Application of the IFN-γ ELISPOT assay to quantify T cell responses against proteins

2001 ◽  
Vol 247 (1-2) ◽  
pp. 17-24 ◽  
Author(s):  
Alexander Schmittel ◽  
Ulrich Keilholz ◽  
Sandra Bauer ◽  
Ulrike Kuhne ◽  
Stefan Stevanovic ◽  
...  
Keyword(s):  
T Cell ◽  
Elispot Assay ◽  
T Cell Responses ◽  
Cell Responses ◽  
Ifn Γ

scholarly journals Naturally Exposed Populations Differ in Their T1 and T2 Responses to the Circumsporozoite Protein of Plasmodium falciparum

2002 ◽  
Vol 70 (3) ◽  
pp. 1468-1474 ◽  
Author(s):  
W. H. H. Reece ◽  
M. Plebanski ◽  
P. Akinwunmi ◽  
P. Gothard ◽  
K. L. Flanagan ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
T Cell ◽  
Affect Regulation ◽  
Elispot Assay ◽  
T Cell Responses ◽  
Circumsporozoite Protein ◽  
Interleukin 4 ◽  
Cell Responses ◽  
Ifn Γ ◽  
T1 And T2

ABSTRACT T-cell responses directed against the circumsporozoite protein (CS) of Plasmodium falciparum can mediate protection against malaria. We determined the frequency of T cells reactive to different regions of the CS in the blood of donors naturally exposed to P. falciparum by examining T1 (gamma interferon [IFN-γ] ELISPOT assay), T2 (interleukin 4 [IL-4] ELISPOT assay), and proliferative T-cell responses. The proliferative responses were weak, which confirmed previous observations. The responses to the CS in the IL-4 and IFN-γ ELISPOT assays were also weak (<40 responding cells per 106 cells), much weaker than the response to the purified protein derivative of Mycobacterium tuberculosis in the same donors. Moreover, a response in one assay could not be used to predict a response in either of the other assays, suggesting that although these assays may measure different responding cells, all of the responses are weakly induced by natural exposure. Interestingly, the two different study populations used had significantly different T1 and T2 biases in their responses in the C terminus of the protein, suggesting that the extent of P. falciparum exposure can affect regulation of the immune system.

scholarly journals Enzyme-Linked Immunospot Assay for Detection of Human Respiratory Syncytial Virus F Protein-Specific Gamma Interferon-Producing T Cells

2014 ◽  
Vol 21 (5) ◽  
pp. 628-635 ◽  
Author(s):  
Kathryn Patton ◽  
Shahin Aslam ◽  
Jim Lin ◽  
Li Yu ◽  
Stacie Lambert ◽  
...  
Keyword(s):  
T Cells ◽  
Respiratory Syncytial Virus ◽  
T Cell ◽  
Elispot Assay ◽  
T Cell Responses ◽  
The Elderly ◽  
Assay Sensitivity ◽  
Cell Responses ◽  
Ifn Γ ◽  
Syncytial Virus

ABSTRACTRespiratory syncytial virus (RSV) causes significant disease in elderly adults, and we have previously reported that individuals 65 years of age and older have reduced RSV F protein-specific gamma interferon (IFN-γ)-producing T cells compared to healthy younger adults. To measure RSV F-specific memory T cell responses in the elderly following infection or vaccination, we optimized and qualified an IFN-γ enzyme-linked immunospot (ELISPOT) assay. Since peripheral blood mononuclear cells (PBMC) from the elderly could be more fragile, we established optimal cryopreservation techniques and minimal viability acceptance criteria. The number of cells per well, types and concentrations of stimulation antigens, and incubation times were evaluated to maximize assay sensitivity and precision. The optimized assay uses 300,000 cells/well, 2 μg/ml of an RSV F peptide pool (RSV Fpp), and incubation for 22 ± 2 h in serum-free CTL-Test medium. The assay was qualified by 3 analysts using 3 RSV F-responding donor PBMC samples (high, medium, and low responders) tested on 5 different assay days. The assay sensitivity or limit of detection (LOD) was determined to be 21 spot-forming cells (SFC) per 106PBMC, and the lower limit of quantitation (LLOQ) was estimated to be 63 SFC/106PBMC. The intra- and interassay percent coefficients of variation (CV) were <10.5% and <31%, respectively. The results of the qualification study demonstrate that a robust, precise, and sensitive IFN-γ ELISPOT assay has been developed that is fit for measuring RSV F-specific IFN-γ T cell responses in subjects enrolled in a vaccine clinical trial or in epidemiology studies.

