Subcutaneous insulin B:9-23/IFA immunisation induces Tregs that control late-stage prediabetes in NOD mice through IL-10 and IFNgamma

Abstract

Aims/hypothesis: Subcutaneous immunisation with the 9-23 amino acid region of the insulin B chain (B:9-23) in incomplete Freund's adjuvant (IFA) can protect the majority of 4- to 6-week-old prediabetic NOD mice and is currently in clinical trials. Here we analysed the effect of B:9-23/IFA immunisation at later stages of the disease and the underlying mechanisms.

Methods: NOD mice were immunised once s.c. with B:9-23/IFA at 5 or 9 weeks of age, or when blood glucose reached 10 mmol/l or higher. Diabetes incidence was followed in addition to variables such as regulatory T cell (Treg) induction, cytokine production (analysed by Elispot) and emergence of pathogenic CD8(+)/NRP-V7(+) cells.

Results: A single B:9-23/IFA immunisation protected the majority of NOD mice at advanced stages of insulitis, but not after blood glucose reached 13.9 mmol/l. It increased Treg numbers and lost its protective effect after IFNgamma or IL-10 neutralisation, but not in the absence of IL-4. CD4(+)CD25(+) and to a lesser extent IFNgamma-producing cells from mice protected by B:9-23/IFA induced tolerance upon transfer into new NOD animals, indicating that a dominant Treg-mediated effect was operational. Reduced numbers of CD8(+)/NRP-V7(+) memory T cells coincided with protection from the disease.

Conclusions/interpretation: Protection from diabetes after B:9-23/IFA immunisation cannot be achieved once diabetes is fully established, but can be achieved at most prediabetic stages of the disease. Protection is mediated by Tregs that require IFNgamma and IL-10. These findings should provide important guidance for ongoing human trials, especially for the development of suitable T cell biomarkers.

Fig. 1

Insulin B:9-23/IFA treatment given at all prediabetic stages protects from diabetes. a Percentage of NOD mice developing diabetes after a single 100 μg B:9-23/IFA s.c. injection (blue line) at 9 weeks of age (n = 14) vs mice that were left untreated (black line, n = 14) or were treated with PBS/IFA (red line, n = 12). p < 0.001. b As above (a), but with immunisation at 5 weeks of age (n = 12 per group). All mice were monitored for blood glucose from 10 to >35 weeks of age. Each immunisation protocol was repeated in at least two independent experiments. p < 0.01 compared with non-treated mice. Red arrows, time of immunisation. c Individual blood glucose represented by differently coloured lines after B:9-23/IFA treatment at 9 weeks of age. One mouse that remained hyperglycaemic for more than three consecutive measurements was killed at 18 weeks of age. d Individual mice were treated with a single s.c. B:9-23/IFA injection when blood glucose was >10 mmol/l. Red triangles, individual entry blood glucose of mice progressing to diabetes; blue squares, entry blood glucose of mice that maintained normoglycaemia. Dashed line (c, d) indicates cut-off blood glucose value by which mice were considered diabetic

Fig. 2

Insulin B:9-23/IFA treatment decreases insulitis without enhancing beta cell proliferation. a Pancreatic sections from 12-week old normoglycaemic control and B:9-23/IFA-treated mice were stained for insulin/CD4 and scored for insulitis as described. Percentages represent the number of islets of a given score (white, score 0–1; light grey, score 2–3; dark grey, score 4–5) over the total number of islets examined; at least six islets were examined per mouse (four mice per group). p < 0.01 for B:9-23/IFA vs PBS/IFA. b Pancreatic histology. Sections were co-stained for insulin (blue) and CD8 or CD4 as indicated (both red) and analysed at 10× magnification. c Pancreatic sections from same mice as above (a, b) were stained for Ki-67 (green), insulin (blue) and CD3 (red) and analysed with confocal microscopy at 20× magnification. Representative Ki-67/insulin microphotographs showing the presence of Ki-67 cells within the pancreas with several degrees of insulitis (as shown) were analysed per group. White arrows, Ki-67⁺/Ins⁺ double-stained cells

Fig. 3

Insulin B:9-23/IFA treatment induces Tregs that protect from diabetes. a Treg (CD4⁺CD25⁺FOXP3⁺CD127^low) frequency in the PDLN, (b) spleen and (c) blood of NOD mice immunised with B:9-23/IFA (white bars) or PBS/IFA (grey bars) s.c. at 9 weeks of age or left untreated (black bars). Treg frequency in the blood was monitored for 3 consecutive weeks after immunisation (as indicated), whereas in PDLN and spleen Treg frequency was monitored for 2 consecutive weeks as shown. The experiment was repeated at least twice with three to four mice per group (ND, not done). p < 0.05 for PDLN at 12 weeks and p < 0.01 for spleen at 11 weeks of age when B:9-23/IFA is compared with controls. d Total Treg numbers in PDLN and (e) the spleen. The Treg percentage for each individual mouse was multiplied with its respective total spleen or PDLN lymphocyte number. Trypan blue staining was used to exclude dead cells. p < 0.01 for PDLN at 12 weeks, p < 0.05 and p < 0.01 for spleen at 12 and 11 weeks of age respectively (B:9-23/IFA vs controls). Mice analysed were those analysed above (a–c). f Representative flow cytometry plots as indicated in the PDLN and spleen at 12 weeks of age, indicating the CD25⁺FOXP3⁺ cells after gating on CD4⁺CD8⁻CD127^low lymphocytes. g, h CD8-depleted lymphocytes from B:9-23/IFA-treated and protected mice (>35 weeks) were pooled and cultured for 3 days in the presence of B:9-23 (10 μg/ml) and rhIL-2 (50 U/ml). Subsequently, CD4⁺CD25⁺ (Tregs) or CD4⁺CD25⁻ T cells were isolated and adoptively transferred to 8- to 9-week-old new prediabetic NOD animals. The CD4/CD25 profile of the cells prior to purification is shown above (g). h Diabetes incidence in NOD animals after receiving >1 × 10⁶ CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells i.v. Donor cells were derived from mice treated at either 5 or 9 weeks of age; CD25⁺ n = 8, CD25⁻ n = 14; p < 0.05. i Mean percentage and (j) total number of NRP-V7⁺CD8⁺ cells in the spleen after gating on live CD4⁻CD19⁻ lymphocytes from 12-week-old mice immunised at 9 weeks of age with B:9-23/IFA and control. p < 0.05

