N5021 Bergen, Norway
Geir Bredholt Influenza Centre, Department of Clinical Science, University of Bergen, N5021 Bergen, Norway
Rebecca Jane Cox Influenza Centre, Department of Clinical Science, University of Bergen, N5021 Bergen, Norway
Frédéric Clement Center for Vaccinology (CEVAC), Ghent University and University Hospital, Ghent, Belgium

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Law JC, Watts TH. Considerations for Choosing T Cell Assays during a Pandemic. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2023;211:169-174. [PMID: 37399079 DOI: 10.4049/jimmunol.2300129] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/21/2023] [Accepted: 03/15/2023] [Indexed: 07/05/2023]

Sereda AD, Kazakova AS, Namsrayn SG, Vlasov ME, Sindryakova IP, Kolbasov DV. Subsequent Immunization of Pigs with African Swine Fever Virus (ASFV) Seroimmunotype IV Vaccine Strain FK-32/135 and by Recombinant Plasmid DNA Containing the CD2v Derived from MK-200 ASFV Seroimmunotype III Strain Does Not Protect from Challenge with ASFV Seroimmunotype III. Vaccines (Basel) 2023;11:vaccines11051007. [PMID: 37243111 DOI: 10.3390/vaccines11051007] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Revised: 05/15/2023] [Accepted: 05/19/2023] [Indexed: 05/28/2023] Open

Lehmann PV, Roen DR, Lehmann AA. Unbiased, High-Throughput Identification of T Cell Epitopes by ELISPOT. Methods Mol Biol 2023;2673:69-88. [PMID: 37258907 DOI: 10.1007/978-1-0716-3239-0_5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/02/2023]

Yang TY, Braun M, Lembke W, McBlane F, Kamerud J, DeWall S, Tarcsa E, Fang X, Hofer L, Kavita U, Upreti VV, Gupta S, Loo L, Johnson AJ, Chandode RK, Stubenrauch KG, Vinzing M, Xia CQ, Jawa V. Immunogenicity assessment of AAV-based gene therapies: An IQ consortium industry white paper. Mol Ther Methods Clin Dev 2022;26:471-494. [PMID: 36092368 PMCID: PMC9418752 DOI: 10.1016/j.omtm.2022.07.018] [Citation(s) in RCA: 34] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]

Janssens Y, Joye J, Waerlop G, Clement F, Leroux-Roels G, Leroux-Roels I. The role of cell-mediated immunity against influenza and its implications for vaccine evaluation. Front Immunol 2022;13:959379. [PMID: 36052083 PMCID: PMC9424642 DOI: 10.3389/fimmu.2022.959379] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2022] [Accepted: 07/27/2022] [Indexed: 12/25/2022] Open

Immunological Assessment of Lung Responses to Inhalational Lipoprotein Vaccines Against Bacterial Pathogens. Methods Mol Biol 2021. [PMID: 34784043 DOI: 10.1007/978-1-0716-1900-1_16] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]

Ranieri E, Netti GS, Gigante M. CTL ELISPOT Assay and T Cell Detection. Methods Mol Biol 2021;2325:65-77. [PMID: 34053051 DOI: 10.1007/978-1-0716-1507-2_5] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]

Abstract

Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T-cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T-cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T-cell (CTL) studies have taken advantage with this high-throughput technology by providing insights of quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T-cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T-cells is of relevance for immune diagnostic. The most utilized Elispot assay is the Interferon-gamma (IFN-γ) test, a marker for CD8⁺ CTL activation, but Elispot can be also used to distinguish different subsets of activated T-cells by using other cytokines such as T-helper (Th) 1 type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21 and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10 and IL-13), and Th17 (IL-17) cells.The reliability of Elispot generated data, by the evaluation of T-cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot Assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Milteny cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T-cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot Assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and Perforin Elispot assays will be described.

