A simple and efficient method for the monitoring of antigen-specific T cell responses using peptide pool arrays in a modified ELISpot assay

2001 ◽  
Vol 254 (1-2) ◽  
pp. 59-66 ◽  
Author(s):  
Timothy W Tobery ◽  
Su Wang ◽  
Xin-Min Wang ◽  
Michael P Neeper ◽  
Kathrin U Jansen ◽  
...  
Keyword(s):  
T Cell ◽  
Efficient Method ◽  
Elispot Assay ◽  
T Cell Responses ◽  
Peptide Pool ◽  
Cell Responses

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.

A phase I/II randomized trial using GM-CSF-producing and CD40L-expressing bystander cell line (GM.CD40L) vaccine in combination with CCL21 in stage IV lung adenocarcinoma: Preliminary results.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3091-3091
Author(s):  
Jhanelle Elaine Gray ◽  
Alberto Chiappori ◽  
Charles C. Williams ◽  
Mary Colleen Pinder ◽  
Eric B. Haura ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
Phase I ◽  
Lung Adenocarcinoma ◽  
Cell Line ◽  
Phase Ii ◽  
Elispot Assay ◽  
T Cell Responses ◽  
Bystander Cell ◽  
Cell Responses

3091 Background: Our GM.CD40L vaccine (an allogeneic tumor cell-based vaccine generated from human bystander cell line) recruits and activates dendritic cells, which then migrate to regional lymph nodes, where T cell activation occurs, leading to systemic tumor cell killing. The CCL21 chemokine helps to recruit T cells and leads to enhanced T cell responses. The GM.CD40L.CCL21 combination has demonstrated additive effects in NSCLC mouse models. Methods: We initiated a phase I/II randomized study to evaluate GM.CD40L (Arm A) vs. GM.CD40L.CCL21 (Arm B) in patients with lung adenocarcinoma who had failed first-line therapy. Primary endpoints were safety and tolerability of Arm B in phase I and progression-free survival (PFS) in phase II; secondary endpoints included anti-tumor immune responses/T-cell responses by ELISpot assay on PBMC. Immune-related response criteria as determined by the investigator served to determine discontinuation from study treatment. Intradermal vaccines were administered every 14 days for 3 doses and then monthly X3. A two-stage minimax design was used. Results: In phase I, 3 patients received GM.CD40L.CCL21; no dose-limiting toxicities occurred. Between 4/2012 and 12/2012, Arm A enrolled 11 and Arm B enrolled 16 patients, including those in phase I (median age: 70/67.5 years, females: 45.5%/37.5%, PS1: 54.5%/75%, median prior regimens: 3/5 for Arm A vs. Arm B, respectively). Most common toxicities for Arm A vs. Arm B were injection site reaction (45.5%/43.8%), fatigue (9.1%/37.5%), anorexia (0%/12.5%), and pain in extremity (0%/12.5%). Median PFS for Arm A vs. B was 4.4 vs. 4.4 months (p=0.37). Of the 6 patients who remained on study post RECIST v1.1 progression, all demonstrated further progression on subsequent scans and were removed from the study. Of patients evaluable for efficacy, stable disease was 3/7 and progressive disease was 6/7 for Arm A vs. Arm B, respectively. Analyses of ELISpot assay on the PBMC are underway. Conclusions: GM.CD40L.CCL21 vaccine is well tolerated; thus far, median PFS results are similar to GM.CD40L vaccine. Updated results of the phase II trial will be presented. Clinical trial information: NCT01433172.

scholarly journals T cell mediated immunity against influenza H5N1 nucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors and non-H5N1 subjects

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11021
Author(s):  
Pirom Noisumdaeng ◽  
Thaneeya Roytrakul ◽  
Jarunee Prasertsopon ◽  
Phisanu Pooruk ◽  
Hatairat Lerdsamran ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Influenza Virus ◽  
T Cell ◽  
Avian Influenza ◽  
Influenza Vaccine ◽  
Elispot Assay ◽  
H5n1 Virus ◽  
T Cell Responses ◽  
Cell Responses ◽  
Influenza H5n1

Background Protection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising. Methods This study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN-γ ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection. Results IFN-γ ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN-γ ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN-γ and TNF-α, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry. Conclusions This study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.