scholarly journals Human Immunodeficiency Virus-Specific Gamma Interferon Enzyme-Linked Immunospot Assay Responses Targeting Specific Regions of the Proteome during Primary Subtype C Infection Are Poor Predictors of the Course of Viremia and Set Point

2008 ◽  
Vol 83 (1) ◽  
pp. 470-478 ◽  
Author(s):  
Clive M. Gray ◽  
Mandla Mlotshwa ◽  
Catherine Riou ◽  
Tiyani Mathebula ◽  
Debra de Assis Rosa ◽  
...  
Keyword(s):  
T Cell ◽  
Elispot Assay ◽  
T Cell Responses ◽  
Subtype C ◽  
Course Of Disease ◽  
Set Point ◽  
Cell Responses ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv 1

ABSTRACT It is unknown whether patterns of human immunodeficiency virus (HIV)-specific T-cell responses during acute infection may influence the viral set point and the course of disease. We wished to establish whether the magnitude and breadth of HIV type 1 (HIV-1)-specific T-cell responses at 3 months postinfection were correlated with the viral-load set point at 12 months and hypothesized that the magnitude and breadth of HIV-specific T-cell responses during primary infection would predict the set point. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses across the complete proteome were measured in 47 subtype C HIV-1-infected participants at a median of 12 weeks postinfection. When corrected for amino acid length and individuals responding to each region, the order of recognition was as follows: Nef > Gag > Pol > Rev > Vpr > Env > Vpu > Vif > Tat. Nef responses were significantly (P < 0.05) dominant, targeted six epitopic regions, and were unrelated to the course of viremia. There was no significant difference in the magnitude and breadth of responses for each protein region with disease progression, although there was a trend of increased breadth (mean, four to seven pools) in rapid progressors. Correlation of the magnitude and breadth of IFN-γ responses with the viral set point at 12 months revealed almost zero association for each protein region. Taken together, these data demonstrate that the magnitude and breadth of IFN-γ ELISPOT assay responses at 3 months postinfection are unrelated to the course of disease in the first year of infection and are not associated with, and have low predictive power for, the viral set point at 12 months.

Protective T-Cell Responses to Several Recombinant Aspergillus Antigens May Be Detected Since the Onset of the Infection in Patients with Invasive Aspergillosis, and May Be Exploited for Therapeutic Purposes,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3229-3229
Author(s):  
Leonardo Potenza ◽  
Daniela Vallerini ◽  
Patrizia Barozzi ◽  
Giovanni Riva ◽  
Forghieri Fabio ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Invasive Aspergillosis ◽  
Elispot Assay ◽  
T Cell Responses ◽  
Lytic Activity ◽  
Recombinant Antigens ◽  
Cell Responses ◽  
Ifn Γ