Fig. 4

Insulin B:9-23/IFA treatment induces IFNγ/IL-4 production in the spleen and IL-10/IL-4 in the PDLN. In B:9-23/IFA immunised mice (white bars), laboratory measurements showed an increase in IL-10 (p < 0.05) and IL-4 (p < 0.05) cytokine-producing cell numbers in the PDLN (a), in IFNγ in the blood (p < 0.05) (b), and in IFNγ and IL-4 in the spleen (p < 0.01 and p < 0.05 respectively) (c) vs no treatment (black bars) and PBS/IFA treatement (grey bars). In the spleen (d), the IFNγ:IL-17 ratio was increased more than tenfold (p < 0.01) The number of B:9-23-reactive CD4⁺ T cells producing IFNγ, IL-17, IL-4 and IL-10 in all secondary lymphoid organs analysed was calculated by deducting the number of spot-forming units (SFU) without stimulation from the number of SFU after B:9-23 re-stimulation. In each condition, 50 U/ml of rhIL-2 was added to the culture, which lasted for 3 days. In the spleen, Elispot using CD8-depleted lymphocytes was performed, whereas in the blood and PDLN, T cell-depleted splenocytes from age-matched non-treated NOD mice were added as APCs. The numbers of SFU without stimulation (background), which were subtracted to generate the experimental results for IFNγ, IL-17, IL-4 and IL-10, were: for spleen 80 ± 20, 150 ± 45, 115 ± 30 and 25 ± 15; for PDLN 43 ± 13, 75 ± 25, 48 ± 12 and 45 ± 15; and for blood: 35 ± 12, 75 ± 15, 60 ± 25 and 6 ± 5 respectively. The analysis shown is from 12-week-old immunised or non-treated NOD animals; similar results were obtained from two independent experiments with three mice per group. One representative experiment is shown

Fig. 5

The protective effect of B:9-23/IFA is mediated by IFNγ and IL-10, but not by IL-4 production. a Percentage of mice developing diabetes upon in vivo neutralisation of IFNγ (light grey line) or IL-10 (dark grey line) after s.c. treatment with insulin B:9-23/IFA of 9-week old NODs. Control, B:9-23/IFA treated with the isotype anti-rat IgG (black line) or PBS/IFA alone (dashed black line) are also depicted. Mice were treated for both neutralising antibodies with two intraperitoneal injections per week, at 10 and 11 weeks of age. Anti-IFNγ (clone XMG1.2), anti-IL-10 (clone JES5-2A5) or isotype control were given to mice (150 µg per animal per injection) i.p. The mice received a total of four antibody injections. b Diabetes progresses normally in Il-4 ^−/− NOD mice (light grey line), similarly to Il-4 ^−/− NOD mice treated with PBS/IFA (dashed black line) and IL4 wild type (WT) controls (black line). Upon B:9-23/IFA immunisation tolerance is achieved irrespective of endogenous IL-4 levels (black line). At least 12 mice were included in each experimental group. p < 0.01 compared with WT

Fig. 6

CD4⁺CD25⁺ insulin B:9-23/IFA-induced cells (Tregs) mediate protection against diabetes through IFNγ production. a Lymphocytes from protected B:9-23/IFA immunised mice were pooled and cultured for 3 days in the presence of B:9-23 (10 μg/ml) and rhIL-2 (50 U/ml). Subsequently, cells were stimulated for 4 h with plate-bound aCD3 (5 μg/ml) and soluble aCD28 (2.5 μg/ml), and assayed for cytokine production. The IL-10/IFNγ as well as the FOXP3/IFNγ profile after gating on the CD4⁺CD25⁺ and CD4⁺CD25⁻ populations is shown. The isotype control for both cytokines is also shown for the CD4⁺CD25⁺ population. b Upon adoptive transfer of CD4⁺CD25⁺ cells from protected mice, some (n = 6) received two anti-IFNγ (clone XMG1.2) neutralising antibody injections (150 μg/each), one at 10 and the other at 11 weeks of age. At the time of adoptive transfer, recipients were 8 to 9 weeks old. Total i.p. injections per recipient, n = 4. c Cells from above procedure (a) were sorted into CD4⁺IFNγ⁺ (black line) or CD4⁺IFNγ⁻ (grey line) populations as described. d Approximately 1 × 10⁶ of cells were adoptively transferred to 8- to 9-week-old prediabetic NOD mice and diabetes incidence was followed; n = 14 for IFNγ⁺ cells (grey line), n = 12 for no treatment (black line) and n = 10 for IFNγ⁻ cells (black dashed line)