Performance characteristics of common immunologic biomarkers used in clinical trials

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 620-620
Author(s):  
V. Goodell ◽  
B. Macleod ◽  
M. Slota ◽  
M. L. Disis
Keyword(s):  
T Cell ◽  
Cell Response ◽  
Elispot Assay ◽  
T Cell Responses ◽  
T Cell Response ◽  
Breast Cancer Patients ◽  
Cell Responses ◽  
Her 2 ◽  
High Level ◽  
Limiting Dilution

620 Background: Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of 2 cell-based assays widely used in immune monitoring, the ELISPOT assay and a modified limiting dilution assay based on titrated thymidine incorporation, to predict T cell responses to a HER-2/neu protein vaccine in breast cancer patients. We also assessed the ability of these assays to predict T cell response to tetanus toxoid and CMV, and analyzed the correlation between results from the ELISPOT and modified limiting dilution assays. Methods: 27 Stage II, II, and IV HER-2/neu positive breast cancer patients were vaccinated against the HER-2/neu protein and tt. PBMC were collected before and after vaccination. Samples were analyzed by mLDA and ELISPOT for T cell response to HER-2/neu (a low level response), response to tt (moderate level) and T cell response to CMV (high level). Results: Correlation analysis indicates that there is a strong, significant association between results from the ELISPOT assay and the mLDA for HER-2/neu specific T cell response (Rs=0.547, p=0.02). ROC curves plotted to assess the diagnostic performance of the mLDA and ELISPOT assay indicate that T cell proliferation measurements are a significant indicator of T cell response to the HER-2/neu vaccine (p=0.05), as well as responses to tt (p=0.01) and CMV (p=0.016), respectively. Precursor frequency, as measured by ELISPOT, is a significant indicator of high level T cell response to CMV (p=0.03), but not of a moderate tt response (p=0.09), or HER-2/neu (p=0.09) T cell responses. Conclusion: The mLDA achieves greater assay accuracy in measuring low level T cell responses to HER-2/neu and moderate responses to tt than the ELISPOT assay. No significant financial relationships to disclose.

scholarly journals Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses

2009 ◽  
Vol 344 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Coral-Ann M. Almeida ◽  
Steven G. Roberts ◽  
Rebecca Laird ◽  
Elizabeth McKinnon ◽  
Imran Ahmed ◽  
...  
Keyword(s):  
T Cell ◽  
High Throughput ◽  
Elispot Assay ◽  
T Cell Responses ◽  
Cell Responses ◽  
High Throughput Detection

scholarly journals Robust Stimulation of Humoral and Cellular Immune Responses following Vaccination with Antigen-Loaded β-Glucan Particles

mBio ◽  
2010 ◽  
Vol 1 (3) ◽  
Author(s):  
Haibin Huang ◽  
Gary R. Ostroff ◽  
Chrono K. Lee ◽  
Charles A. Specht ◽  
Stuart M. Levitz
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Elispot Assay ◽  
Cellular Response ◽  
T Cell Responses ◽  
Minor Role ◽  
Mouse Bone Marrow ◽  
Antigen Delivery ◽  
Vaccine Platform ◽  
Cell Responses

ABSTRACTβ-Glucan particles (GPs) are purifiedSaccharomyces cerevisiaecell walls treated so that they are primarily β1,3-d-glucans and free of mannans and proteins. GPs are phagocytosed by dendritic cells (DCs) via the Dectin-1 receptor, and this interaction stimulates proinflammatory cytokine secretion by DCs. As the hollow, porous GP structure allows for high antigen loading, we hypothesized that antigen-loaded GPs could be exploited as a receptor-targeted vaccine delivery system. Ovalbumin (OVA) was electrostatically complexed inside the hollow GP shells (GP-OVA). Incubation of C57BL/6J mouse bone marrow-derived DCs with GP-OVA resulted in phagocytosis, upregulation of maturation markers, and rapid proteolysis of OVA. Compared with free OVA, GP-OVA was >100-fold more potent at stimulating the proliferation of OVA-reactive transgenic CD8+OT-I and CD4+OT-II T cells, as measured byin vitro[3H]thymidine incorporation using DCs as antigen-presenting cells. Next, immune responses in C57BL/6J mice following subcutaneous immunizations with GP-OVA were compared with those in C57BL/6J mice following subcutaneous immunizations with OVA absorbed onto the adjuvant alum (Alum/OVA). Vaccination with GP-OVA stimulated substantially higher antigen-specific CD4+T-cell lymphoproliferative and enzyme-linked immunospot (ELISPOT) responses than that with Alum/OVA. Moreover, the T-cell responses induced by GP-OVA were Th1 biased (determined by gamma interferon [IFN-γ] ELISPOT assay) and Th17 biased (determined by interleukin-17a [IL-17a] ELISPOT assay). Finally, both the GP-OVA and Alum/OVA formulations induced strong secretions of IgG1 subclass anti-OVA antibodies, although only GP-OVA induced secretion of Th1-associated IgG2c antibodies. Thus, the GP-based vaccine platform combines adjuvanticity and antigen delivery to induce strong humoral and Th1- and Th17-biased CD4+T-cell responses.IMPORTANCEMost licensed vaccines work by promoting protective antibody responses. However, for many infectious diseases, antibody-mediated protection appears to play a relatively minor role, and vaccination has met with limited success. While live-attenuated organisms generally elicit T-cell responses, their use in vaccines is limited by the potential for causing disease. Thus, there is an urgent need for new vaccine platforms that deliver antigens in such a manner as to promote strong T-cell-mediated responses. Here we designed a novel vaccine platform consisting of yeast-derived β-glucan particles (GPs) that combines antigen delivery and adjuvant activity. GPs loaded with the model antigen ovalbumin (OVA) stimulated robust humoral and T-cell responses in mice. In addition, the cellular response was Th1 and Th17 biased. This work has implications for the design of vaccines that stimulate biased T-cell responses as well as for understanding how immunity to fungal pathogens develops.

