In Vivo Control of Diabetogenic T-Cells by Regulatory CD4+CD25+ T-Cells Expressing Foxp3
Article Figures & Tables
Figures
- FIG. 1.
Prevention of accelerated diabetes by prior transfer of unpurified leukocytes. DP-BB rats (age 26–33 days) received 10 × 106 diabetogenic T-cells (D); one group (n = 34) received no protective transfer. Unpurified protective cells containing 64 × 106 T-cells (P) significantly affected diabetes onset compared with no protective cells when protective cells were transferred 2 (n = 8; P < 0.05), 3 (n = 4; P < 0.002), or 4 (n = 10; P < 0.0001) days before transfer of diabetogenic cells. Data are pooled from four experiments. The dashed line (n = 491) is the spontaneous diabetes incidence in DP-BB rats reared within the same room at the same time.
- FIG. 2.
Tracking of CTO-labeled diabetogenic T-cells and CFDA-SE−labeled protective CD4+ T-cells after transfer. A: Ratio of diabetogenic donor T-cells to recipient T-cells on day 2 or 4 after transfer of diabetogenic cells (n = 6). *P < 0.05 vs. cLNs and spleen; †P < 0.05 vs. mLNs, cLNs, and spleen. B: Representative fluorescence-activated cell sorter (FACS) dot plot (of n = 9) displaying all live T-cells in panLNs 2 days after transfer of diabetogenic cells. Recipient T-cells were double negative. C: Representative FACS dot plot (of n = 3) from recipient that had received CD4+ protective T-cells 7 days earlier.
- FIG. 3.
Prevention of accelerated diabetes by CD25+ and CD25− subsets. Rats received 3 × 106 diabetes-inducing T-cells. A: Transfer 4 days before. Purified CD4+CD25+ or CD4+CD25− T-cells significantly affected diabetes onset (P < 0.0004 and P < 0.006, respectively) when transferred to prediabetic DP-BB rats age 29–33 days. The rats received no protective transfer (n = 8), 20 × 106 CD4+CD25− T-cells (n = 8), or 2 × 106 CD4+CD25+ T-cells (n = 6). Data are pooled from two experiments. B: Simultaneous transfer. Purified CD4+CD25+ T-cells significantly affected diabetes onset (P < 0.001), whereas CD4+CD25− T-cells did not (NS) when transferred to prediabetic DP-BB rats age 27–29 days. The rats received no protective transfer (n = 9), 20 × 106 CD4+CD25− T-cells (n = 11), or 2 × 106 CD4+CD25+ T-cells (n = 12). Data are pooled from two experiments.
- FIG. 4.
Prevention of spontaneous diabetes by CD25+ and CD25− subsets. DP-BB rats (n = 52) received protective transfers of purified CD4+CD25+ or CD4+CD25− T-cells. Data are pooled from three experiments. The dashed line (n = 491) is the spontaneous diabetes incidence in DP-BB rats reared within the same room at the same time. A: Transfer of protective subsets at age 27 ± 2 days. Transfer of 2 × 106 CD4+CD25+ T-cells (n = 14; P < 0.0001) and 20 × 106 CD4+CD25− T-cells (n = 18; P < 0.0001) significantly affected diabetes onset compared with no transfer. There was no significant difference between the two test groups. B: Transfer of protective subsets at age 42 days. Transfer of 2 × 106 CD4+CD25+ T-cells (n = 10; P < 0.0001) and 20 × 106 CD4+CD25− T-cells (n = 10; P < 0.0004) significantly affected diabetes onset compared with no transfer. There was a significant difference between the two test groups (P < 0.004).
- FIG. 5.
Semiquantitative RT-PCR of rat Foxp3 in CD4+CD25+ or CD4+CD25− subsets. Data are means + SD of four different sets of purified cells (each set tested in triplicate).
Tables
- TABLE 1
Accelerated diabetes after transfer of purified, diabetogenic T-cells
No transfer 3 × 106 cells 5 × 106 cells 10 × 106 cells 23 × 106 cells Diabetes incidence 455 of 491 (93) 18 of 18 (100) 30 of 30 (100) 32 of 34 (94) 3 of 3 (100) Median age (days) at diabetes onset 76 (72–81)* 58 (56–61)† 52 (50–54)‡ 50 (46–53)§ 47 Data are n (%) or median (interquartile range) and were pooled from 14 experiments.
- *
* P < 0.0001 vs. 3, 5, 10, and 23 × 106 cells;
- †
† P < 0.0004 vs. 5 × 106 cells, P < 0.0023 vs. 10 × 106 cells, and P < 0.0001 vs. 23 × 106 cells;
- ‡
‡ P < 0.0001 vs. 23 × 106 cells, but NS vs. 10 × 106 cells;
- §
§ NS vs. 23 × 106 cells.
- TABLE 2
Prevention of accelerated diabetes by prior transfer of pure CD4+ T cells
Group Diabetogenic T-cells (×106) Protective CD4+ T-cells (×106) Rats Rats rendered diabetic before age 120 days Degree of protection against diabetes (%) Age at diabetes onset (days) 1A 5 None 12 12 0* 52 (49–53) 1B None 43 10 0 100 — 1C 5 43 11 1 91 82 1D 5 21.5 2 0 100 — 2A 3 None 10 10 0† 58 (58–59) 2B 3 21.5 3 0 100 — 2C 3 10 6 1 83 109 Data are median (interquartile range) unless otherwise indicated. DP-BB rats (age 32–35 days) received CD4+ T-cells in the indicated amounts. After 4 days, diabetes-inducing T-cells were transferred in the indicated amounts. Data are pooled from four experiments.
- *
* P < 0.0001 vs. groups 1B and 1C, P < 0.0175 vs. group 1D;
- †
† P < 0.0045 vs. group 2B, P < 0.0003 vs. group 2C.
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