The Ex Vivo IFN-γ Enzyme-Linked Immunospot (ELISpot) Assay

2015 ◽  
pp. 197-205 ◽  
Author(s):  
Martha Sedegah
Keyword(s):  
Elispot Assay ◽  
Ex Vivo ◽  
Ifn Γ

scholarly journals Evaluation of the Modified ELISPOT Assay for Gamma Interferon Production in Cancer Patients Receiving Antitumor Vaccines

2000 ◽  
Vol 7 (2) ◽  
pp. 145-154 ◽  
Author(s):  
Tadao Asai ◽  
Walter J. Storkus ◽  
Theresa L. Whiteside
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocyte ◽  
Elispot Assay ◽  
Ex Vivo ◽  
Peptide Vaccine ◽  
Precursor Cells ◽  
Gamma Interferon ◽  
Patients With Cancer ◽  
Ifn Γ

ABSTRACT Frequencies of vaccine-responsive T-lymphocyte precursors in peripheral blood mononuclear cells (PBMC) prior to and after administration of peptide-based vaccines in patients with cancer can be measured by limiting-dilution assays (LDA) or by ELISPOT assays. We have used a modified version of the ELISPOT assay to monitor changes in the frequency of gamma interferon (IFN-γ)-producing T cells in a population of lymphocytes responding to a relevant peptide or a nonspecific stimulator, such as phorbol myristate acetate-ionomycin. Prior to its use for monitoring of patient samples, the assay was validated and found to be comparable to the LDA performed in parallel, using tumor-reactive cytolytic T-lymphocyte (CTL) lines. The sensitivity of the ELISPOT assay was found to be 1/100,000 cells, with an interassay coefficient of variation of 15%, indicating that it could be reliably used for monitoring of changes in the frequency of IFN-γ-secreting responder cells in noncultured or cultured lymphocyte populations. To establish that the assay is able to detect the T-cell precursor cells responsive to the vaccine, we used CD8+T-cell populations positively selected from PBMC of HLA-A2+patients with metastatic melanoma, who were treated with dendritic cell-based vaccines containing gp100, MELAN-A/MART-1, tyrosinase, and influenza virus matrix peptides. The frequency of peptide-specific responder T cells ranged from 0 to 1/2,600 before vaccination and increased by at least 1 log unit after vaccination in two patients, one of whom had a clinical response to the vaccine. However, no increases in the frequency of peptide-responsive T cells were observed in noncultured PBMC or PBMC cultured in the presence of the relevant peptides after the melanoma patients enrolled in another trial were treated with the intramuscular peptide vaccine plus MF59 adjuvant. Thus, while the ELISPOT assay was found to be readily applicable to assessments of frequencies of CTL precursors of established CTL lines and ex vivo-amplified PBMC, its usefulness for monitoring of fresh PBMC in patients with cancer was limited. In many of these patients antitumor effector T cells are present at frequencies of lower than 1/100,000 in the peripheral circulation. Serial monitoring of such patients may require prior ex vivo amplification of specific precursor cells.

scholarly journals Early Gamma Interferon and Interleukin-2 Responses to Vaccination Predict the Late Resting Memory in Malaria-Naïve and Malaria-Exposed Individuals

2006 ◽  
Vol 74 (11) ◽  
pp. 6331-6338 ◽  
Author(s):  
Philip Bejon ◽  
Sheila Keating ◽  
Jedidah Mwacharo ◽  
Oscar K. Kai ◽  
Susanna Dunachie ◽  
...  
Keyword(s):  
Elispot Assay ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Gamma Interferon ◽  
Long Term Memory ◽  
Effector Cells ◽  
Ifn Γ ◽  
Relationship Of ◽  
The Relationship