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Wang C, Wang Z, Rosner GL, Huh WK, Roden RBS, Bae S. A batch-effect adjusted Simon's two-stage design for cancer vaccine clinical studies. Biometrics 2020;77:1075-1088. [PMID: 32822525 DOI: 10.1111/biom.13358] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2019] [Revised: 07/25/2020] [Accepted: 08/10/2020] [Indexed: 11/29/2022]

Immunization of Pigs with Recombinant Plasmids Containing Genes of Ubiquitinated p30, p54 and CD2v Proteins of African Swine Fever Virus. ACTA VET-BEOGRAD 2020. [DOI: 10.2478/acve-2020-0007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open

Wilcox CR, Jones CE. Beyond Passive Immunity: Is There Priming of the Fetal Immune System Following Vaccination in Pregnancy and What Are the Potential Clinical Implications? Front Immunol 2018;9:1548. [PMID: 30061881 PMCID: PMC6054988 DOI: 10.3389/fimmu.2018.01548] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2018] [Accepted: 06/22/2018] [Indexed: 02/06/2023] Open

Karulin AY, Megyesi Z, Caspell R, Hanson J, Lehmann PV. Multiplexing T- and B-Cell FLUOROSPOT Assays: Experimental Validation of the Multi-Color ImmunoSpot^® Software Based on Center of Mass Distance Algorithm. Methods Mol Biol 2018;1808:95-113. [PMID: 29956177 DOI: 10.1007/978-1-4939-8567-8_9] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]

Abstract

Over the past decade, ELISPOT has become a highly implemented mainstream assay in immunological research, immune monitoring, and vaccine development. Unique single cell resolution along with high throughput potential sets ELISPOT apart from flow cytometry, ELISA, microarray- and bead-based multiplex assays. The necessity to unambiguously identify individual T and B cells that do, or do not co-express certain analytes, including polyfunctional cytokine producing T cells has stimulated the development of multi-color ELISPOT assays. The success of these assays has also been driven by limited sample/cell availability and resource constraints with reagents and labor. There are few commercially available test kits and instruments available at present for multi-color FLUOROSPOT. Beyond commercial descriptions of competing systems, little is known about their accuracy in experimental settings detecting individual cells that secrete multiple analytes vs. random overlays of spots. Here, we present a theoretical and experimental validation study for three and four color T- and B-cell FLUOROSPOT data analysis. The ImmunoSpot^® Fluoro-X™ analysis system we used includes an automatic image acquisition unit that generates individual color images free of spectral overlaps and multi-color spot counting software based on the maximal allowed distance between centers of spots of different colors or Center of Mass Distance (COMD). Using four color B-cell FLUOROSPOT for IgM, IgA, IgG1, IgG3; and three/four color T-cell FLUOROSPOT for IL-2, IFN-γ, TNF-α, and GzB, in serial dilution experiments, we demonstrate the validity and accuracy of Fluoro-X™ multi-color spot counting algorithms. Statistical predictions based on the Poisson spatial distribution, coupled with scrambled image counting, permit objective correction of true multi-color spot counts to exclude randomly overlaid spots.

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Multi-Color FLUOROSPOT Counting Using ImmunoSpot^® Fluoro-X™ Suite. Methods Mol Biol 2018;1808:115-131. [PMID: 29956178 DOI: 10.1007/978-1-4939-8567-8_10] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]

A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool. Cells 2017;6:cells6040047. [PMID: 29215584 PMCID: PMC5753071 DOI: 10.3390/cells6040047] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2017] [Revised: 12/01/2017] [Accepted: 12/03/2017] [Indexed: 12/29/2022] Open

Rojas JM, Avia M, Pascual E, Sevilla N, Martín V. Vaccination with recombinant adenovirus expressing peste des petits ruminants virus-F or -H proteins elicits T cell responses to epitopes that arises during PPRV infection. Vet Res 2017;48:79. [PMID: 29157291 PMCID: PMC5697415 DOI: 10.1186/s13567-017-0482-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2017] [Accepted: 10/26/2017] [Indexed: 12/22/2022] Open

Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity. Viruses 2015;7:4414-37. [PMID: 26258786 PMCID: PMC4576189 DOI: 10.3390/v7082828] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2015] [Revised: 07/15/2015] [Accepted: 07/27/2015] [Indexed: 11/23/2022] Open

Trück J, Mitchell R, Thompson AJ, Morales-Aza B, Clutterbuck EA, Kelly DF, Finn A, Pollard AJ. Effect of cryopreservation of peripheral blood mononuclear cells (PBMCs) on the variability of an antigen-specific memory B cell ELISpot. Hum Vaccin Immunother 2015;10:2490-6. [PMID: 25424961 DOI: 10.4161/hv.29318] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open