Abstract Abstract 3229 Introduction: Several studies have reported that different components of fungi of the genera Aspergillus spp may induce protective T-cell responses in either mouse models of invasive aspergillosis (IA) or in human healthy subjects. We evaluated the occurrence of Aspergillus-specific T-cell responses to different Aspergillus recombinant antigens in patients with proven IA, during the course of the IA, to identify the antigens most frequently targeted by protective immune responses. We characterized phenotypically and functionally such specific T cells. Finally, from peripheral blood (PB) samples of the same IA proven patients, also collected during the active infection phase, we sought to expand such Aspergillus-specific T cells. Methods: 15 patients with proven IA, according to the EORTC/MSG criteria, have been enrolled into the study. Specific immune responses producing interleukin-10 (IL-10), interferon-gamma (IFN-γ), IL-4 and IL-17A were detected and characterized by enzyme linked immunospot (ELISpot) assay and cytokine secretion assay (CSA), in all the patients, during the entire course of the IA. The recombinant antigens used were GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, and galactomannan (GM). Cytotoxicity has been investigated by means of the colorimetric assay with (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxyanilide) sodium salt plus coenzyme Q0 (XTT assay). Aspergillus-specific T cells were obtained by culturing PB mononuclear cells with a mixture containing PEP1p, GEL1p, α1–3 glucan and β1–3 glucan. The infection course were divided into 4 phases, defined from t1 to t4, each of fifteen days interval, starting from the radiological diagnosis of IA. Results: Aspergillus-specific T cells producing either IL-10 or IFN-γ were detected to all the antigens, but GM. The number of antigens targeted by IFN-γ producing specific T cells progressively increased along the course of IA, being such protective responses to 3 out of 7 antigens at t1 and to 6 out of 7 antigens at t4. At t1, IFN-γ producing specific T cells were only detected to GEL1p, α1–3 glucan and β1–3 glucan. GEL1p and α1–3 glucan resulted the antigens most constantly targeted by IFN-γ producing specific T cells, persisting the responses to these antigens in all the phases of IA. No Aspergillus-specific T cells producing IL-4 to any antigens were detected by the ELISpot assay. Aspergillus-specific T cells producing IL-17A were detected in only one out of 15 patients, and targeted CRF1p. Specific T cells to GM and specific T cells producing IL-4 to all the antigens could be shown only by CSA, suggesting that they are present only at very low frequencies during the infection. After 13-day cultures, Aspergillus-specific T cells were expanded from five out of five patients. The specific T cells tested for lytic activity included a median of 95.8% CD3+ cells, either CD4+ or CD8+ T cells, and showed a median lytic activity of 9.45% either at 3/1 or at 5/1 effector/target cells ratios. Conclusions: In patients with IA, protective immune responses may be detected since the onset and increase during the infection. At the onset of IA, specific T cells producing IFN-γ target antigens involved in the cell wall biosynthesis of Aspergillus. On the other hand, at the same phase, specific protective immune responses to CRF1p, PEP1p, and SOD1p, which are all putative virulence factors for Aspergillus, are absent. Aspergillus-specific T cells may be expanded by a mixture of antigens, even in the course of IA and are able to directly kill fungal hyphae. The above mentioned antigens and the corresponding protective T cells may be exploited for therapeutic strategies of either vaccine or autologous cytotoxic cell infusions in patients at high risk for IA. Disclosures: Luppi: MSD; GILEAD: Research Funding.

scholarly journals Batf3-Dependent Dendritic Cells Promote Optimal CD8 T Cell Responses against Respiratory Poxvirus Infection

2018 ◽  
Vol 92 (16) ◽  
Author(s):  
Pritesh Desai ◽  
Vikas Tahiliani ◽  
Georges Abboud ◽  
Jessica Stanfield ◽  
Shahram Salek-Ardakani
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Vaccinia Virus ◽  
Respiratory Infection ◽  
Host Resistance ◽  
T Cell Responses ◽  
Cell Responses ◽  
Ifn Γ ◽  
The Impact

