Activation of Insulin-Reactive CD8 T-Cells for Development of Autoimmune Diabetes
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Figures
- FIG. 1.
Comparison of thymocytes and splenocytes from G9NOD transgenic mice and nontransgenic NOD mice. A–C: Cells from G9NOD transgenic mice. D–F: Cells from nontransgenic NOD mice. A and D: Thymocytes single-positive CD4 and anti-CD8. B and E: Splenocytes stained with anti-CD4 and anti-CD8. C and F: Splenocytes stained with anti-CD8 and a pool of anti-Vα2, -3, -8, and -11. The results are representative of 15 transgenic mice in more than five independent experiments. The mean (SD) values for the cell populations are as follows: A: CD4 3.46 (1.71); CD8 5.89 (2.28). B: CD4 17.38 (4.91); CD8 20.35 (3.77). C: CD8Vα2, -3, -8, -11 negative 20.0 (4.1); CD8Vα2, -3, -8, -11 positive 3.8 (1.9). D: CD4 10.2 (1.9); CD8 4.7 (0.7). E: CD4 40.63 (5.6); CD8 17.07(1.9). F: CD8Vα2, -3, -8, -11 negative 15.36 (1.03); CD8Vα2, -3, -8, -11 positive 1.85 (0.08).
- FIG. 2.
Phenotype of G9Cα−/−.NOD mice and G9RAG−/−.NOD mice. G9Cα−/−.NOD thymocytes (A), splenocytes (B), and PLN cells (C) were stained with anti-CD4 and -CD8. D: PLN cells were stained with anti-Vβ6 and CD8. E: Gated CD8 T-cells were stained with Kd-B15-23 (bold line) and control Kd-LLO 91–99 (dotted line) tetramer, respectively. G9RAG−/−.NOD thymocytes (F), splenocytes (G), and PLN cells (H) were stained with anti-CD4 and -CD8. I: PLN cells were stained with anti-Vβ6 and CD8. The results represent three independent experiments. The mean (SD) values for the distribution of cell populations are as follows: A: CD4 1.43 (0.42); CD8 5.1 (1.6). B: CD4 8.28 (1.35); CD8 21.14 (4.62). C: CD4 6.28 (0.57); CD8 52.18 (9.26). F: CD4 0.85 (0.44); CD8 2.93 (1.22). G: CD4 2.21 (2.16); CD8 33.42 (6.94). H: CD4 1.18 (0.66); CD8 68.7 (13.32).
- FIG. 3.
Functional responses of purified CD8 T-cells from G9Cα−/−.NOD mice. A: Cytotoxic assay of insulin-reactive CD8 T-cells directly ex vivo (○) from G9Cα−/−.NOD mice compared with precultured cells (●) in response to increasing B15-23 peptide concentrations at an E:T ratio of 10:1. B: Intracellular IFN-γ and TNF-α production in response to increasing concentrations of insulin B15-23 peptide after a 40-h culture. C: 3H-Thymidine incorporation proliferation assay of splenocytes from G9Cα−/−.NOD mice in response to B15-23 peptide and to influenza hemagglutinin (flu HA) control peptide. ●, Ins B15-23; ○, flu HA. D: 3H-Thymidine incorporation proliferation assay of splenocytes from G9Cα−/−.NOD mice in response to cross-presented insulin protein or control KLH. The results were from one of four independent experiments.
- FIG. 4.
The role of CD4 T-cells. A: 3H-Thymidine incorporation proliferation assay of CD8, CD4, and mixed CD8 and CD4 splenocytes from G9Cα−/−.NOD mice in response to B15-23 peptide G9Cα−/−. B: Adoptive transfer of purified CD4 T-cells of splenocytes from diabetic NOD intravenously into 4-week-old G9Cα−/−.NOD mice (n = 16). No diabetes occurred in mice adoptively transferred with CD4 T-cells purified from young NOD mice (n = 5). P < 0.018 when both groups were compared. The mice were tested weekly for glycosuria and diabetes confirmed by blood glucose of >13.9mmol/l.
- FIG. 5.
Activated CD8 T-cells from G9Cα−/−.NOD mice cause diabetes. A: G9Cα−/−.NOD mice were immunized with B15-23 peptide (50 μg) together with CpG oligonucleotides at different doses (two injections, 9–21 days apart). Diabetes was seen after the second injection in the majority of mice and the graph illustrates the mice, following the second injection, immunized with peptide plus 65 μg CpG (●), peptide plus 100 μg CpG (○), and peptide plus 195 μg CpG (▵). Two of four mice immunized with peptide, and high-dose CpG (195 μg) actually developed diabetes before the second injection and the remaining two mice are shown in the graph. None of the control mice injected with peptide alone (n = 11) or the various doses of CpG alone (n = 13) developed diabetes (data not shown). P < 0.0001 when all groups were compared. B: Flow cytometry of spleen cells from a nonimmunized G9Cα−/−.NOD mouse, gated on CD8 T-cells, shown in the thin line, compared with cells from a diabetic mouse after CpG and peptide immunization, shown in the thick line. The upper panel shows upregulation of CD44 and the lower panel illustrates downregulation of CD62L. C: Histology showing sections from diabetic G9Cα−/−.NOD mouse immunized with CpG and peptide (top row), CpG alone (middle row), and peptide alone (bottom row), stained with anti-CD8, anti-CD4, and anti-B220 (B-cells). (A high-quality representation of this figure is available in the online issue.)
- FIG. 6.
Adoptive transfer of purified activated G9Cα−/−.NOD CD8 T-cells into NOD.scid mice. G9Cα−/−.NOD CD8 T-cells were purified and cultured with matured bone marrow–derived dendritic cells together with 0.5 μg/ml insulin B15-23 peptide for 3 days. After washing, the cells were intravenously injected into 6-week-old NOD.scid mice (n = 8). Mice were tested daily for glycosuria and diabetes confirmed by blood glucose >13.9mmol/l.
- FIG. 7.
Diabetes incidence in G9Cα−/−.RIP-B7.1 NOD mice. G9Cα−/−.NOD mice that also expressed RIP-B7.1 transgene (●, n = 18) were compared with nontransgenic G9Cα−/−.NOD mice (○, n = 20) and observed for diabetes. Mice were tested weekly for glycosuria and diabetes confirmed by blood glucose >13.9 mmol/l (P < 0.0001).
- FIG. 8.
Insulin-reactive CD8 T-cells proliferate only in PLNs but not in the spleen, MLN, or ALN when adoptively transferred into NOD mice. CFSE-labeled purified G9Cα−/−.NOD CD8 T-cells were injected iv into 4-week-old NOD mice and the lymphoid tissue extracted and cells analyzed in PLNs (A) and spleen (B). The cells were gated on CD8 T-cells, and CFSE-labeled cells, costained with anti-CD69, are shown from PLNs (A) and spleen (B). Percentage proliferation of divided cells is shown after gating on CD8 T-cells from PLNs (C) and spleen (D). Proliferation in MLN and ALN was similar to spleen (data not shown). All groups were compared by ANOVA P = 0.014.
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