Characterization of CD4+ T cells specific for glutamic acid decarboxylase (GAD65) and proinsulin in a patient with stiff-person syndrome but without type 1 diabetes

Abstract

Background: Glutamic acid decarboxylase (GAD) is a rate-limiting enzyme in the synthesis of gamma-amino butyric acid (GABA) and an important autoantigen both in patients with type 1 diabetes (T1D) and stiff-person syndrome (SPS). Autoantibodies (GADA) to the 65-kDa isoform of GAD are a characteristic feature in both diseases. Approximately 30% of patients with SPS develop diabetes, yet, it is unclear to which extent co-existing autoimmunity to GAD65 and other islet autoantigens determines the risk of developing T1D.

Methods: In this study, we monitored CD4+ T-cell responses to GAD65 and proinsulin in a patient with SPS who remained normoglycaemic during the 46-month follow-up.

Results: Fluctuating but persistent T-cell reactivity to GAD65 was identified, as well as T-cell reactivity to proinsulin at one time point. The majority of the T-cell clones isolated from the patient with SPS produced high levels of Th2 cytokines (IL-13, IL-5 and IL-4). We also examined levels of GADA, insulin and IA-2 autoantibodies, and epitope specificity of GADA. In both serum and cerebrospinal fluid (CSF), GADA levels were high, and GADA persisted throughout the follow-up. Despite T-cell reactivity to both GAD65 and proinsulin, autoantibodies to other islet autoantigens did not develop.

Conclusions: Further follow-up will determine whether the beta-cell autoimmunity observed in this patient will eventually lead to T1D.

Figure 1. GAD65Ab titer and inhibition of GAD65 enzymatic activity remains stable over time

Longitudinal serum samples taken at the indicated time points were tested for their capacity to inhibit GAD65 enzymatic activity (white squares, right Y-axis) and their GAD65Ab titer (black squares, left Y-axix). GAD65 enzyme activity was measured in the absence and presence of serum samples. Inhibition of GAD65 enzyme activity is shown as percent inhibition. GAD65Ab titer was determined in a RBA and is reported as GAD65Ab index.

Figure 2. Epitope analysis using GAD65-specific rFab

Longitudinal samples taken at the indicated time points were analyzed for their epitope binding pattern in our epitope-specific RBA using GAD65-specific rFab b78 (black squares), b96.11 (white squares), MICA-3 (white circles), DPD (diamonds), and N-GAD65 mAb (black circles). Binding to radiolabled GAD65 by serum samples was tested in the absence (100%) and presence of each of these rFab at half-maximal concentration.

Figure 3. DR301-tetramer staining of the PBMC from the SPS patient

PBMC at 14-day post-stimulation with GAD65 339–352, GAD65 247–266 or proinsulin B24-C36 peptide (10 μg/ml) were stained with both specific DR301 tetramers containing the cognate peptide (upper panels in each section), and DR301-NS1 negative control tetramer (lower panel in each section). The cells were gated on live lymphocytic population and CD3+ cells. The frequency of CD4 and tetramer-positive cells is shown in the quadrant. The tetramer-positive cells were single cell sorted on 96-well plates.

Figure 4. Tetramer staining and cytokine profiles of the T-cell clones

The DR301-GAD339–352 tetramer was used to stain T-cell clones single cell sorted from the PBMC samples obtained at three time points (#1–3, upper panel). Similarly, DR301-GAD 247–266 and proinsulin B24-C36 tetramers were used to stain clones derived from the first time point (middle, lower left panels). In all cases tetramer staining was evaluated on the viable lymphocytic population as gated based on the forward/side scatter profile. The frequency of CD4/tetramer-positive cells is noted in the upper right quadrant. Staining with the control tetramer containing DR301 binding irrelevant peptide (flu, NS-1) was set at 0.2%. Cytokines (pg/ml, Y axis) were determined from the supernatants collected from triplicate cultures of the T cell clones at 48hrs post stimulation with DR301+ irradiated PBMC pulsed with the cognate peptides (middle-lower right panel).