ABSTRACTRespiratory infection with vaccinia virus (VacV) elicits robust CD8+T cell responses that play an important role in host resistance. In the lung, VacV encounters multiple tissue-resident antigen-presenting cell (APC) populations, but which cell plays a dominant role in priming of virus-specific CD8+effector T cell responses remains poorly defined. We used Batf3−/−mice to investigate the impact of CD103+and CD8α+dendritic cell (DC) deficiency on anti-VacV CD8+T cell responses. We found that Batf3−/−mice were more susceptible to VacV infection, exhibiting profound weight loss, which correlated with impaired accumulation of gamma interferon (IFN-γ)-producing CD8+T cells in the lungs. This was largely due to defective priming since early in the response, antigen-specific CD8+T cells in the draining lymph nodes of Batf3−/−mice expressed significantly reduced levels of Ki67, CD25, and T-bet. These results underscore a specific role for Batf3-dependent DCs in regulating priming and expansion of effector CD8+T cells necessary for host resistance against acute respiratory VacV infection.IMPORTANCEDuring respiratory infection with vaccinia virus (VacV), a member ofPoxviridaefamily, CD8+T cells play important role in resolving the primary infection. Effector CD8+T cells clear the virus by accumulating in the infected lungs in large numbers and secreting molecules such as IFN-γ that kill virally infected cells. However, precise cell types that regulate the generation of effector CD8+T cells in the lungs are not well defined. Dendritic cells (DCs) are a heterogeneous population of immune cells that are recognized as key initiators and regulators of T-cell-mediated immunity. In this study, we reveal that a specific subset of DCs that are dependent on the transcription factor Batf3 for their development regulate the magnitude of CD8+T cell effector responses in the lungs, thereby providing protection during pulmonary VacV infection.

scholarly journals Peripheral T-Cell Reactivity to Heat Shock Protein 70 and Its Cofactor GrpE from Tropheryma whipplei Is Reduced in Patients with Classical Whipple's Disease

2017 ◽  
Vol 85 (8) ◽  
Author(s):  
Lucia Trotta ◽  
Kathleen Weigt ◽  
Katina Schinnerling ◽  
Anika Geelhaar-Karsch ◽  
Gerrit Oelkers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
T Cell Responses ◽  
Tropheryma Whipplei ◽  
Content Type ◽  
Cell Responses ◽  
Ifn Γ

ABSTRACT Classical Whipple's disease (CWD) is characterized by the lack of specific Th1 response toward Tropheryma whipplei in genetically predisposed individuals. The cofactor GrpE of heat shock protein 70 (Hsp70) from T. whipplei was previously identified as a B-cell antigen. We tested the capacity of Hsp70 and GrpE to elicit specific proinflammatory T-cell responses. Peripheral mononuclear cells from CWD patients and healthy donors were stimulated with T. whipplei lysate or recombinant GrpE or Hsp70 before levels of CD40L, CD69, perforin, granzyme B, CD107a, and gamma interferon (IFN-γ) were determined in T cells by flow cytometry. Upon stimulation with total bacterial lysate or recombinant GrpE or Hsp70 of T. whipplei, the proportions of activated effector CD4+ T cells, determined as CD40L+ IFN-γ+, were significantly lower in patients with CWD than in healthy controls; CD8+ T cells of untreated CWD patients revealed an enhanced activation toward unspecific stimulation and T. whipplei-specific degranulation, although CD69+ IFN-γ+ CD8+ T cells were reduced upon stimulation with T. whipplei lysate and recombinant T. whipplei-derived proteins. Hsp70 and its cofactor GrpE are immunogenic in healthy individuals, eliciting effective responses against T. whipplei to control bacterial spreading. The lack of specific T-cell responses against these T. whipplei-derived proteins may contribute to the pathogenesis of CWD.

scholarly journals IFN-γ and IL-10 islet-antigen-specific T cell responses in autoantibody-negative first-degree relatives of patients with type 1 diabetes

