Guidelines for the automated evaluation of Elispot assays

Janetzki, Sylvia; Price, Leah; Schroeder, Hélène; Britten, Cedrik M.; Welters, Marij J.P.; Hoos, Axel

doi:10.1038/nprot.2015.068

Cited by 73 publications

(59 citation statements)

References 47 publications

(61 reference statements)

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“…ELISpot plates coated with mouse IgG1 anti-human IFN-γ monoclonal antibody were incubated overnight at 37°C, 5% CO 2 , washed with PBS/Tween-20, incubated with biotinylated mouse IgG1 anti-human IFN-γ for 1 hour at room temperature, washed with PBS, incubated with avidin-peroxidase complex for 1 hour at room temperature, washed, and incubated with substrate (3-amino-9-ethylcarbazole) for 4 minutes at room temperature. Spot enumeration was performed in a blinded fashion by Zellnet Consulting, Inc. (Fort Lee, NJ) using a KS ELISpot reader (Zeiss Inc., Thornwood, NY), software version KS 4.9.16 using established guidelines (22). Results were expressed as the mean spot-forming cells (SFC)/10 6 PBMC after subtraction of background counts from PBMCs cultured without peptide.…”

Section: Methodsmentioning

confidence: 99%

Long-term Survival in Glioblastoma with Cytomegalovirus pp65-Targeted Vaccination

Batich

Reap

Archer

et al. 2017

Clinical Cancer Research

236

167

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Purpose Patients with glioblastoma (GBM) have a <15 month median survival despite surgical resection, high-dose radiation and chemotherapy with temozolomide (TMZ). We previously demonstrated that targeting Cytomegalovirus (CMV) pp65 using dendritic cells (DCs) can extend survival and, in a separate study, that dose-intensified (DI) TMZ and adjuvant GM-CSF potentiates tumor-specific immune responses in patients with GBM. Here, we evaluated pp65-specific cellular responses following DI-TMZ with pp65-DCs and determined the effects on long-term progression-free survival (PFS) and overall survival (OS). Experimental Design Following standard of care, 11 patients with newly diagnosed GBM received DI-TMZ (100 mg/m2/day × 21 days per cycle) with at least three vaccines of pp65-lysosome-associated membrane glycoprotein (LAMP) mRNA-pulsed DCs admixed with GM-CSF on Day 23 ± 1 of each cycle. Thereafter, monthly DI-TMZ cycles and pp65-DCs were continued if patients had not progressed. Results Following DI-TMZ cycle 1 and three doses of pp65-DCs, pp65 cellular responses significantly increased. After DI-TMZ, both the proportion and proliferation of regulatory T-cells (TRegs) increased and remained elevated with serial DI-TMZ cycles. Median PFS and OS were 25.3 months (CI95: 11.0-∞) and 41.1 months (CI95: 21.6-∞), exceeding survival using recursive partitioning analysis and matched historical controls. Four patients remained progression-free at 59 to 64 months from diagnosis. No known prognostic factors (age, KPS, IDH-1/2 mutation, and MGMT promoter methylation) predicted more favorable outcomes for the patients in this cohort. Conclusions Despite increased TReg proportions following DI-TMZ, patients receiving pp65-DCs showed long-term PFS and OS, confirming prior studies targeting CMV in GBM.

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Section: Methodsmentioning

confidence: 99%

Long-term Survival in Glioblastoma with Cytomegalovirus pp65-Targeted Vaccination

Batich

Reap

Archer

et al. 2017

Clinical Cancer Research

236

167

View full text Add to dashboard Cite

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“…Efforts to harmonize classic single-cell immune monitoring assays have included the identification of critical assay steps, and guidelines for harmonized assay conduct have been made available (ELISpot [123–125], multimer staining [126, 127], intracellular cytokine staining [128–130] and Immunoscore [131, 132]). These efforts have been shown to dramatically reduce the variability among laboratories and provide a basis for the comparison of immune assay results obtained at different sites, or even across trials [133].…”

Section: Pre-analytical and Analytical Validationmentioning

confidence: 99%

Validation of biomarkers to predict response to immunotherapy in cancer: Volume I — pre-analytical and analytical validation