ABSTRACT Two different cell populations respond to potent T-cell-inducing vaccinations. The induction and loss of effector cells can be seen using an ex vivo enzyme-linked immunospot (ELISPOT) assay, but the more durable resting memory response is demonstrable by a cultured ELISPOT assay. The relationship of the early effector response to durable resting memory is incompletely understood. Effector phenotype is usually identified by gamma interferon (IFN-γ) production, but interleukin-2 (IL-2) has been specifically linked to the differentiation of memory cells. Here, IFN-γ- and IL-2-secreting effector cells were identified by an ex vivo ELISPOT assay 1 week after vaccination and compared with the resting memory responses detected by a cultured ELISPOT assay 3 months later. The different kinetics and induction of IL-2 by different vaccines and natural exposure are described. Furthermore, both early IFN-γ and IL-2 production independently predicted subsequent memory responses at 3 months in malaria-naïve volunteers, but only IFN-γ predicted memory in malaria-exposed volunteers. However, dual ELISPOT assays were also performed on malaria-exposed volunteers to identify cells producing both cytokines simultaneously. This demonstrated that double-cytokine-producing cells were highly predictive of memory. This assay may be useful in predicting vaccinations most likely to generate stable, long-term memory responses.

scholarly journals Frequencies of Ex Vivo-Activated Human Immunodeficiency Virus Type 1-Specific Gamma-Interferon-Producing CD8+ T Cells in Infected Children Correlate Positively with Plasma Viral Load

2002 ◽  
Vol 76 (24) ◽  
pp. 12414-12422 ◽  
Author(s):  
Florence Buseyne ◽  
Daniel Scott-Algara ◽  
Françoise Porrot ◽  
Béatrice Corre ◽  
Nassima Bellal ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Viral Load ◽  
Elispot Assay ◽  
Ex Vivo ◽  
Cell Subset ◽  
Gamma Interferon ◽  
Functional Assay ◽  
Immunodeficiency Virus ◽  
Ifn Γ

ABSTRACT HIV-specific CD8+ T cells are critical in controlling human immunodeficiency virus (HIV) replication. We present the evaluation of a gamma-interferon (IFN-γ)-based enzyme linked immunospot (ELISPOT) assay for the quantification of HIV-specific CD8+ T cells from HIV-infected children. We studied 20 HLA-A∗0201-positive HIV-infected children. The IFN-γ production in response to stimulation with two HLA-A∗0201-restricted immunodominant CD8 epitopes (SLYNTVATL [SL9] in Gag and ILKEPVHGV [IV9] in Pol) was tested using the ELISPOT assay. The results were compared to labeling with the corresponding tetramers. Among the 20 children, 18 had detectable responses against the SL9 and/or the IV9 epitope using the ELISPOT assay (medians, 351 and 134 spot-forming cells/106 peripheral blood mononuclear cells, respectively). Comparison of results from the tetramer and ELISPOT assays suggests that only a fraction of HIV-specific CD8+ T cells were able to produce IFN-γ. Most importantly, we found that the frequencies of IFN-γ-producing CD8+ T cells were positively correlated with the viral load whereas the frequencies of tetramer-binding CD8+ T cells were not. The high sensitivity of the ELISPOT assay and the fact that this functional assay provided information different from that of tetramer labeling support its use for measurement of HIV-specific CD8+ T cells. In conclusion, our results show that the ex vivo-activated IFN-γ-producing HIV-specific CD8+-T-cell subset is dependent upon continuous antigenic stimulation.

scholarly journals Dose Dependent Antimicrobial Cellular Cytotoxicity—Implications for ex vivo Diagnostics

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana Copaescu ◽  
Phuti Choshi ◽  
Sarah Pedretti ◽  
Effie Mouhtouris ◽  
Jonathan Peter ◽  
...  
Keyword(s):  
Cell Viability ◽  
Elispot Assay ◽  
Ex Vivo ◽  
Cellular Cytotoxicity ◽  
Cell Cytotoxicity ◽  
Viability Staining ◽  
Drug Concentrations ◽  
Ldh Assay ◽  
Ifn Γ ◽  
Dose Dependent

