Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8⁺T Effector Memory Subset

Abstract

CD26 is a T cell activation marker consisting in a type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain. It has been described that DPPIV inhibition delays the onset of type 1 diabetes and reverses the disease in non-obese diabetic (NOD) mice. The aim of the present study was to assess the effect of MK626, a DPPIV inhibitor, in type 1 diabetes incidence and in T lymphocyte subsets at central and peripheral compartments. Pre-diabetic NOD mice were treated with MK626. Diabetes incidence, insulitis score, and phenotyping of T lymphocytes in the thymus, spleen and pancreatic lymph nodes were determined after 4 and 6 weeks of treatment, as well as alterations in the expression of genes encoding β-cell autoantigens in the islets. The effect of MK626 was also assessed in two in vitro assays to determine proliferative and immunosuppressive effects. Results show that MK626 treatment reduces type 1 diabetes incidence and after 6 weeks of treatment reduces insulitis. No differences were observed in the percentage of T lymphocyte subsets from central and peripheral compartments between treated and control mice. MK626 increased the expression of CD26 in CD8+ T effector memory (TEM) from spleen and pancreatic lymph nodes and in CD8+ T cells from islet infiltration. CD8+TEM cells showed an increased proliferation rate and cytokine secretion in the presence of MK626. Moreover, the combination of CD8+ TEM cells and MK626 induces an immunosuppressive response. In conclusion, treatment with the DPPIV inhibitor MK626 prevents experimental type 1 diabetes in association to increase expression of CD26 in the CD8+ TEM lymphocyte subset. In vitro assays suggest an immunoregulatory role of CD8+ TEM cells that may be involved in the protection against autoimmunity to β pancreatic islets associated to DPPIV inhibitor treatment.

Fig 1. DPPIV inhibitor MK626 decreases T1D incidence and reduces the insulitis score in NOD mice.

A) Cumulative incidence (percentage) of diabetes in NOD mice treated with MK626 (black circles, n = 15) and in the control group (white circles, n = 15). Significant differences were found when compared MK626-treated mice versus control group (*p≤0.05, Kaplan-Meier log-rank analysis). B) Insulitis score from treated and untreated mice at each time-point. Pancreases from 4 mice/group were analyzed by two independent observers at 2, 4 and 6 weeks after treatment. Insulitis score was significantly lower in treated mice than in controls after 6 weeks of treatment. Results are expressed as mean ± SEM (*p≤0.05, Mann-Whitney test). Lower panel: sections of pancreas from haematoxylin and eosin illustrating islets taken from a treated and control mice at 6 weeks. c) Percentage of islets in each of the five infiltration categories in treated and control mice at 2, 4 and 6 weeks: White = 0, no insulitis; Dotted = 1, peri-insular; Striped = 2, mild insulitis (<25% of the infiltrated islet); Squared = 3, moderate insulitis (25–75% of the islet infiltrated); Black = 4, severe insulitis (>75% islet infiltration).

Fig 2. Serum TGF-β concentration from NOD mice after MK626 treatment.

Serum level of TGF-β from control (white bars) and treated (black bars) after 4 and 6 weeks of treatment. Results are expressed as mean ± SD from 10 mice/time-point. No statistical differences were observed between groups (Mann-Whitney test).

Fig 3. Effects of the DPPIV inhibitor MK626 on splenic T- lymphocytes.

Splenocytes were obtained at 4 and 6 weeks after treatment. A) Percentage of T effector memory (EM)_, T central memory (CM) and naïve T lymphocytes (CD8⁺and CD4⁺) in control (white circles) and treated mice (black circles). B) Representative FACS plots of the naïve (CD3⁺CD4⁺/CD8⁺CD44^lowCD62L⁺), T_CM (CD3⁺CD4⁺/CD8⁺CD44^highCD62L⁺) and T_EM (CD3⁺CD4⁺/CD8⁺CD44^highCD62L^neg) both for CD4⁺ and CD8⁺ T lymphocytes from spleen in treated and control mice at 4 weeks of treatment. C) MFI for CD26 expression on T_EM, T_CM and naïve T lymphocytes (CD8⁺ and CD4⁺) in control (white circles) and treated mice (black circles). Lines represent the mean of 8–10 mice. Comparison between treated and untreated mice showed an increased expression of CD26 on CD8⁺ T_EM cells at any of the study’s time-points. (*p<0.05, **p<0.01, three way Anova).

Fig 4. Effects of the DPPIV inhibitor MK626 on T lymphocytes from pancreatic lymph nodes (PLNs).

T cells from PLNs were obtained from control (white circles) and treated (black circles) NOD mice at 4 weeks after treatment. A) Percentage of T effector memory (EM)_, T central memory (CM) and naïve T lymphocytes (CD8⁺and CD4⁺). B) MFI for CD26 expression on T_EM, T_CM and naïve T lymphocytes (CD8⁺and CD4⁺). The expression of CD26 was significantly increased on CD4⁺ T_EM and CD8⁺ T_EM cells in MK626-treated mice when compared to controls. Lines represent the mean of 6 mice (*p<0.05, ***p<0.001, three way Anova).

Fig 5. Percentage of T lymphocytes and CD26 membrane expression in the leukocytic infiltrate of mice treated 4 weeks with MK626.

A) Percentage of CD8⁺ and CD4⁺ T lymphocytes infiltrating pancreatic islets in untreated (white circles) and treated (black circles) mice assessed by flow cytometry. Lines represent the mean of 6–9 mice. No statistical differences were observed between groups (Mann-Whitney test). B) Expression of CD26 determined by flow cytometry (MFI) in CD8⁺ and CD4⁺ T lymphocytes from control (white circles) and treated (black circles) mice. Lines represent the mean of 4–8 mice. (*p≤0.05, Mann-Whitney test).

Fig 6. β-cell specific gene expression in the islets of MK626 NOD treated mice.

Quantitative RT-PCR results for Ins2, Iapp, Gcg and CD45 genes in control (white bars) and treated group (black bars) determined using Taqman assays. Gene expression signals were normalized to Gadph. All data are expressed as mean ± SD from 5 mice. Significance was tested using Mann-Whitney test.

Fig 7. Analysis of CD8⁺ T_EM cell proliferation and cytokine production in vitro induced by MK626 treatment.

CD8⁺ T_EM cell from control (n = 9) and treated (n = 9) mice obtained at 4 weeks of treatment were stimulated with anti–CD3 in vitro. A) Proliferative rate in CD8⁺ T_EM lymphocytes from MK626-treated mice (black circles) and in control group (white circles). T cell proliferation was expressed as counts per minute (c.p.m) using ³H-TdR. B) Cytokine concentration (IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IFN-γ, TNF-α and TGF-β) in supernatant from proliferation assays in treated mice (black bars) compared to control mice (white bars). Results are expressed as mean ± SEM. Mann-Whitney test was used for the evaluation of statistical significance (*p<0.05;**p<0.01).

Fig 8. Suppressive effect of MK626 and CD8⁺ T_EM lymphocytes in vitro.

A) Representative dot plots showing CFSE-T lymphocytes, stimulated with insulin-loaded mDCs, in the presence or absence of CTV-CD8⁺ T_EM lymphocytes (ratio 1:1) and MK626 (10μM). B) Percentage of proliferating CFSE^low T lymphocytes versus all T lymphocytes, with insulin-loaded mDCs (circles and triangles), in the presence of CTV-CD8⁺ T_EM lymphocytes (triangles) and MK626 (open symbols) for 3 days. Plots show the mean (line) of three independent experiments.