Assessing the effectiveness of Interleukin-2 therapy in experimental type 1 diabetes

Abstract

Aim: Much focus of immunotherapy for type 1 diabetes (T1D) has been devoted on selectively boosting regulatory T (Treg) cells using low dose IL-2 due to their constitutive expression of IL-2Rα, CD25. However, several clinical trials using a low dose of IL-2 only showed a limited improvement of metabolic control. It can therefore be hypothesized that further decreasing IL-2 dosage may increase the selective responsiveness of Treg cells.

Methods: We induced experimental T1D using multiple low dose streptozotocin (STZ) injections and treated the mice with an ultra-low dose IL-2 (uIL-2, approximately 7-fold lower than low dose). Immune response was studied using multicolor flow cytometry.

Results: We found that uIL-2 did not protect STZ mice from developing hyperglycemia. It did neither increase Treg cell proportions, nor did it correct the phenotypic shift of Treg cells seen in T1D. It only partially decreased the proportion of IFN-γ⁺ T cells. Likewise, uIL-2 also did not protect the dysfunction of regulatory B (Breg) cells. Strikingly, when administered in combination with an anti-inflammatory cytokine IL-35, uIL-2 abrogated IL-35's protective effect. Low dose IL-2, on the other hand, protected half of the STZ mice from developing hyperglycemia. No difference was found in the Treg and Breg response, and it only tended to decrease CD80 expression in macrophages and dendritic cells.

Conclusion: In conclusion, further decreasing IL-2 dosage may not be a suitable approach for T1D therapy, and the limited success suggests that an alternative low dose IL-2 therapy strategy or other immunotherapies should be considered.

Keywords: Interleukin-2 (IL-2); Regulatory B (Breg) cells; Regulatory T (Treg) cells; Streptozotocin; Type 1 diabetes (T1D).

Fig. 1

Ultra-low dose IL-2 does not prevent hyperglycemia in STZ mice. Male CD-1 mice received saline or STZ injection for 5 consecutive days, and PBS, uIL-2 or uIL-2 + IL-35 for the next 8 days. Blood glucose was monitored daily after day 5. A Blood glucose levels of mice. Repeated two-way ANOVA followed by Tukey’s test was performed, asterisks show the difference between Vehicle (n = 6) and STZ (n = 6), uIL-2 (n = 4), and uIL-2 + IL-35 (n = 6). B Percentage of diabetes free mice. Log-rank test followed by Bonferroni correction was performed. C Insulitis scores of the mice. Kruskal-Wallis test followed by Dunn’s test was used. Results are shown as mean ± SEM. The experiment was performed twice with n = 2 or 3 for each group each time. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 respectively

Fig. 2

Ultra-low dose IL-2 does not prevent the phenotypic shift of Treg cells in STZ mice. Male CD-1 mice received saline or STZ injection for 5 consecutive days, and PBS, uIL-2 or uIL-2 + IL-35 for the next 8 days. A CD4⁺CD25⁺Foxp3⁺ Treg cell proportions. B Proportions of IL-35⁺ cells among Treg cells. C Proportions of IFN-γ⁺ cells among Treg cells. One-way ANOVA followed by Tukey’s test was performed. Results are shown as mean ± SEM. The experiment was performed twice with n = 2 or 3 for each group each time. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 respectively

Fig. 3

Ultra-low dose IL-2 partially lowers IFN-γ production in T cells. Male CD-1 mice received saline or STZ injection for 5 consecutive days, and PBS, uIL-2 or uIL-2 + IL-35 for the next 8 days. A Proportions of CD4⁺CD25⁻ T cells. B Proportions of IFN-γ⁺ cells among CD4⁺CD25⁻ T cells. C Proportions of CD8⁺ T cells. D Proportions of IFN-γ⁺ cells among CD8⁺ T cells. One-way ANOVA followed by Tukey’s test was performed. Results are shown as mean ± SEM. The experiment was performed twice with n = 2 or 3 for each group each time. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 respectively

Fig. 4

Ultra-low dose IL-2 does not prevent Breg cell dysfunction in STZ mice. Male CD-1 mice received saline or STZ injection for 5 consecutive days, and PBS, uIL-2 or uIL-2 + IL-35 for the next 8 days. A Proportions of CD19⁺CD1d⁺CD5⁺ Breg cells. B Proportions of IL-35⁺ cells among Breg cells. C Proportions of IL-10⁺ cells among Breg cells. One-way ANOVA followed by Tukey’s test was performed. Results are shown as mean ± SEM. The experiment was performed twice with n = 2 or 3 for each group each time. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 respectively

Fig. 5

Low dose IL-2 partially protects STZ mice from hyperglycemia. Male CD-1 mice received saline or STZ injection for 5 consecutive days, and PBS, uIL-2 or ldIL-2 for the next 8 days. Blood glucose was monitored daily after day 5. A Blood glucose levels of mice. Repeated two-way ANOVA followed by Tukey’s test was performed, ** and *** denote p < 0.01 and p < 0.001 respectively between low dose IL-2 (ldIL-2, n = 8) and uIL-2 (n = 6), ‡ denotes p < 0.05 between ldIL-2 and STZ (n = 14). B Percentage of diabetes free mice. Log-rank test followed by Bonferroni correction was performed. * denotes p < 0.05. C Insulitis of the mice. Kruskal-Wallis test followed by Dunn’s test was used. * denotes p < 0.05. Results are shown as mean ± SEM

Fig. 6

Low dose IL-2 does not change Treg and Breg cell phenotype. Male CD-1 mice received STZ injection for 5 consecutive days, and PBS or low dose IL-2 (ldIL-2) for the next 8 days. A Proportions of CD4⁺CD25⁺Foxp3⁺ Treg cells. B Proportions of IL-35⁺ cells among Treg cells. C Proportions of CD19⁺CD1d⁺CD5⁺ Breg cells. D Proportions of IL-35⁺ cells among Breg cells. Unpaired t test was performed. Results are shown as mean ± SEM. The experiment was performed twice with n = 2 for each group each time

Fig. 7

Low dose IL-2 tentatively decreases CD80 expression in antigen presenting cells. Male CD-1 mice received STZ injection for 5 consecutive days, and PBS or ldIL-2 for the next 8 days. Single splenocytes were stained and investigated using flow cytometry. A Fluorescence intensity of CD80 in CD11b⁺F4/80⁺ macrophages, MHC-II⁺CD11c⁺F4/80^-XCR1⁺ cDC1s and MHC-II⁺CD11c⁺F4/80^-CD172a⁺ cDC2s. B Proportions of CD80⁺ cells among macrophages, cDC1s and cDC2s. Unpaired t-test was performed. Results are shown as mean ± SEM. The experiment was performed twice with n = 2 for each group each time