CD25⁺ T cells play a role in diabetes prevention in NOD mice. Female NOD mice were treated i.p. when 8, 11 and 15 days old with 1 mg of anti-CD25 or with the isotype control antibody. The incidence of diabetes in anti-CD25-treated mice (n = 11) was significantly higher than in either control antibody-treated (n = 13) (P < 0·0005, log rank analysis) or untreated female NOD mice (n =13) (P < 0·05, log rank analysis). Administration of isotype control antibody significantly reduced the incidence of diabetes compared to untreated NOD mice (P < 0·005, log rank analysis).

CD4⁺ CD25⁺ T cells from the spleens of either 7-week-old, 16-week-old or diabetic female NOD mice can suppress proliferation of their own splenic CD4⁺ CD25^– T cells in vitro. (a) 1 × 10⁵ CFSE-labelled splenic CD4⁺ CD25^– T effector cells together with 5 × 10⁵ irradiated, CD4-depleted splenocytes and anti-CD3 (1 μg/ml) were cultured with either no CD4⁺ CD25⁺ cells (0 : 1 ratio) or with various numbers of splenic CD4⁺ CD25⁺ T cells from the same mouse ranging from 1 × 10⁵ (1 : 1 ratio) to 1·25 × 10⁴ (1 : 8 ratio) for 72 hr. Representative FACS histogram plots from triplicate wells of CFSE and CD4⁺ cells show a marked reduction of effector proliferation in 7-week (i), 16-week (ii) and diabetic (iii) NOD mice when regulatory T cells are added at a 1 : 1 ratio compared to effectors alone. The suppressive effect of the CD4⁺ CD25⁺ T cells was reduced as the ratio of CD4⁺ CD25⁺ T cells to CD4⁺ CD25^– T cells decreased. (b) The ability of NOD CD4⁺ CD25⁺ T cells from both diabetic and non-diabetic mice to suppress the proliferation of CD4⁺ CD25^– T cells from the same individual was also demonstrated by repeating the above assay using thymidine incorporation as the measure of proliferation. 5 × 10⁴ splenic CD4⁺ CD25^– T effector cells together with 2·5 × 10⁵ irradiated, CD4-depleted splenocytes and anti-CD3 (1 μg/ml) were cultured with either no CD4⁺ CD25⁺ cells (0 : 1 ratio) or various numbers of splenic CD4⁺ CD25⁺ T cells from the same mouse ranging from 5 × 10⁴ (1 : 1 ratio) to 6·25 × 10³ (1 : 8 ratio) for 72 hr. The addition of CD4⁺ CD25⁺ T cells at Treg : T effector ratios of 1 : 1 and 1 : 2 caused a significant reduction in proliferation in cocultures of Tregs and T effectors from 7-week-old non-diabetic mice (i), 16-week-old non-diabetic mice (ii) and diabetic NOD mice (iii). Thymidine incorporation was less than 5·0 × 10² c.p.m. in all triplicates of 5 × 10⁴ CD4⁺ CD25^– T cells cultured alone and in wells with irradiated splenocytes with anti-CD3 alone. (c) IFN-γ was measured in the supernatants from the experiment described in (a). The addition of CD4⁺ CD25⁺ cells to their own CD4⁺ CD25^– cells caused a significant reduction in IFN-γ production in 7-week-old (i), 16-week-old (ii) and diabetic (iii) NOD mice at all four dilutions of Tregs compared to T effectors cultured with irradiated APCs and anti-CD3 (Student t-test, P < 0·05 in all cases).

CD4⁺ CD25⁺ T cells from the PLNs of 5-week-old female NOD mice and diabetic female NOD mice can suppress the proliferation of CD4⁺ CD25^– T cells from PLNs in vitro. (a) 2·5 × 10⁴ CFSE-labelled CD4⁺ CD25^– T effector cells from a pool of PLNs from either 5-week-old (i) or diabetic (ii) donor NOD donors together with 1·25 × 10⁵ of irradiated, CD4-depleted splenocytes and anti-CD3 (1 μg/ml) were cultured with either no CD4⁺ CD25⁺ cells (0 : 1 ratio) or various numbers of their own PLN CD4⁺ CD25⁺ cells ranging from 2·5 × 10⁴ (1 : 1 ratio) to 3·125 × 10³ (1 : 8 ratio). Representative FACS histogram plots from triplicate wells of CFSE and CD4⁺ cells show a marked reduction of effector T-cell proliferation when regulatory T cells are added at a 1 : 1 ratio compared to effectors alone. The suppressive effect of the CD4⁺ CD25⁺ T cells was gradually reduced as the ratio of CD4⁺ CD25⁺ T cells to CD4⁺ CD25^– T cells decreased in both regulatory T-cell groups. (b) 1 × 10⁵ CFSE-labelled CD4⁺ CD25^– T effector cells from a pool of PLNs from diabetic NOD donors together with 5 × 10⁵ irradiated, CD4⁺-depleted diabetic splenocytes and anti-CD3 (1 μg/ml) were cultured with either no CD4⁺ CD25⁺ cells (0 : 1 ratio) or various numbers of PLNs CD4⁺ CD25⁺ cells ranging from 1 × 10⁵ (1 : 1 ratio) to 1·25 × 10⁴ (1 : 8 ratio) from either 11-week-old (i) or diabetic NOD (ii) mice. Representative FACS histogram plots from triplicate wells of CFSE and CD4⁺ cells show a marked reduction of effector T-cell proliferation when CD4⁺ CD25⁺ T cells are added at a 1 : 1 ratio compared to effectors alone. The suppressive effect of the CD4⁺ CD25⁺ T cells was gradually reduced as the ratio of CD4⁺ CD25⁺ T cells to CD4⁺ CD25^– T cells decreased in both regulatory T-cell groups.