Introduction:Ex vivo and in vitro diagnostics, such as interferon-γ (IFN-γ) release enzyme linked ImmunoSpot (ELISpot) and flow cytometry, are increasingly employed in the research and diagnostic setting for severe T-cell mediated hypersensitivity. Despite an increasing use of IFN-γ release ELISpot for drug causality assessment and utilization of a range of antimicrobial concentrations ex vivo, data regarding antimicrobial-associated cellular cytotoxicity and implications for assay performance remain scarcely described in the literature. Using the measurement of lactate dehydrogenase (LDH) and the 7-AAD cell viability staining, we aimed via an exploratory study, to determine the maximal antimicrobial concentrations required to preserve cell viability for commonly implicated antimicrobials in severe T-cell mediated hypersensitivity.Method: After an 18-h incubation of patient peripheral blood monocytes (PBMCs) and antimicrobials at varying drug concentrations, the cell cytotoxicity was measured in two ways. A colorimetric based assay that detects LDH activity and by flow cytometry using the 7-AAD cell viability staining. We used the PBMCs collected from three healthy control participants with no known history of adverse drug reaction and two patients with a rifampicin-associated drug reaction with eosinophilia and systemic symptoms (DRESS), confirmed on IFN-γ ELISpot assay. The PBMCs were stimulated for the investigated drugs at the previously published drug maximum concentration (Cmax), and concentrations 10- and 100-fold above.Results: In a human immunodeficiency virus (HIV) negative and a positive rifampicin-associated DRESS with positive ex vivo IFN-γ ELISpot assay, use of 10- and 100-fold Cmax drug concentrations decreased spot forming units/million cells by 32–100%, and this corresponded to cell cytotoxicity of more than 40 and 20% using an LDH assay and 7-AAD cell viability staining, respectively. The other antimicrobials (ceftriaxone, flucloxacillin, piperacillin/tazobactam, and isoniazid) tested in healthy controls showed similar dose-dependent increased cytotoxicity using the LDH assay, but cytotoxicity remained lower than 40% for all Cmax and 10-fold Cmax drug concentrations except flucloxacillin. All 100-fold Cmax concentrations resulted in cell death >40% (median 57%), except for isoniazid. 7-AAD cell viability staining also confirmed an increase in lymphocyte death in PBMCs incubated with 10-fold and 100-fold above Cmax for ceftriaxone, and flucloxacillin; however, piperacillin/tazobactam and isoniazid indicated no differences in percentages of viable lymphocytes across concentrations tested.Conclusion: The LDH cytotoxicity and 7-AAD cell viability staining techniques both demonstrate increased cell death corresponding to a loss in ELISpot sensitivity, with use of higher antimicrobial drug concentrations for ex vivo diagnostic IFN-γ ELISpot assays. For all the antimicrobials evaluated, the use of Cmax and 10-fold Cmax concentrations impacts cell viability and potentially affects ELISpot performance. These findings inform future approaches for ex vivo diagnostics such as IFN-γ release ELISpot.

scholarly journals Comparison of Cytomegalovirus-Specific Immune Cell Response to Proteins versus Peptides Using an IFN-γ ELISpot Assay after Hematopoietic Stem Cell Transplantation

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 312
Author(s):  
Eva Wagner-Drouet ◽  
Daniel Teschner ◽  
Christine Wolschke ◽  
Kerstin Schäfer-Eckart ◽  
Johannes Gärtner ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Response ◽  
Elispot Assay ◽  
Cmv Infection ◽  
Hematopoietic Stem ◽  
Ifn Γ ◽  
Antigen Presenting

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8+ counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients.

scholarly journals Checkpoint blockade accelerates a novel switch from an NKT-driven TNFα response toward a T cell driven IFN-γ response within the tumor microenvironment

2021 ◽  
Vol 9 (6) ◽  
pp. e002269
Author(s):  
Shota Aoyama ◽  
Ryosuke Nakagawa ◽  
Satoshi Nemoto ◽  
Patricio Perez-Villarroel ◽  
James J Mulé ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Ex Vivo ◽  
T Cell Response ◽  
Effector Function ◽  
Checkpoint Blockade ◽  
Necrosis Factor Alpha ◽  
Ifn Γ

