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Many assays to evaluate the nature, breadth, and quality of antigen-specific T cell responses are currently applied in human medicine. In most cases, assay-related protocols are developed on an individual laboratory basis, resulting in a large number of different protocols being applied worldwide. Together with the inherent complexity of cellular assays, this leads to unnecessary limitations in the ability to compare results generated across institutions. Over the past few years a number of critical assay parameters have been identified which influence test performance irrespective of protocol, material, and reagents used. Describing these critical factors as an integral part of any published report will both facilitate the comparison of data generated across institutions and lead to improvements in the assays themselves. To this end, the Minimal Information About T Cell Assays (MIATA) project was initiated. The objective of MIATA is to achieve a broad consensus on which T cell assay parameters should be reported in scientific publications and to propose a mechanism for reporting these in a systematic manner. To add maximum value for the scientific community, a step-wise, open, and field-spanning approach has been taken to achieve technical precision, user-friendliness, adequate incorporation of concerns, and high acceptance among peers. Here, we describe the past, present, and future perspectives of the MIATA project. We suggest that the approach taken can be generically applied to projects in which a broad consensus has to be reached among scientists working in fragmented fields, such as immunology. An additional objective of this undertaking is to engage the broader scientific community to comment on MIATA and to become an active participant in the project. [ABSTRACT FROM AUTHOR]

The choice of serum for supplementation of media for T cell assays and in particular, Elispot has been a major challenge for assay performance, standardization, optimization, and reproducibility. The Assay Working Group of the Cancer Vaccine Consortium (CVC-CRI) has recently identified the choice of serum to be the leading cause for variability and suboptimal performance in large international Elispot proficiency panels. Therefore, a serum task force was initiated to compare the performance of commercially available serum-free media to laboratories’ own medium/serum combinations. The objective of this project was to investigate whether a serum-free medium exists that performs as well as lab-own serum/media combinations with regard to antigen-specific responses and background reactivity in Elispot. In this way, a straightforward solution could be provided to address the serum challenge. Eleven laboratories tested peripheral blood mononuclear cells (PBMC) from four donors for their reactivity against two peptide pools, following their own Standard Operating Procedure (SOP). Each laboratory performed five simultaneous experiments with the same SOP, the only difference between the experiments was the medium used. The five media were lab-own serum-supplemented medium, AIM-V, CTL, Optmizer, and X-Vivo. The serum task force results demonstrate compellingly that serum-free media perform as well as qualified medium/serum combinations, independent of the applied SOP. Recovery and viability of cells are largely unaffected by serum-free conditions even after overnight resting. Furthermore, one serum-free medium was identified that appears to enhance antigen-specific IFNγ-secretion. [ABSTRACT FROM AUTHOR]

The Cancer Immunotherapy Immunoguiding Program has conducted an IFN-γ ELISPOT proficiency panel to examine the influence of serum supplementation of test media on assay performance. Sixteen European laboratories analyzed the same PBMC samples using different locally established protocols. Participants generated two simultaneous data sets—one using medium supplemented with serum and one without serum. Performances of the two test conditions were compared by quantifying: (1) the number of viable cells, (2) background spot formation induced in the medium only control and (3) the ability to detect antigen-specific T cell responses. The study demonstrated that the number of viable cells recovered and the overall background spot production were not significantly different between the two conditions. Furthermore, overall laboratory performance was equivalent for the two test conditions; 11 out of 16 laboratories reported equal or greater detection rates using serum-free medium, while 5 laboratories reported decreased detections rates under serum-free conditions. These results show that good performance of the IFN-γ ELISPOT assay can be achieved under serum-free conditions. Optimization of the protocol for serum-free conditions should result in excellent detection rates and eliminate the requirement of serum batch and stability testing, allowing further harmonization of the assay. [ABSTRACT FROM AUTHOR]