Onset of diabetes in NOD.scid mice is faster when recipients are given splenocytes from diabetic NOD donors and anti-CD25. (a) 2 × 10⁷ splenocytes from diabetic female NOD donors were injected i.v. into 7-week-old male NOD.scid mice together with either 2 mg anti-CD25 antibody i.p., 2 mg isotype control antibody i.p. or 200 μl PBS i.p. Recipient NOD.scid mice were tested for the presence of urinary glucose. Onset of diabetes was faster in recipients treated with anti-CD25 compared with isotype control antibody or PBS (log rank analysis, P < 0·05 in both cases). There was no significant difference in the incidence of diabetes between PBS-treated and control antibody-treated recipients (log rank analysis, P = 0·06). (b) 5 × 10⁶ T cells or 5 × 10⁶ T cells depleted of CD25⁺ cells from diabetic females NOD mice were injected i.v. into 8-week-old male NOD.scid mice. Onset of diabetes was faster in recipients given CD25-depleted T cells than in those given non-depleted T cells (log rank analysis, P < 0·05).

When using CD4 and CD25 expression to purify T regs, the phenotype of the contaminating CD25^– population is different in diabetic NOD mice. (a) CD4⁺ CD25⁺ T cells from individual NOD mice which were either 6 weeks old or diabetic were purified on MACS columns and the proportion of cells which were CD25⁺ or Foxp3⁺ post purification was analysed by FACS. There was no significant difference in the proportion of cells expressing CD25 (i) or Foxp3 (ii) post MACS CD4⁺ CD25⁺ purification from 6-week-old mice compared to diabetic NOD mice. (b) As the MACS purification method could not achieve a totally pure population of CD25⁺ T cells, the phenotype of the contaminating CD25^– population was analysed by costaining with CD44 or CD62L. The proportion of CD25^– cells which were CD62L^lo (i) and CD44^hi (ii) was significantly higher in diabetic mice.

There is no age-related decline in the proportion of Tregs in the spleen, PLNs or pancreas. Single-cell suspensions from the spleen and PLNs of female NOD mice were costained for CD4 and CD25 and/or CD4 and Foxp3. The proportions of CD4⁺ T cells which expressed CD25 (i) and expressed Foxp3 (ii) were calculated as a percentage for the spleen (a), PLNs (b) and pancreas (c). (a) There was no significant correlation between age and proportion of CD4⁺ T cells expressing CD25 and expressing Foxp3 in non-diabetic NOD mice in the spleen (CD25 r = 0·14, P = 0·23; Foxp3 r = 0·27, P = 0·19, Spearman rank correlation). The proportion of CD4⁺ cells that expressed CD25 in diabetic mice was not significantly different to that in non-diabetic mice (Mann–Whitney U-test, P = 0·64). Similarly, the proportion of CD4⁺ cells that expressed Foxp3 was not significantly different in diabetic NOD mice compared to non-diabetic NOD mice (Mann–Whitney U-test, P = 0·15). (b) There was no significant correlation between age and proportion of CD4⁺ T cells expressing CD25 in non-diabetic NOD mice in the PLNs (r = 0·24, P = 0·09, Spearman rank correlation). There was a small but significant positive correlation between age and the proportion of CD4⁺ T cells that expressed Foxp3 in the PLNs of non-diabetic mice (r = 0·48, P < 0·05, Spearman rank correlation). A significantly higher proportion of CD4⁺ cells expressed CD25 in diabetic mice compared to in all non-diabetic mice (Mann–Whitney U-test, P < 0·001). The proportion of CD4⁺ T cells that expressed Foxp3 was not significantly different in diabetic NOD mice compared to non-diabetic NOD mice (Mann–Whitney U-test, P =0·08). (c) The proportion of CD4⁺ T cells expressing CD25 in 11-week-old, non-diabetic mice was not significantly different from that in diabetic NOD mice (Mann–Whitney U-test, P = 0·11). There was no significant difference between the proportion of CD4⁺ T cells expressing Foxp3 in 11-week-old non-diabetic mice compared to diabetic NOD mice (Mann–Whitney U-test, P = 0·58).