BackgroundThe temporal response to checkpoint blockade (CB) is incompletely understood. Here, we profiled the tumor infiltrating lymphocyte (TIL) landscape in response to combination checkpoint blockade at two distinct timepoints of solid tumor growth.MethodsC57BL/6 mice bearing subcutaneous MC38 tumors were treated with anti-PD-1 and/or anti-CTLA-4 antibodies. At 11 or 21 days, TIL phenotype and effector function were analyzed in excised tumor digests using high parameter flow cytometry. The contributions of major TIL populations toward overall response were then assessed using ex vivo cytotoxicity and in vivo tumor growth assays.ResultsThe distribution and effector function among 37 distinct TIL populations shifted dramatically between early and late MC38 growth. At 11 days, the immune response was dominated by Tumor necrosis factor alpha (TNFα)-producing NKT, representing over half of all TIL. These were accompanied by modest frequencies of natural killer (NK), CD4+, or CD8+ T cells, producing low levels of IFN-γ. At 21 days, NKT populations were reduced to a combined 20% of TIL, giving way to increased NK, CD4+, and CD8+ T cells, with increased IFN-γ production. Treatment with CB accelerated this switch. At day 11, CB reduced NKT to less than 20% of all TIL, downregulated TNFα across NKT and CD4+ T cell populations, increased CD4+ and CD8+ TIL frequencies, and significantly upregulated IFN-γ production. Degranulation was largely associated with NK and NKT TIL. Blockade of H-2kb and/or CD1d during ex vivo cytotoxicity assays revealed NKT has limited direct cytotoxicity against parent MC38. However, forced CD1d overexpression in MC38 cells significantly diminished tumor growth, suggesting NKT TIL exerts indirect control over MC38 growth.ConclusionsDespite an indirect benefit of early NKT activity, CB accelerates a switch from TNFα, NKT-driven immune response toward an IFN-γ driven CD4+/CD8+ T cell response in MC38 tumors. These results uncover a novel NKT/T cell switch that may be a key feature of CB response in CD1d+ tumors.

scholarly journals BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation

2021 ◽  
Vol 12 ◽  
Author(s):  
Molly Javier Uyeda ◽  
Robert A. Freeborn ◽  
Brandon Cieniewicz ◽  
Rosa Romano ◽  
Ping (Pauline) Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
Ex Vivo ◽  
Surface Expression ◽  
Surface Molecules ◽  
Ifn Γ ◽  
And Function ◽  
Tr1 Cells

Type 1 regulatory T (Tr1) cells are subset of peripherally induced antigen-specific regulatory T cells. IL-10 signaling has been shown to be indispensable for polarization and function of Tr1 cells. However, the transcriptional machinery underlying human Tr1 cell differentiation and function is not yet elucidated. To this end, we performed RNA sequencing on ex vivo human CD49b+LAG3+ Tr1 cells. We identified the transcription factor, BHLHE40, to be highly expressed in Tr1 cells. Even though Tr1 cells characteristically produce high levels of IL-10, we found that BHLHE40 represses IL-10 and increases IFN-γ secretion in naïve CD4+ T cells. Through CRISPR/Cas9-mediated knockout, we determined that IL10 significantly increased in the sgBHLHE40-edited cells and BHLHE40 is dispensable for naïve CD4+ T cells to differentiate into Tr1 cells in vitro. Interestingly, BHLHE40 overexpression induces the surface expression of CD49b and LAG3, co-expressed surface molecules attributed to Tr1 cells, but promotes IFN-γ production. Our findings uncover a novel mechanism whereby BHLHE40 acts as a regulator of IL-10 and IFN-γ in human CD4+ T cells.

scholarly journals An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible toMycobacterium tuberculosis

1998 ◽  
Vol 66 (10) ◽  
pp. 4981-4988 ◽  
Author(s):  
Irina Lyadova ◽  
Vladimir Yeremeev ◽  
Konstantin Majorov ◽  
Boris Nikonenko ◽  
Sergei Khaidukov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Antimycobacterial Activity ◽  
Enzymatic Digestion ◽  
Interleukin 4 ◽  
T Cell Clones ◽  
Cell Clones ◽  
Ifn Γ

