GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes

Abstract

Insulin-dependent type 1 diabetes is an autoimmune disease mediated by T lymphocytes recognizing pancreatic islet cell antigens. Glutamic acid decarboxylase 65 (GAD65) appears to be an important autoantigen in the disease. However, T cells from both patients with type 1 diabetes and healthy subjects vigorously proliferate in response to GAD65 stimulation ex vivo, leading us to postulate that the critical event in the onset of human diabetes is the activation of autoreactive T cells. Thus, we investigated whether GAD65-reactive T cells in patients with diabetes functioned as previously activated memory T cells, no longer requiring a second, costimulatory signal for clonal expansion. We found that in patients with new-onset type 1 diabetes, GAD65-reactive T cells were strikingly less dependent on CD28 and B7-1 costimulation to enter into cell cycle and proliferate than were equivalent cells derived from healthy controls. We hypothesize that these autoreactive T cells have been activated in vivo and have differentiated into memory cells, suggesting a pathogenic role in type 1 diabetes. In addition, we observed different effects with selective blockade of either B7-1 or B7-2 molecules; B7-1 appears to deliver a negative signal by engaging CTLA-4, while B7-2 engagement of CD28 upregulates T cell proliferation and cytokine secretion.

Figure 1

Response to GAD65 in individual type 1 diabetes patients and normal control subjects with blocking costimulatory signals. PBMCs from type 1 diabetes patients and normal control subjects were cultured with different concentrations of GAD65 (0.1–20 μg/ml) or TT (0.01–1 Lf/ml) in the presence or absence of anti-CD28 F(ab′) fragments. After 5 days of culture, rhIL-2 was added to each of ten replicates established for each antigen concentration with or without blocking Ab’s. After an additional 5–7 days of culture, supernatants were collected for cytokine detection, and proliferation was assessed by [³H]thymidine incorporation. (a) T cell proliferation in 19 separate experiments. Each symbol represents the mean thymidine uptake of ten replicate cultures ± SE, stimulated only with the antigen (open squares) or with antigen plus anti–CD28 F(ab′) fragment (filled squares). T cells from both patients and controls proliferated in response to GAD65, and blockade of CD28 costimulatory pathway inhibited GAD65 proliferation and (b and c) cytokine secretion in healthy subjects but not in patients with type 1 diabetes. A summary of all the new-onset patients and controls is shown in Figure 2. (d) Proliferation and cytokine secretion in five long-term diabetic patients.

Figure 2

Proliferative response to GAD65 is not dependent on CD28/B7 co-stimulatory pathway in type 1 diabetes patients. The mean percentage of inhibition of GAD65-specific proliferation and cytokine secretion with CD28 blockade, as described in Figure 1, is shown. Each symbol is representative of ten diabetic patients (open circles) or nine normal controls (filled circles). The Mann-Whitney test was used to estimate the difference in the CD28 requirement between diabetic patients and controls. The difference between the two groups was significant at any concentration of GAD65 (diabetic patients vs. controls, proliferation: GAD, 5 μg/ml and 10 μg/ml, P = 0.0019; GAD 20 μg/ml, P = 0.0047; IFN-γ: GAD 5 μg/ml, P = 0.049; GAD 10 μg/ml, P = 0.0019; GAD 20 μg/ml, P = 0.001; IL-13: GAD 5 μg/ml, P = 0.049; GAD 10 μg/ml, P = 0.03; GAD 20 μg/ml, P = 0.0019) showing that antigen-specific T cells from type 1 diabetes patients are CD28 independent. No inhibition was detected in TT-specific T cells at any concentration of antigen either in the patients or in the normal controls.

Figure 3

Differential effects of anti–B7-1 and anti–B7-2. PBMCs were cultured with different concentration of GAD65 in the presence or absence of anti–B7-1 F(ab′) or anti–B7-2 F(ab′) mAb’s. Anti–B7-1 F(ab′) fragments inhibited the proliferative and cytokine response to GAD65 at all antigen concentrations in healthy subjects (filled circles), similar to anti-CD28, but only at low antigen concentration in patients with type 1 diabetes (open circles). The difference between the two groups was significant (patients vs. controls, proliferation: GAD 10 μg/ml, P = 0.0079; GAD 20 μg/ml, P = 0.001; IFN-γ: GAD 10 μg/ml, P = 0.01; GAD 20 μg/ml, P = 0.001; IL-13: GAD 10 μg/ml, P = 0.01; GAD 20 μg/ml, P = 0.001). In contrast anti–B7-2 decreased the proliferative and cytokine response in both normal controls and diabetic patients.

Figure 4

Blocking CD28 or CTLA-4 counter-receptors in combination with B7-1 or B7-2. PBMCs (1.5 × 10⁵/well) from five healthy subjects and five patients with type 1 diabetes were stimulated with 10 μg/ml GAD65 in the presence or absence of each one of the blocking Ab’s or with different combinations of Ab’s. Ten replicates were established for each condition and harvested after 10–12 days of culture, and counts per minute per well were determined. Each column represents the mean percentage of inhibition of proliferation to GAD65 in the presence of single blocking Ab’s or combinations of them in five patients with recent onset of type 1 diabetes and five normal control subjects. As shown previously in Figures 2 and 3, the blockade of CD28, B7-1, or B7-2 caused strong inhibition of proliferation in normal subjects. (a and b) Blocking both B7-1 and B7-2 molecules at the same time induced a more significant decrease of the response than blocking just one of the molecules alone in both patients and controls. While anti-CD28 in combination with anti–B7-2 had almost no effect on proliferation or cytokine secretion (data not shown), anti-CD28 in combination with anti–B7-1 induced significant decreases of responses to GAD65. In contrast, coblockade with anti–CTLA-4 and anti–B7-1 enhanced T cell proliferation, while anti–CTLA-4 in combination with anti–B7-2 suppressed GAD-specific response. While anti-CD28 induced significantly more suppression of GAD responses in the normal subjects, the effects of combining the B7-1, B7-2, CD28, and CTLA-4 F(ab′) mAb’s were not different as compared with patients with diabetes.