The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays

Abstract

The interpretation of the results obtained from immunomonitoring of clinical trials is a diYcult task due to the variety of methods and protocols available to detect vaccine-speciWc T-cell responses. This heterogeneity as well as the lack of standards has led to signiWcant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-speciWc T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pretested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-speciWc T-cells in each sample using tetramer staining and one functional assay. The results of the Wrst testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-speciWc CD8 + T-cells by tetramer staining. Analysis by ELISPOT was inXuenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the Wrst phase. 123The recommendations improved the number of antigenspeciWc T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identiWcation of distinct variables that inXuence the sensitivity of diVerent T-cell assays and to formally show that a deWned correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could deWne rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

The candidate immunotherapeutical target, the receptor for hyaluronic acid-mediated motility, is associated with proliferation and shows prognostic value in B-cell chronic lymphocytic leukemia

“…4 ELISpot assays were performed as recommended by the Cancer Immunotherapy Monitoring Panel that our institution is a part of. 13 …”

The importance of correctly timing cancer immunotherapy

“…Therefore, an important aspect is to ensure that the measurement and reporting of the results obtained are comparable between laboratories so that it is easier to interpret and value the data generated. In the last decade a huge effort, undertaken by the Association of Cancer ImmunoTherapy's (CIMT's) immunoguiding program [125] and Cancer Immunotherapy Consortium (CIC) [126], has focused on this in the realm of human studies, and this has successfully led to: (i) a strong awareness of the need to harmonize or standardize measurements, (ii) to technical improvements and harmonization of the assays used, and (iii) to more transparent reporting [127][128][129]. It can be envisaged that similar efforts by scientists performing studies in mouse tumor models will lead to a quicker design of immunotherapeutic strategies, which expedite the translation to the treatment of patients.…”

Identification of HLA-A*01- and HLA-A*02-restricted CD8+ T-cell epitopes shared among group B enteroviruses

“…In the ELISPOT assay, 500 000 pre-sensitized cells were stimulated for 24 h with 1 mg ml 21 per peptide and as a positive control 10 mg phytohaemagglutinin (PHA)-L ml 21 was used. IFN-c was detected using the Human IFN-c ELISpot kit (MabTech) according to the manufacturer's recommendation and the cancer immunotherapy monitoring panel (Britten et al, 2008). Reactions were regarded as positive if the mean number of spots per well was at least 10 and more than three times the mean number of spots of the negative control using an HIV peptide.…”