Diabetologia ◽  
2010 ◽  
Vol 53 (7) ◽  
pp. 1451-1460 ◽  
Author(s):  
L. G. Petrich de Marquesini ◽  
J. Fu ◽  
K. J. Connor ◽  
A. J. Bishop ◽  
N. E. McLintock ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
T Cell ◽  
T Cell Responses ◽  
Cell Responses ◽  
First Degree Relatives ◽  
Ifn Γ ◽  
Islet Antigen

scholarly journals PD-L1 – PD-1 interactions limit effector Treg cell populations at homeostasis and during infection

2020 ◽  
Author(s):  
J.A. Perry ◽  
J.T. Clark ◽  
J. Gullicksrud ◽  
J. DeLong ◽  
L. Shallberg ◽  
...  
Keyword(s):  
T Cell ◽  
Treg Cell ◽  
Cell Activity ◽  
T Cell Responses ◽  
Treg Cells ◽  
Whole Body ◽  
Cell Responses ◽  
Early Production ◽  
Ifn Γ ◽  
Specific Deletion

AbstractWhile much is known about the factors that promote the development of diverse Treg cell responses, less is known about the pathways that constrain Treg cell activities. The studies presented here reveal that at homeostasis there is a population of effector Treg cells that express PD-1, and that blockade of PD-L1 or loss of PD-1 results in increased Treg cell activity. In response to infection with the parasite T. gondii, the early production of IFN-γ results in widespread upregulation of PD-L1. Moreover, blockade of PD-L1, whole body deletion of PD-1, or lineage-specific deletion of PD-1 in Foxp3+ cells prevented the loss of the effector Treg cells but resulted in reduced pathogen specific CD4+ T cell responses during infection. Thus, at homeostasis basal PD-L1 expression constrains and tunes the pool of Treg cells, but during infection the upregulation of PD-L1 provides a mechanism to contract the Treg cell population required to maximize the development of pathogen specific CD4+ T cell responses.

Nitric oxide-producing CD11b+Ly-6G(Gr-1)+CD31(ER-MP12)+cells in the spleen of cyclophosphamide–treated mice: implications for T-cell responses in immunosuppressed mice

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 212-220 ◽  
Author(s):  
Iñigo Angulo ◽  
Federico Gómez de las Heras ◽  
José F. Garcı́a-Bustos ◽  
Domingo Gargallo ◽  
M. Angeles Muñoz-Fernández ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Proliferation ◽  
T Cell ◽  
T Cell Responses ◽  
T Cell Proliferation ◽  
Intensive Chemotherapy ◽  
Suppressive Activity ◽  
Cell Responses ◽  
Nos Inhibitors ◽  
Ifn Γ

Abstract During recovery from intensive chemotherapy with cyclophosphamide (CTX), mice suffer a severe but transitory impairment in spleen cell proliferation to T-cell mitogens (Con A or anti-CD3 plus IL-2). Although CTX treatment reduced spleen T-cell cellularity, this cannot fully account for T-cell unresponsiveness. The results showed that CTX induces the colonization of spleen by an immature myeloid CD11b+Ly-6G+CD31+ population. Its presence closely correlated with the maximum inhibition of T-cell proliferation. Moreover, this suppressive activity was dependent on nitric oxide (NO) production in cultures since (1) higher amounts of nitric oxide and inducible nitric oxide synthase (iNOS) mRNA were produced in CTX spleen cells (CTX-SC) than in control splenocyte cultures and (2) NOS inhibitors greatly improved the proliferation of T lymphocytes. Nitric oxide production and suppressive activity were also dependent on endogenous interferon-γ (IFN-γ) production since anti–IFN-γ abrogated both effects. Finally, iNOS protein expression was restricted to a heterogeneous population of CD31+cells in which CD11b+Ly-6G+ cells were required to suppress T-cell proliferation. These results indicated that CTX might also cause immunosuppression by a mechanism involving the presence of immature myeloid cells with suppressor activity. This may have implications in clinical praxis since inappropriate immunotherapies in patients treated with intensive chemotherapy could lead to deleterious T-cell responses. (Blood. 2000;95:212-220)

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