Coexpression of CD25 and OX40 (CD134) Receptors Delineates Autoreactive T-cells in Type 1 Diabetes

Characterization of long-term T-cell lines. A: GAD65 responses of T-cell lines derived from HLA-DRB1*0401–positive control subjects (CP) and type 1 diabetic patients. B: FACS analysis of surface marker expression of GAD65-specific T-cell line (TCL) after being stimulated with corresponding peptides. C: Insulin- and proinsulin-specific responses of two T-cell lines from an HLA-DRB1*0401–positive type 1 diabetic patient. D: Surface phenotype of proinsulin-specific T-cell line after being stimulated with corresponding peptides. The incorporation of radioactive thymidine is shown in A and C. The ratio of mean fluorescence intensities of T-cell blasts versus those of resting T-cell cluster are indicated in B. D: The percentage of T-cells expressing the indicated activation markers after being stimulated with corresponding peptides divided by the percentage of activation marker–positive T-cells after being stimulated with medium alone is given. MFI, mean fluorescence intensity.

Surface marker analysis of stimulated PBMCs. A: Marker expression of type 1 diabetic patient T1D-7 and healthy control subject CP-10 after the PBMCs were stimulated with GAD65 peptides. Lymphocytes pregated for CD3 expression were further analyzed for the expression of CD45RA and the activation markers CD25 and CD134. Shown is the percentage of memory (CD45RA⁻) and naïve (CD45RA⁺) T-cells expressing CD25 alone (green dots) or in combination with CD134 (red dots). Peptide-reactive T-cells of the type 1 diabetic patient were derived mainly from the CD45RA⁻ memory T-cell cluster and were of a predominant CD25⁺CD134⁺ double-positive phenotype. T-cells of the healthy control subject upregulated CD25 only. Stimulation with the recall antigen TT induced double-positive T-cells in both the patient and the control subject. B: Activation marker analysis of a twin pair discordant for type 1 diabetes. Depicted are the activation marker–positive memory T-cells against control antisense peptide p256a and GAD65 peptide p266.

Prevalence of CD25⁺CD134⁺ double-positive memory T-cells in PBMCs of type 1 diabetic (T1D) patients and healthy control subjects. CD25⁺CD134⁺ memory T-cell responses against GAD65 peptides in pre–type 1 diabetic and type 1 diabetic probands (A) and healthy control subjects (B) and against preproinsulin/insulin proteins and peptides in pre–type 1 diabetic and type 1 diabetic probands (C) and type 2 diabetic patients and healthy control subjects (D). Responses reaching the cutoff level ≥0.3% are shown in red (see Fig. 3). In terms of GAD65 peptide stimulation, two pre-diabetic individuals and 9 of 12 type 1 diabetic patients responded with dual-activation marker expression, recognizing several GAD65 peptides (P < 0.01 vs. control subjects). After PBMCs were stimulated with PPI antigen/peptides, one of three pre-diabetic individuals and 18 of 20 type 1 diabetic patients showed coexpression of CD25⁺CD134⁺ (P < 0.0001 for patients vs. control subjects). Also shown is the frequency of CD25⁺ (E) and combined CD25⁺CD134⁺ (F) memory T-cells of all study subjects against that of peptide libraries derived from both autoantigens (17 GAD65 and/or 24 proinsulin stimuli). Using the cutoff value, the number of cultures with CD25 single-positive cells did not differ significantly between type 1 diabetic patients and control subjects, whereas the frequency of CD25⁺CD134⁺ T-cells differed significantly between cultures testing pre-diabetic and control PBMCs (P < 0.01) and PBMCs of type 1 diabetic patients and control subjects (P < 0.001).