Rekers NV, von Herrath MG, Wesley JD. Immunotherapies and immune biomarkers in Type 1 diabetes: A partnership for success. Clin Immunol 2015;161:37-43. [PMID: 26122172 DOI: 10.1016/j.clim.2015.05.021] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2015] [Revised: 05/13/2015] [Accepted: 05/17/2015] [Indexed: 12/16/2022]

Measuring Cellular Immunity to Influenza: Methods of Detection, Applications and Challenges. Vaccines (Basel) 2015;3:293-319. [PMID: 26343189 PMCID: PMC4494351 DOI: 10.3390/vaccines3020293] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2015] [Revised: 03/27/2015] [Accepted: 03/30/2015] [Indexed: 12/11/2022] Open

Hope CM, Coates PTH, Carroll RP. Immune profiling and cancer post transplantation. World J Nephrol 2015;4:41-56. [PMID: 25664246 PMCID: PMC4317627 DOI: 10.5527/wjn.v4.i1.41] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/11/2014] [Revised: 11/03/2014] [Accepted: 11/07/2014] [Indexed: 02/06/2023] Open

ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells. Cells 2015;4:71-83. [PMID: 25643292 PMCID: PMC4381210 DOI: 10.3390/cells4010071] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2014] [Revised: 11/18/2014] [Accepted: 12/01/2014] [Indexed: 12/03/2022] Open

ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting. Cells 2015;4:56-70. [PMID: 25612115 PMCID: PMC4381209 DOI: 10.3390/cells4010056] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Accepted: 01/14/2015] [Indexed: 11/24/2022] Open

High Reproducibility of ELISPOT Counts from Nine Different Laboratories. Cells 2015;4:21-39. [PMID: 25585297 PMCID: PMC4381207 DOI: 10.3390/cells4010021] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2014] [Accepted: 11/26/2014] [Indexed: 11/17/2022] Open

Serial Measurements of Apoptotic Cell Numbers Provide Better Acceptance Criterion for PBMC Quality than a Single Measurement Prior to the T Cell Assay. Cells 2015;4:40-55. [PMID: 25585298 PMCID: PMC4381208 DOI: 10.3390/cells4010040] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2014] [Accepted: 12/05/2014] [Indexed: 11/17/2022] Open

Bacchetta R, Lucarelli B, Sartirana C, Gregori S, Lupo Stanghellini MT, Miqueu P, Tomiuk S, Hernandez-Fuentes M, Gianolini ME, Greco R, Bernardi M, Zappone E, Rossini S, Janssen U, Ambrosi A, Salomoni M, Peccatori J, Ciceri F, Roncarolo MG. Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells. Front Immunol 2014;5:16. [PMID: 24550909 PMCID: PMC3907718 DOI: 10.3389/fimmu.2014.00016] [Citation(s) in RCA: 116] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2013] [Accepted: 01/09/2014] [Indexed: 11/21/2022] Open

Affiliation(s)

Rosa Bacchetta Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, San Raffaele Telethon Institute for Gene Therapy , Milan , Italy
Barbarella Lucarelli Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, San Raffaele Telethon Institute for Gene Therapy , Milan , Italy
Claudia Sartirana Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, San Raffaele Telethon Institute for Gene Therapy , Milan , Italy
Silvia Gregori Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, San Raffaele Telethon Institute for Gene Therapy , Milan , Italy
Maria T Lupo Stanghellini Hematology and BMT Unit, San Raffaele Hospital , Milan , Italy
Patrick Miqueu TcLand Expression S. A. , Nantes , France
Stefan Tomiuk Miltenyi Biotec GmbH , Bergisch-Gladbach , Germany
Maria Hernandez-Fuentes MRC Center for Transplantation, King's College London , London , UK
Monica E Gianolini Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, San Raffaele Telethon Institute for Gene Therapy , Milan , Italy
Raffaella Greco Hematology and BMT Unit, San Raffaele Hospital , Milan , Italy
Massimo Bernardi Hematology and BMT Unit, San Raffaele Hospital , Milan , Italy
Elisabetta Zappone Hematology and BMT Unit, San Raffaele Hospital , Milan , Italy
Silvano Rossini Unit of Immunohaematology and Transfusion Medicine Service, San Raffaele Hospital , Milan , Italy
Uwe Janssen Miltenyi Biotec GmbH , Bergisch-Gladbach , Germany
Alessandro Ambrosi Center for Statistics in Biomedical Sciences, San Raffaele Scientific Institute , Milan , Italy
Monica Salomoni MolMed SpA , Milan , Italy
Jacopo Peccatori Hematology and BMT Unit, San Raffaele Hospital , Milan , Italy
Fabio Ciceri Hematology and BMT Unit, San Raffaele Hospital , Milan , Italy
Maria-Grazia Roncarolo Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, San Raffaele Telethon Institute for Gene Therapy , Milan , Italy ; Vita-Salute San Raffaele University , Milan , Italy

