The effector T cells of diabetic subjects are resistant to regulation via CD4+ FOXP3+ regulatory T cells

Abstract

Defects in immune regulation have been implicated in the pathogenesis of diabetes in mouse and in man. In vitro assays using autologous regulatory (Treg) and responder effector (Teff) T cells have shown that suppression is impaired in diabetic subjects. In this study, we addressed whether the source of this defect is intrinsic to the Treg or Teff compartment of diabetic subjects. We first established that in type 1 diabetes (T1D) individuals, similar levels of impaired suppression were seen, irrespective of whether natural (nTreg) or adaptive Treg (aTreg) were present. Then using aTreg, we examined the ability of T1D aTreg to suppress Teff of healthy controls, as compared with the ability of control aTreg to suppress Teff of diabetic subjects. Taking this approach, we found that the aTregs from T1D subjects function normally in the presence of control Teff, and that the T1D Teff were resistant to suppression in the presence of control aTreg. This escape from regulation was seen with nTreg as well and was not transferred to control Teff coincubated with T1D Teff. Thus, the "defective regulation" in T1D is predominantly due to the resistance of responding T cells to Treg and is a characteristic intrinsic to the T1D Teff. This has implications with respect to pathogenic mechanisms, which underlie the development of disease and the target of therapies for T1D.

Figure 1

Adaptive Treg suppression is impaired in subjects with T1D. A) CSFE based analysis of suppression: FACS plots represent proliferating CFSE-labeled Teff cells when cultured alone and activated with anti-CD3/anti-CD28 coated beads or co-incubated with Treg. Histograms show the % of proliferating Teff cells cultured alone or after gating out the Treg population. The % of inhibition was determined by comparing the % of proliferating Teff cells cultured alone to the % of proliferating Teff cells in co-culture. B) Suppression assays using aTreg of control subjects and either autologous or allogeneic Teff from controls show a consistent level of suppression.( ■ mean of data from all assays, best fit curve by nonlinear regression with 95% confidence intervals shown) (n=12). Suppression when nTreg are used ( ) Representative of n=4. C) Comparison of % inhibition at a ratio of 1:4 aTreg:Teff for control subjects (■) as compared to T1D subjects (△) (n=10) (p= 0.0015). D) % Suppression displayed over a range of aTreg: Teff ratios demonstrates diminished suppression among T1D subjects, data is presented as mean of all assays (◆) and as a curve representing non-linear regression and the 95% confidence interval (black line n=10). This is shown in comparison to the control curve in grey.

Figure 2

Impaired suppression is intrinsic to the T1D Teff population. A) aTreg isolated from a T1D subject were utilized in a suppression assay with Teff from a control subject (dashed) or T1D subject (solid). B) Combined data from multiple experiments demonstrates that the % inhibition seen when T1D aTreg are co-incubated with control Teff (□) (n= 13), Best fit curve shown as a black line with 95% confidence interval and is no different than that seen with control aTreg (grey line). C) Control aTreg were coincubated with Teff from a control subject (black) or Teff from three different T1D subjects (open symbols with dashed lines) (n=3). D) Combined data for suppression assays in which control aTreg are co-incubated with T1D Teff (n=11) in black with 95% confidence intervals. This is shown in comparison to the control curve in grey.

Figure 3

T1D Teff are resistant to nTreg. T1D Teff were co-incubated with nTreg directly isolated from the peripheral blood, %inhibition at a Treg:Teff ratio of 1:1 is shown for, nTreg with control Teff (●) (n=1) and nTreg with T1D Teff (△) (n=4) at a Treg:Teff ratio of 1:1, nTreg with control Teff (■) (n=4) and nTreg with T1D Teff (▽) (n=6) at a ratio of 1:4. For comparison the level of suppression at 1:4 seen with control aTreg co-incubated with the same T1D Teff cells shown in the nTreg assays is shown ( ).

Figure 4

T2D Teff are not resistant to regulation. Control aTreg were co-incubated with allogeneic control Teff or T2D Teff and % inhibition was measured on day 4. Open symbols with dotted lines represent data from T2D Teff, solid symbols with solid lines are data for control Teff. Unpaired t test with Welch’s correction showed no statistically significant differences at a ratio of 1:4, 1:8, 1:16, or at 1:32. At a ratio of 1:64 (p= 0.03).

Figure 5

Impairment in T1D Teff suppression is not transferable to control Teff. Suppression assays were performed with control nTreg, co-cultured at a 1:4 ratio with Teff cells. 50% of Teff cells were labeled with CFSE, while the other Teff were unlabeled. Proliferation of CFSE labeled cells was examined on day 4. Data is shown for cultures containing control Teff only, or T1D Teff and Control Teff in co-culture. % inhibition is calculated based on proliferation of Teff in cultures without Treg. The % inhibition of T1D Teff was consistently lower than control Teff from the same culture p=0.0034.

Figure 6

Impaired aTreg and Teff function in one T1D subject. aTreg were induced from a control and diabetic subject. Suppression assays were performed with autologous Teff from the diabetic subject or allogeneic control Teff. % inhibition is shown for T1D Teff with autologous aTreg (○) is severely impaired, suppression is decreased when control Teff or aTreg are used in combination with T1D aTreg (□) or T1D Teff (▽) respectively and does not attain the level seen with the control aTreg plus control Teff (◆). This represents 1 of two experiments.