Article Figures & Data
Figures
- FIGURE 1.
Dose response kinetics for a given test Ag were initially established in at least 20 patients each. Proinsulin was added (top panels) in the concentrations indicated to cultures of PBMC from 24 patients; 13 of the 24 showed positive (upper left) and 11 no responses (lower left). Positive responses were normalized by calculating each data point as a percentage of maximum for a given donor (upper right panel, heavy line: group mean). Responses (lower panels) to IA-2, ICA69, BSA, and the GAD65, Tep69, and ABBOS peptides were similarly normalized and plotted.
- FIGURE 2.
Blind duplicate series. Blood samples from 7 patients and 5 FDRs were split into two aliquots, each labeled with a different ID and sent to the Toronto assay lab without announcement. Each sample gave 96 replicate responses to the various test and control Ags. Data (mean cpm [3H]TdR) from set 1 and 2 are plotted on x- and y-axes, respectively; many points overlap.
- FIGURE 3.
T cell responses (mean SI) to diabetes-related test Ags in newly diagnosed patients (n = 148), age- and MHC class II (DQ)-matched siblings of index cases with a low risk of developing diabetes (n = 51), and in unrelated, healthy general population controls (n = 40).
- FIGURE 4.
T cell responses to the Ags indicated in 148 patients with recent onset IDDM. Data are presented as overall means + 1 SD, the latter reflecting mainly the distribution of response amplitudes in this cohort. White bars, no IL-2 added; black bars, IL-2 added. ∗, IL-2 effect; p < 0.0001.
- FIGURE 5.
Persistence of T cell autoreactivity past onset of IDDM. A, Of the study index cases, 109 were followed prospectively for T cell responses every 2–4 mo. The prevalence (%) of positive responses is shown. Insert, Stability of T cell responses in each patient. Two thirds show the same response patterns throughout the follow-up period; some gain or lose one or more responses. B, T cell responses (SI) in 31 patients with longstanding (9.2 ± 5.1, range 3–26 yr) IDDM. Not all responses could be measured in all patients.
- FIGURE 6.
Apoptosis resistance in patients, siblings, and controls. Lymphocytes were activated with anti-CD3 Ab, and 0–100 μg/ml of anti-IL-2 was added. The graph indicates the amount of IL-2 required for a 50% reduction in cell survival. Cell death was measured through BCECF catalysis. [3H]TdR incorporation gave similar results in parallel cultures.
Tables
Allele Sibs (%) Patients (%) p DQB1*0201/0302 26 37 0.47 DQB1*0302/not 201 26 19 0.57 DQB1*0201/not 302 30 34 >0.9 DQB1*0602/x 5 2 >0.9 Other 13 8 0.36 Ag Description/Source Derivation, Comments PI Lilly Pharm. E. coli, full-length* IA-2 Bayer Pharm. E. coli, “ICA512”* GAD65 Peptide 524 SRLSKVAPVIKARMMEYGTT* HSP60 Peptide 277 VLGGGCALLRCIPALDSLTPANED* ICA69 β-isoform E. coli* α-isoform Baculovirus† ICA69-36 “Tep69” AFIKATGKKEDE, homolog BSA-148* ICA69-202 KNFDKLKMDVCQ, homolog BSA-394† ICA69-350 SEEGACLGPVAG, homolog BSA-193† BSA Sigma Fraction V* BSA-148 “ABBOS” FKADEKKFWGKYL, homolog ICA69-36* BSA-394 TSVFDKLKHLVD, homolog ICA69-202† BSA-193 EDKGACLLPKIE, homolog ICA69-350† Tetanus toxoid PMC Canada Pharmaceutical grade* OVA Sigma Fraction V* OVA152 EYQDNRVSFLGHFI* Human hemoglob. Sigma Purified† Human cytochr. c Sigma Purified† Human actin Sigma Purified† -
a Description of the test antigens/peptides used, not all could be tested in all study subjects. Eleven Ags (marked with an asterisk) were used routinely; the remainder (marked with a dagger) were tested only in subsets of probands to answer specific questions. Peptide names indicate origin and N-terminal amino acid position. Residues printed in bold identify homologies described in the text.
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Ag Normals (%) (n = 40) Low Risk Sibs (%) (n = 51) IDDM (%) (n = 148) Prevalence IDDM vs Sibs Tetanus toxoid 90.3 (7.8 ± 1.9) 100 (5.4 ± 1.6) 93.8 (5.8 ± 2.1) p = 0.63 PI 2.4 35 (2.3 ± 0.67) 79 (2.7 ± 1.36) p < 0.0001 IA-2/ICA512 0 32 (3.1 ± 0.76) 78 (2.1 ± 0.44) p < 0.0001 ICA69 4.9 (1.8 ± 0.16) 29 (1.9 ± 0.25) 62 (2.3 ± 0.54) p < 0.0001 Tep69 0 22 (1.9 ± 0.37) 51 (2 ± 0.62) p < 0.0001 BSA 9.6 (1.9 ± 0.6) 16 (1.7 ± 0.29) 55 (2 ± 0.51) p < 0.0001 ABBOS 2.4 24 (1.9 ± 0.31) 62 (2.1 ± 0.54) p < 0.0001 GAD65 4.9 (2.3 ± 1.12) 31 (1.9 ± 0.39) 55 (2.3 ± 0.61) p < 0.0001 HSP60 2.4 8 (1.9 ± 0.43) 16 (2.1 ± 1) 0.09 OVA 0 0 0 Other controlsb 0 0 0 -
a Prevalence (%) and amplitudes (mean ± SD) of positive proliferative responses to test Ags.
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b Other control Ags (human hemoglobin, cytochrome c, actin) were tested only in 9 normals, 14 sibs, and 27–36 patients.
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Medium ICA69 Tep69 GAD65 OVA IDDM Medium 1129 IL2 2436 5572 5103 4534 2216 ICA69 1382 1171 Tep69 1440 1420 1295 GAD65 1479 1268 1266 1184 OVA 1289 1098 1306 1240 1105 PI 2980 9431 8181 7860 3113 IA2 3244 7864 7717 7389 3052 Tetanus 5879 12204 11640 11137 5412 Sib Medium 1224 IL2 1998 2403 2218 4584 2149 ICA69 1382 1234 Tep69 1440 1186 1261 GAD65 1379 1322 1468 1244 OVA 1088 1077 1209 1310 1187 PI 1330 1573 1410 1461 1259 IA2 1442 1242 1365 1348 1086 Tetanus 6430 6653 6591 8927 6317 -
a Rescue of anergic T cells by costimulation with the “non-anergic” diabetes-associated Ags, PI or IA2 or with tetanus toxoid. Mean thymidine incorporation (cpm) in cultures stimulated as indicated. Redundant entries are blank. SD were within ±10%. Thirteen additional patients and four sibs were tested with similar results.
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No. of Patients (%) No. of Sibs (%) ABBOS+/ICA69+ 91 (99) 9 (64) ABBOS+/ICA69− 1 (1) 5 (36) -
a Comparison of patients and siblings with positive responses to the ABBOS mimicry epitope in BSA. p = 0.0001; RR = 32.8.
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