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Ranieri E, Popescu I, Gigante M. CTL ELISPOT assay. Methods Mol Biol 2014;1186:75-86. [PMID: 25149304 DOI: 10.1007/978-1-4939-1158-5_6] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]

Abstract

Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and perforin Elispot assays are described.

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Butterfield LH, Buffo MJ. Immunologic monitoring of cancer vaccine trials using the ELISPOT assay. Methods Mol Biol 2014;1102:71-82. [PMID: 24258974 DOI: 10.1007/978-1-62703-727-3_5] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]

Enumeration and characterization of human memory T cells by enzyme-linked immunospot assays. Clin Dev Immunol 2013;2013:637649. [PMID: 24319467 PMCID: PMC3844203 DOI: 10.1155/2013/637649] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2013] [Accepted: 09/07/2013] [Indexed: 11/27/2022]

Jin C, Roen DR, Lehmann PV, Kellermann GH. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi. Cells 2013;2:607-20. [PMID: 24709800 PMCID: PMC3972671 DOI: 10.3390/cells2030607] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2013] [Revised: 08/30/2013] [Accepted: 09/04/2013] [Indexed: 11/17/2022] Open

Bestard O, Lucia M, Crespo E, Van Liempt B, Palacio D, Melilli E, Torras J, Llaudó I, Cerezo G, Taco O, Gil-Vernet S, Grinyó JM, Cruzado JM. Pretransplant immediately early-1-specific T cell responses provide protection for CMV infection after kidney transplantation. Am J Transplant 2013;13:1793-805. [PMID: 23711167 DOI: 10.1111/ajt.12256] [Citation(s) in RCA: 99] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2012] [Revised: 03/11/2013] [Accepted: 03/12/2013] [Indexed: 01/25/2023]

Bestard O, Crespo E, Stein M, Lúcia M, Roelen DL, de Vaal YJ, Hernandez-Fuentes MP, Chatenoud L, Wood KJ, Claas FH, Cruzado JM, Grinyó JM, Volk HD, Reinke P. Cross-validation of IFN-γ Elispot assay for measuring alloreactive memory/effector T cell responses in renal transplant recipients. Am J Transplant 2013;13:1880-90. [PMID: 23763435 DOI: 10.1111/ajt.12285] [Citation(s) in RCA: 78] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2012] [Revised: 02/12/2013] [Accepted: 03/25/2013] [Indexed: 01/25/2023]

Saade F, Gorski SA, Petrovsky N. Pushing the frontiers of T-cell vaccines: accurate measurement of human T-cell responses. Expert Rev Vaccines 2013;11:1459-70. [PMID: 23252389 DOI: 10.1586/erv.12.125] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]

Costa C, Saldan A, Sinesi F, Sidoti F, Balloco C, Simeone S, Piceghello A, Mantovani S, Di Nauta A, Solidoro P, Cavallo R. The Lack and Cytomegalovirus-Specific Cellular Immune Response May Contribute to the Onset of Organ Infection and Disease in Lung Transplant Recipients. Int J Immunopathol Pharmacol 2012;25:1003-1009. [DOI: 10.1177/039463201202500417] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/30/2023] Open

Ramachandran H, Laux J, Moldovan I, Caspell R, Lehmann PV, Subbramanian RA. Optimal thawing of cryopreserved peripheral blood mononuclear cells for use in high-throughput human immune monitoring studies. Cells 2012;1:313-24. [PMID: 24710478 PMCID: PMC3901099 DOI: 10.3390/cells1030313] [Citation(s) in RCA: 76] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2012] [Revised: 06/29/2012] [Accepted: 07/03/2012] [Indexed: 11/20/2022] Open

