Guidelines for analysis of low-frequency antigen-specific T cell results: Dye-based proliferation assay vs 3H-thymidine incorporation.

It is generally recognized that dysregulation of the immune system plays a critical role in many diseases, including autoimmune diseases and cancer. T cells play a crucial role in maintaining self-tolerance, while loss of immune tolerance and T cell activation can lead to severe inflammation and tissue damage. T cell responses have a key role in the effectiveness of vaccination strategies and immunomodulating therapies. Immunomonitoring methods have the ability to elucidate immunological processes, monitor the development of disease and assess therapeutic effects. In this respect, it is of particular interest to evaluate antigen (Ag)-specific T cells by determining their frequency, type and functionality in cellular assays. Nevertheless, Ag-specific T cells are detected infrequently in most diseases using current techniques. Many efforts have been made to develop more sensitive, reproducible, and reliable methods for Ag-specific T cell detection. It has been found that analysis of cellular proliferation can be a useful tool to determine the presence and frequency of Ag-specific T cell and to provides insight into modulation of the T cell response by a specific antigen or therapy. However, the selection of a cut-off value for a positive response and therefore a more accurate interpretation of the data, continues to be a major concern. Here, we provide guidelines to select a proper cut-off for monitoring of Ag-specific CD4+ T cell responses. In vitro Ag-stimulation has been assessed with two methods; a dye-based proliferation assay and 3H-thymidine-based assay. Two cut-off approaches are compared; mean and variance of control wells, and the stimulation index. By evaluating the proliferative response to the in vitro Ag-stimulation using these two methods, we demonstrate the importance of taking into consideration the variability of the control wells to distinguish a positive from a false positive response.

The natalizumab wearing-off effect: End of natalizumab cycle, recurrence of MS symptoms.

OBJECTIVE: Natalizumab is effective in treating relapsing-remitting multiple sclerosis (RRMS). However, many patients report an increase of multiple sclerosis symptoms at the end of the natalizumab cycle: a wearing-off effect. The objective of this study was to evaluate the prevalence of the wearing-off effect in patients with standard and extended intervals and to study possible associations with pharmacokinetic/dynamic measurements and patient characteristics in a prospective, monocenter, cross-sectional cohort study. METHODS: Patients with RRMS, with a minimum of 6 natalizumab infusions, were asked to complete 3 questionnaires: the Multiple Sclerosis Impact Scale, the 36-Item Short Form Health Survey, and a general questionnaire regarding the wearing-off effect. Natalizumab concentration and α4-integrin receptor saturation were measured before redosing. RESULTS: Ninety-three patients were included. A total of 54% experienced a wearing-off effect during natalizumab treatment and 32% experienced a current wearing-off effect at time of measurement. The self-reported wearing-off effect was not associated with natalizumab concentration nor with α4-integrin receptor saturation. The wearing-off effect was more frequently reported in the standard interval group (39%) than in the extended interval group (19%); the duration of symptoms was comparable between both groups. The wearing-off effect was not associated with number of infusions, disease duration, age, or sex. CONCLUSION: The wearing-off effect is a frequently reported phenomenon but is unlikely to reflect a nonoptimal pharmacokinetic/dynamic state. We did not find risk factors predicting the wearing-off effect.

Pharmacodynamic assessment of cell-bound natalizumab on PBMC samples stored in liquid nitrogen.

Natalizumab is a monoclonal IgG4 antibody used for treatment of relapsing remitting MS. Natalizumab interferes with lymphocyte migration by blocking alpha-4 integrin (CD49d). Saturation levels of alpha-4 integrin on circulating T cells by natalizumab have been associated with clinical effectiveness of therapy. However, in most cases, measurements have been carried out using freshly isolated PBMCs. The aim of this study was to set up and evaluate a method to measure relative levels of cell-bound natalizumab using frozen PBMC samples. A new method was set up to measure cell-bound natalizumab by flow cytometry on T cell subsets using fully saturated cells as a 100% reference. A comparison was made between spike samples and samples of natalizumab-treated MS patients freshly isolated and stored in liquid nitrogen. Cell-bound natalizumab could be measured (using an anti-IgG4 antibody) on cells stored in liquid nitrogen. Natalizumab was found to slowly dissociate from the cells during isolation and subsequent sample work-up. This dissociation was more pronounced for monovalent natalizumab resulting from Fab arm exchange (the predominant isoform in patients) than bivalent natalizumab straight from the vial. We established a correction factor to account for this phenomenon. The resulting method has good accuracy compared to assessing fresh cells. The inter-assay precision (%CV) is ca. 12% using frozen cells. In conclusion, we established a method to assess relative levels of cell-bound natalizumab on cells obtained from frozen PBMC samples.