ABSTRACT I/St mice, previously characterized as susceptible toMycobacterium tuberculosis H37Rv, were given 103 or 105 CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44− CD45RB+cells disappeared within 2 weeks postinfection, while the number of CD44+ CD45RB−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3+CD4+ CD8− T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ+ IL-4−) clone and one Th0-like (IFN-γ+ IL-4+IL-10+) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

scholarly journals Assessment of Aspergillus-Specific T Cells for Diagnosis of Invasive Aspergillosis in a Leukemic Child with Liver Lesions Mimicking Hepatosplenic Candidiasis

2008 ◽  
Vol 15 (10) ◽  
pp. 1625-1628 ◽  
Author(s):  
Leonardo Potenza ◽  
Patrizia Barozzi ◽  
Giulio Rossi ◽  
Giovanni Palazzi ◽  
Daniela Vallerini ◽  
...  
Keyword(s):  
T Cells ◽  
Invasive Aspergillosis ◽  
Elispot Assay ◽  
Interleukin 10 ◽  
Diagnostic Tools ◽  
Liver Lesions ◽  
Negative Results ◽  
Hepatosplenic Candidiasis ◽  
Ifn Γ ◽  
Leukemic Child

ABSTRACT A child with acute myeloid leukemia presented with multiple liver lesions mimicking hepatosplenic candidiasis during the neutropenic phase following the induction chemotherapy. All the available diagnostic tools showed repeatedly negative results, including galactomannan. An enzyme-linked immunospot (ELISPOT) assay showed a high number of Aspergillus-specific T cells producing interleukin-10 [TH2(IL-10)] and a low number of Aspergillus-specific T cells producing gamma interferon [TH1(IFN-γ)], revealing invasive aspergillosis (IA) before the confirmatory biopsy. A progressive skewing from the predominance of TH2(IL-10) to a predominance of TH1(IFN-γ) was observed close to the complete resolution of the infection and foreshadowed the outcome. The ELISPOT assay holds promise for diagnosing pediatric IA.

scholarly journals Persistent High Frequencies of Varicella-Zoster Virus ORF4 Protein-Specific CD4+ T Cells after Primary Infection

2006 ◽  
Vol 80 (19) ◽  
pp. 9772-9778 ◽  
Author(s):  
Louise Jones ◽  
Antony P. Black ◽  
Gathsaurie N. Malavige ◽  
Graham S. Ogg
Keyword(s):  
T Cells ◽  
Varicella Zoster Virus ◽  
Peripheral Blood ◽  
Primary Infection ◽  
Ex Vivo ◽  
High Frequencies ◽  
Varicella Zoster ◽  
Early Protein ◽  
Orf4 Protein ◽  
Ifn Γ

ABSTRACT Open reading frame 4 (ORF4) of varicella-zoster virus (VZV) encodes an immediate-early protein that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication, but there are no data addressing the existence of potential ORF4 protein-specific CD4+ T cells. We tested the hypothesis that VZV ORF4 protein-specific CD4+ T cells could be identified and characterized within the peripheral blood of healthy immune donors following primary infection. Gamma interferon (IFN-γ) immunosorbent assays were used to screen peripheral blood mononuclear cells obtained from healthy seropositive donors for responses to overlapping ORF4 peptides, viral lysate, and live vaccine. High frequencies of ORF4 protein-specific T cells were detected ex vivo in individuals up to 52 years after primary infection. Several immunogenic regions of the ORF4 protein were identified, including a commonly recognized epitope which was restricted through HLA-DRB1*07. Total ORF4 protein-specific responses comprised 19.7% and 20.7% of the total lysate and vaccine responses, respectively, and were dominated by CD4+ T cells. Indeed, CD4+ T cells were found to dominate the overall virus-specific IFN-γ cellular immune response both ex vivo and after expansion in vitro. In summary, we have identified an ORF4 protein as a novel target antigen for persistent VZV-specific CD4+ T cells, with implications for disease pathogenesis and future vaccine development.

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