Distinguishing Latent from Active Mycobacterium tuberculosis Infection Using Elispot Assays: Looking Beyond Interferon-gamma. Cells 2012;1:89-99. [PMID: 24710416 PMCID: PMC3901089 DOI: 10.3390/cells1020089] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2012] [Revised: 04/11/2012] [Accepted: 04/17/2012] [Indexed: 12/15/2022] Open

Moralès O, Richard A, Martin N, Mrizak D, Sénéchal M, Miroux C, Pancré V, Rommelaere J, Caillet-Fauquet P, de Launoit Y, Delhem N. Activation of a helper and not regulatory human CD4+ T cell response by oncolytic H-1 parvovirus. PLoS One 2012;7:e32197. [PMID: 22359669 PMCID: PMC3281136 DOI: 10.1371/journal.pone.0032197] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2011] [Accepted: 01/24/2012] [Indexed: 01/01/2023] Open

Abstract

BACKGROUND

H-1 parvovirus (H-1 PV), a rodent autonomous oncolytic parvovirus, has emerged as a novel class of promising anticancer agents, because of its ability to selectively find and destroy malignant cells. However, to probe H-1 PV multimodal antitumor potential one of the major prerequisites is to decipher H-1 PV direct interplay with human immune system, and so prevent any risk of impairment.

METHODOLOGY/PRINCIPAL FINDINGS

Non activated peripheral blood mononuclear cells (PBMCs) are not sensitive to H-1 PV cytotoxic effect. However, the virus impairs both activated PBMC proliferation ability and viability. This effect is related to H-1 PV infection as evidenced by Western blotting detection of H-1 PV main protein NS1. However, TCID50 experiments did not allow newly generated virions to be detected. Moreover, flow cytometry has shown that H-1 PV preferentially targets B lymphocytes. Despite seeming harmful at first sight, H-1 PV seems to affect very few NK cells and CD8+ T lymphocytes and, above all, clearly does not affect human neutrophils and one of the major CD4+ T lymphocyte subpopulation. Very interestingly, flow cytometry analysis and ELISA assays proved that it even activates human CD4+ T cells by increasing activation marker expression (CD69 and CD30) and both effective Th1 and Th2 cytokine secretion (IL-2, IFN-γ and IL-4). In addition, H-1 PV action does not come with any sign of immunosuppressive side effect. Finally, we have shown the efficiency of H-1 PV on xenotransplanted human nasopharyngeal carcinoma, in a SCID mouse model reconstituted with human PBMC.

CONCLUSIONS/SIGNIFICANCE

Our results show for the first time that a wild-type oncolytic virus impairs some immune cell subpopulations while directly activating a Helper CD4+ T cell response. Thus, our data open numerous gripping perspectives of investigation and strongly argue for the use of H-1 PV as an anticancer treatment.

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Lehmann PV, Zhang W. Unique strengths of ELISPOT for T cell diagnostics. Methods Mol Biol 2012;792:3-23. [PMID: 21956497 DOI: 10.1007/978-1-61779-325-7_1] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]

Abstract

The T cell system plays an essential role in infections, allergic reactions, tumor and transplant rejection, as well as autoimmune diseases. It does so by the selective engagement of different antigen-specific effector cell lineages that differentially secrete cytokines and other effector molecules. These T cell subsets may or may not have cytolytic activity, can preferentially migrate to different tissues, and display variable capabilities to expand clonally. The quest of T cell immune diagnostics is to understand which specific effector function and T cell lineage is associated with a given clinical outcome, be it positive or adverse. No single assay can measure all of the relevant parameters. In this chapter, we review the unique contributions that ELISPOT assays can make toward understanding T cell-mediated immunity. ELISPOT assays have an unsurpassed sensitivity in detecting low frequency antigen-specific T cells that secrete effector molecules, including granzyme and perforin. They provide robust, highly reproducible data - even by first time users. Because ELISPOT assays require roughly tenfold less cell material than flow cytometry, ELISPOT is ideally suited for all measurements requiring parallel testing under multiple conditions. These include defining (a) T cell reactivity to individual peptides of extensive libraries, thereby establishing the fine-specificity of the response, and determinant mapping; (b) reactivity to different concentrations of the antigen in serial dilutions to measure the avidity of the T cell response; or (c) different secretory products released by T cells which define their respective effector lineage/functions. Further, because T cells survive ELISPOT assays unaffected, they can be retested for the acquisition of additional information in follow-up assays. These strengths of ELISPOT assays the weaknesses of flow cytometry-based measurements. Thus, the two assays systems compliment each other in the quest to understand T cell-mediated immunity in vivo.