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 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.

Generation of Cytotoxic T Cell Responses Directed to HLA Class I Restricted Epitopes from the Aspergillus f16 Allergen.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1644-1644
Author(s):  
Gamal Ramadan ◽  
Barbara Davies ◽  
Viswanath P. Kurup ◽  
Carolyn A. Keever-Taylor
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Response ◽  
T Cell Responses ◽  
Peptide Pool ◽  
Hla Class I ◽  
T Cell Response ◽  
Class I ◽  
Cell Responses

Abstract Invasive pulmonary aspergillosis is a primary cause of morbidity and mortality in immunocompromised patients such as hematopoietic progenitor cell transplant patients. Studies both in patients with allergic bronchopulmonary aspergillosis and murine models demonstrated the importance of a CD4+ Th1 T cell response in conferring protection from infection or preventing disease progression. The role of CD8+ T cell response to A. fumigatus is less clear. Our efforts to develop effective immunotherapeutic approaches against A. fumigatus included preparation of 104 overlapping pentadecapeptides spanning the 427 aa coding region of the aspergillus allergen, Asp f16 previously shown to induce T cell responses. Each 15 aa peptide overlaps the preceding peptide by 11 aa. Monocytes from healthy donors were treated with GM-CSF and IL-4 for 2-3 days to generate immature dendritic cells (fast DC), pulsed with a pool containing 1 μg of each pentadecapeptide, then matured with inflammatory cytokines (IL-1β, IL-6, PGE2 and TNF-alpha) for 2 days. Mature, pulsed fast DC were used to prime proliferative and CTL responses (weekly primings). T cells from 5/5 donors proliferated to the peptide pool. CTL lines were obtained from each of the first two donors that were primed. After 4 weeks the line from donor #2 was strongly cytotoxic to autologous peptide pool-pulsed and aspergillus culture extract-pulsed DC and peptide pool pulsed HLA Class I matched BLCL. Supernatant from this line killed fresh aspergillus conidia. Six of 21 smaller pools of 4-11 peptides showed reactivity. Specificity could be narrowed by screening peptides shared by the pools to 3 candidate peptides. Pool-pulsed BLCL matched for only 1 or 2 HLA alleles were used to demonstrate CTL restriction by HLA-B-3501. A database search of peptides likely to be restricted to B3501 identified the likely sequences as YFKYTAAAL, LPLCSAQTW, and GTRFPQTPM. Each induced similar reactivity when pulsed onto B-3501+ targets. CD8+ T cells steadily increased from 5.2% at week 3 to 19.0% after the 7th priming. CTL activity and IFNγ production were exclusively mediated by CD8+ T cells and CD107a was expressed by 42% of the CD8+ T cells in response to pool-pulsed BLCL indicating degranulation. CTL cross-reacted with pool pulsed B3503+ BLCL but not B3502+, or B3508+ BLCL. B3503+ BLCL presented YFKYTAAAL and to a lesser extent GTRFPQTPM but not peptide LPLCSAQTW. Our data show that DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing a CD8+, HLA-Class I restricted Aspergillus-specific T cell response directed to multiple peptides contained within the pool. Further characterization of this system is in progress to identify additional immunogenic peptides from Asp f16 that might be useful in clinical immunotherapy protocols to prime protective immune responses to prevent or treat aspergillus infection.

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