Regulatory T cells are an important component of the immune system that plays a key role in maintaining homeostasis. Identification of distinct regulatory T cell subsets is essential to understand their function. Mass cytometry or CyTOF is a technology that enables the simultaneous measurement of up to 50 markers in single cells by using antibodies tagged with heavy metals, which are then detected with time-of-flight mass spectrometry. This chapter describes a mass cytometry approach for phenotypic characterization of regulatory T cells and determination of their master transcription factor Foxp3.

The frequency of apoptotic cells in a given phenotypically defined population is usually calculated the apoptotic index (AI), i.e., the percentage of apoptotic cells displaying a specific linage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. Firstly, apoptotic cells fragment into apoptotic bodies that later disintegrate. Secondly, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. The present chapter will describe a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of lineage antigen expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures.

Aims: Immunomodulation with autoantigens potentially constitutes a specific and safe treatment for type 1 diabetes (T1D). Studies with GAD-alum administrated subcutaneously have shown to be safe, but its efficacy has been inconclusive. Administration of GAD-alum into the lymph nodes, aimed to optimise antigen presentation, has shown promising results in an open-label clinical trial. Herein, we compared the immune response of the individuals included in the trial with a group who received GAD-alum subcutaneously in a previous study.

Materials and methods: Samples from T1D individuals collected 15 months after administration of either three doses 1 month apart of 4 mu g GAD-alum into lymph nodes (LN, n = 12) or two doses 1 month apart of 20 mu g subcutaneously (SC, n = 12) were studied. GADA, GADA subclasses, GAD(65)-induced cytokines, peripheral blood mononuclear cell proliferation, and T cells markers were analysed.

Results: Low doses of GAD-alum into the lymph nodes induced higher GADA levels than higher doses administrated subcutaneously. Immune response in the LN group was characterised by changes in GADA subclasses, with a relative reduction of IgG1 and enhanced IgG2, IgG3, and IgG4 proportion, higher GAD(65)-induced secretion of IL-5, IL-10, and TNF-alpha, and reduction of cell proliferation and CD8(+) T cells. These changes were not observed after subcutaneous (SC) injections of GAD-alum.

Conclusions: GAD-specific immune responses 15 months after lymph node injections of GAD-alum differed from the ones induced by SC administration of the same autoantigen.

Aim To evaluate the long-term effect of intra-lymphatic administration of GAD-alum and a booster dose 2.5 years after the first intervention (DIAGNODE Extension study) in patients with recent-onset type 1 diabetes. Methods DIAGNODE-1: Samples were collected from 12 patients after 30 months who had received 3 injections of 4 mu g GAD-alum into a lymph node with one-month interval. DIAGNODE Extension study: First in human, a fourth booster dose of autoantigen (GAD-alum) was given to 3 patients at 31.5 months, who were followed for another 12 months. C-peptide was measured during mixed meal tolerance tests (MMTTs). GADA, IA-2A, GADA subclasses, GAD(65)-induced cytokines, PBMCs proliferation and T cells markers were analyzed. Results After 30-month treatment, efficacy was still seen in 8/12 patients (good responders, GR). Partial remission (IDAA1c < 9) had decreased compared to 15 months, but did not differ from baseline, and HbA1c remained stable. GAD(65)-specific immune responses induced by the treatment started to wane after 30 months, and most changes observed at 15 months were undetectable. GADA subclasses IgG2, IgG3 and IgG4 were predominant in the GR along with IgG1. A fourth intra-lymphatic GAD-alum dose to three patients after 31.5 months gave no adverse events. In all three patients, C-peptide seemed to increase the first 6 months, and thereafter, C-peptide, HbA1c, insulin requirement and IDAA1c remained stable. Conclusion The effect of intra-lymphatic injections of GAD-alum had decreased after 30 months. Good responders showed a specific immune response. Administration of a fourth booster dose after 31.5 months was safe, and there was no decline in C-peptide observed during the 12-month follow-up.