CD4⁺ CD25⁺ T cells from the spleens of both 6-week, 12-week and diabetic female NOD mice can suppress CD4⁺ CD25^– T cells from diabetic NOD female mice in vitro. 1 × 10⁵ CFSE-labelled splenic CD4⁺ CD25^– T effector cells from a diabetic donor together with 5 × 10⁵ irradiated, CD4-depleted splenocytes from the same diabetic donor and anti-CD3 (1 μg/ml) were cultured with either no CD4⁺ CD25⁺ cells (0 : 1 ratio) or with various numbers of splenic CD4⁺ CD25⁺ cells ranging from 1 × 10⁵ (1 : 1 ratio) to 1·25 × 10⁴ (1 : 8 ratio) from either a 6-week-old (a), 12-week-old (b) or diabetic (c) NOD donor. Representative FACS histogram plots from triplicate wells of CFSE and CD4⁺ cells show a marked reduction of diabetic effector T-cell proliferation when CD4⁺ CD25⁺ T cells are added at a 1 : 1 ratio compared to effectors alone irrespective of the source of the CD4⁺ CD25⁺ T cells. The suppressive effect of the CD4⁺ CD25⁺ T cells was gradually reduced as the ratio of CD4⁺ CD25⁺ T cells to CD4⁺ CD25^– T cells decreased in all three regulatory T-cell groups.

Pretreatment with anti-CD25 antibody increases BDC2.5 NOD T-cell proliferation in the PLNs of both 7-week-old and 18-week-old NOD mice. The 7- or 18-week-old NOD mice were injected with 2 mg anti-CD25 or isotype control antibody i.p. and 7 days later 1 × 10⁷ CFSE-labelled BDC2.5 NOD T cells were transferred i.v. Pancreatic lymph nodes were harvested 72 hr later and BDC2.5 NOD T-cell proliferation was measured by assessing CFSE dilution on Vβ4⁺ cells. (a) Representative histograms show CFSE staining gated on Vβ4⁺ T cells in NOD PLNs pretreated with (i) isotype control antibody and (ii) anti-CD25 antibody. (iii) There is a significant difference between the percentage of BDC2.5 T cells which have divided in the PLNs of NOD mice pretreated with control antibody (n = 12) compared to the PLNs of NOD mice pretreated with anti-CD25 antibody (n = 13) (P < 0·0001 Mann–Whitney U-test). (b) The same experiment was repeated using non-diabetic 18-week-old NOD female mice. Representative histograms show CFSE staining gated on Vβ4⁺ T cells in the PLNs of NOD mice pretreated with (i) the control antibody and (ii) the anti-CD25 antibody. (iii) There is a significant difference between the percentage of BDC2.5 NOD T cells which have divided in the PLNs of NOD mice pretreated with isotype control antibody (n = 7) compared to the PLNs of NOD mice pretreated with anti-CD25 antibody (n = 7) (P < 0·05, Mann–Whitney U-test). (c) NOD mice were injected with 2 mg anti-CD25 antibody or control antibody and the proportion of CD4⁺ T cells in the spleen and PLNs which express Foxp3 was assessed by FACS analysis. Treatment with anti-CD25 caused a significant, although not absolute, decrease in the proportion of CD4⁺ T cells expressing Foxp3.

CD4⁺ CD25⁺ cells from 6-week-old female NOD mice but not from diabetic NOD mice can delay, although not prevent, the transfer of disease when cotransferred with splenocytes from diabetic NOD donors into NOD.scid recipients. (a) 1 × 10⁷ splenocytes from diabetic NOD donors were cotransferred into NOD.scid recipients either alone or with 5 × 10⁵ CD4⁺ CD25⁺ T cells, 1 × 10⁶ CD4⁺ CD25⁺ T cells or 5 × 10⁵ CD4⁺ CD25^– T cells from 6-week-old female, non-diabetic NOD donors. At day 48 post transfer, there was a significant delay in the onset of diabetes between the NOD.scid recipients administered diabetic splenocytes alone compared to recipients given diabetic splenocytes and 1 × 10⁶ CD4⁺ CD25⁺ T cells (log rank analysis, P < 0·05). However, by day 70 there was no statistically significant difference in the onset of diabetes between the diabetic splenocytes alone and 5 × 10⁵ CD4⁺ CD25⁺ T cells (log rank analysis, P = 0·86), diabetic splenocytes and 1 × 10⁶ CD4⁺ CD25⁺ T cells (log rank analysis, P = 0·30) or diabetic splenocytes and 5 × 10⁵ CD4⁺ CD25^– T cells (log rank analysis, P = 0·56). (b) 1 × 10⁷ diabetic splenocytes were cotransferred into NOD.scid recipients either alone or with 5 × 10⁵ CD4⁺ CD25⁺ T cells, 1 × 10⁶ CD4⁺ CD25⁺ T cells or 5 × 10⁵ CD4⁺ CD25^– T cells from diabetic female NOD donors. There was no statistically significant difference in the onset of diabetes between the diabetic splenocytes alone and 5 × 10⁵ CD4⁺ CD25⁺ T cells (log rank analysis, P = 0·69), diabetic splenocytes and 1 × 10⁶ CD4⁺ CD25⁺ T cells (log rank analysis, P = 0·43). The onset of diabetes was faster in NOD.scid recipients given diabetic splenocytes and 5 × 10⁵ CD4⁺ CD25^– T cells compared to diabetic splenocytes alone (log rank analysis, P < 0·05).