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Zhang W, Lehmann PV. Objective, user-independent ELISPOT data analysis based on scientifically validated principles. Methods Mol Biol 2012;792:155-71. [PMID: 21956509 DOI: 10.1007/978-1-61779-325-7_13] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]

Biomarkers. Ther Drug Monit 2012. [DOI: 10.1016/b978-0-12-385467-4.00016-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]

Dittrich M, Lehmann PV. Statistical analysis of ELISPOT assays. Methods Mol Biol 2012;792:173-83. [PMID: 21956510 DOI: 10.1007/978-1-61779-325-7_14] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]

Slota M, Lim JB, Dang Y, Disis ML. ELISpot for measuring human immune responses to vaccines. Expert Rev Vaccines 2011;10:299-306. [PMID: 21434798 DOI: 10.1586/erv.10.169] [Citation(s) in RCA: 116] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]

Butterfield LH, Palucka AK, Britten CM, Dhodapkar MV, Håkansson L, Janetzki S, Kawakami Y, Kleen TO, Lee PP, Maccalli C, Maecker HT, Maino VC, Maio M, Malyguine A, Masucci G, Pawelec G, Potter DM, Rivoltini L, Salazar LG, Schendel DJ, Slingluff CL, Song W, Stroncek DF, Tahara H, Thurin M, Trinchieri G, van Der Burg SH, Whiteside TL, Wigginton JM, Marincola F, Khleif S, Fox BA, Disis ML. Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers. Clin Cancer Res 2011;17:3064-76. [PMID: 21558394 DOI: 10.1158/1078-0432.ccr-10-2234] [Citation(s) in RCA: 85] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]

Abstract

PURPOSE

To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements.

EXPERIMENTAL DESIGN

The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here, we review the major challenges, give examples of approaches and solutions, and present our recommendations.

RESULTS AND CONCLUSIONS

Although specific immune parameters and assays are not yet validated, we recommend following standardized (accurate, precise, and reproducible) protocols and use of functional assays for the primary immunologic readouts of a trial; consideration of central laboratories for immune monitoring of large, multi-institutional trials; and standardized testing of several phenotypic and functional potential potency assays specific to any cellular product. When reporting results, the full QA (quality assessment)/QC (quality control) should be conducted and selected examples of truly representative raw data and assay performance characteristics should be included. Finally, to promote broader analysis of multiple aspects of immunity, and gather data on variability, we recommend that in addition to cells and serum, RNA and DNA samples be banked (under standardized conditions) for later testing. We also recommend that sufficient blood be drawn to allow for planned testing of the primary hypothesis being addressed in the trial, and that additional baseline and posttreatment blood is banked for testing novel hypotheses (or generating new hypotheses) that arise in the field.

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Costa C, Astegiano S, Terlizzi M, Sidoti F, Curtoni A, Solidoro P, Baldi S, Bergallo M, Cavallo R. Evaluation and Significance of Cytomegalovirus-Specific Cellular Immune Response in Lung Transplant Recipients. Transplant Proc 2011;43:1159-61. [DOI: 10.1016/j.transproceed.2011.03.024] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]

Auletta JJ, Zale EA, Welter JF, Solchaga LA. Fibroblast Growth Factor-2 Enhances Expansion of Human Bone Marrow-Derived Mesenchymal Stromal Cells without Diminishing Their Immunosuppressive Potential. Stem Cells Int 2011;2011:235176. [PMID: 21437189 PMCID: PMC3062108 DOI: 10.4061/2011/235176] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2010] [Accepted: 01/13/2011] [Indexed: 12/11/2022] Open

Mander A, Gouttefangeas C, Ottensmeier C, Welters MJP, Low L, van der Burg SH, Britten CM. Serum is not required for ex vivo IFN-gamma ELISPOT: a collaborative study of different protocols from the European CIMT Immunoguiding Program. Cancer Immunol Immunother 2010;59:619-27. [PMID: 20052465 PMCID: PMC2813523 DOI: 10.1007/s00262-009-0814-4] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2009] [Accepted: 12/21/2009] [Indexed: 02/04/2023]