Masucci

Cesano

Hawtin

et al. 2016

j. immunotherapy cancer

Self Cite

168

131

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Immunotherapies have emerged as one of the most promising approaches to treat patients with cancer. Recently, there have been many clinical successes using checkpoint receptor blockade, including T cell inhibitory receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death-1 (PD-1). Despite demonstrated successes in a variety of malignancies, responses only typically occur in a minority of patients in any given histology. Additionally, treatment is associated with inflammatory toxicity and high cost. Therefore, determining which patients would derive clinical benefit from immunotherapy is a compelling clinical question.Although numerous candidate biomarkers have been described, there are currently three FDA-approved assays based on PD-1 ligand expression (PD-L1) that have been clinically validated to identify patients who are more likely to benefit from a single-agent anti-PD-1/PD-L1 therapy. Because of the complexity of the immune response and tumor biology, it is unlikely that a single biomarker will be sufficient to predict clinical outcomes in response to immune-targeted therapy. Rather, the integration of multiple tumor and immune response parameters, such as protein expression, genomics, and transcriptomics, may be necessary for accurate prediction of clinical benefit. Before a candidate biomarker and/or new technology can be used in a clinical setting, several steps are necessary to demonstrate its clinical validity. Although regulatory guidelines provide general roadmaps for the validation process, their applicability to biomarkers in the cancer immunotherapy field is somewhat limited. Thus, Working Group 1 (WG1) of the Society for Immunotherapy of Cancer (SITC) Immune Biomarkers Task Force convened to address this need. In this two volume series, we discuss pre-analytical and analytical (Volume I) as well as clinical and regulatory (Volume II) aspects of the validation process as applied to predictive biomarkers for cancer immunotherapy. To illustrate the requirements for validation, we discuss examples of biomarker assays that have shown preliminary evidence of an association with clinical benefit from immunotherapeutic interventions. The scope includes only those assays and technologies that have established a certain level of validation for clinical use (fit-for-purpose). Recommendations to meet challenges and strategies to guide the choice of analytical and clinical validation design for specific assays are also provided.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-016-0178-1) contains supplementary material, which is available to authorized users.

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“…A second limitation is the focus on antigens that are presented during the first 48 hours of in vitro infection. An interesting issue was that the frequency of epitope-specific T cells differed between assays 21, 31, 32 . Key differences in methodologies and the timing (see methods) make direct comparisons difficult.…”

Section: Discussionmentioning

confidence: 99%

Limited recognition ofMycobacterium tuberculosis-infected macrophages by polyclonal CD4 and CD8 T cells from the lungs of infected mice

Patankar

Sutiwisesak

Boyce

et al. 2019

Preprint

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20Immune responses following Mycobacterium tuberculosis (Mtb) infection or vaccination 21 are frequently assessed by measuring T cell recognition of crude Mtb antigens, 22 recombinant proteins, or peptide epitopes. We previously showed that not all Mtb-23 specific T cells recognize Mtb-infected macrophages. Thus, an important question is 24 what proportion of T cells elicited by Mtb infection recognize Mtb-infected macrophages. 25We answer this question by developing a modified elispot assay using viable Mtb-26 infected macrophages, a low multiplicity of infection and purified T cells. In C57BL/6 27 mice, CD4 and CD8 T cells were classically MHC restricted. Comparable frequencies of 28 T cells that recognize Mtb-infected macrophages were determined using interferon-γ 29 elispot and intracellular cytokine staining, and lung CD4 T cells more sensitively 30 recognized Mtb-infected macrophages than lung CD8 T cells. Compared to the numbers 31 of Mtb antigen-specific T cells for antigens such as ESAT-6 and TB10.4, low frequencies 32 of pulmonary CD4 and CD8 T cells elicited by aerosolized Mtb infection recognize Mtb-33 infected macrophages. Finally, we demonstrate that BCG vaccination elicits T cells that 34 recognize Mtb-infected macrophages. We propose that the frequency of T cells that 35 recognize infected macrophages could correlate with protective immunity and may be an 36 alternative approach to measuring T cell responses to Mtb antigens. 37with the failure of T cells specific for those antigens to recognize Mtb-infected 64 macrophages 4 . Importantly, following aerosol infection, Mtb disseminates to the 65 mediastinal lymph node, where T cells are first primed by dendritic cells, which then 66 expand and traffic to the lung 9, 10 . We speculate that there may be a mismatch in the 67 antigens presented (or cross-presented) by uninfected DC in the lymph nodes and 68 antigens presented by infected macrophages in the lung. Thus, T cells primed in the lymph 69 nodes during natural infection may not necessarily recognize antigens presented by Mtb-70 infected macrophages in the lung 11 . Regardless of the mechanism, we wondered whether 71 the inability of some T cells to recognize Mtb-infected macrophages might explain why the 72 number of antigen-specific T cells may not necessarily correlate with vaccine-induced 73 protection. 74To assess T cell recognition of Mtb-infected macrophages we developed a 75 modified elispot assay based on interferon (IFN)-g spot forming cells (SFC). Using a low 76 multiplicity of infection (MOI), we quantify the frequency of T cells that recognize Mtb-77 infected macrophages during primary infection in mice. We find that an unexpectedly low 78 frequency of ex vivo CD8 and CD4 T cells recognizes Mtb-infected macrophages. We 79 demonstrate that majority of the T cells from C57BL/6 mice that recognize Mtb-infected 80 macrophages are conventionally MHC-restricted T cells. Our data shows that CD4 T 81 cells efficiently detect Mtb-infected macrophages at a lower MOI, whereas CD8 T cells 82 on...

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Guidelines for the automated evaluation of Elispot assays

Cited by 73 publications

References 47 publications

Long-term Survival in Glioblastoma with Cytomegalovirus pp65-Targeted Vaccination

Long-term Survival in Glioblastoma with Cytomegalovirus pp65-Targeted Vaccination

Validation of biomarkers to predict response to immunotherapy in cancer: Volume I — pre-analytical and analytical validation

Limited recognition ofMycobacterium tuberculosis-infected macrophages by polyclonal CD4 and CD8 T cells from the lungs of infected mice

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