Multiple immuno-regulatory defects in type-1 diabetes

Abstract

Susceptibility to immune-mediated diabetes (IMD) in humans and NOD mice involves their inherently defective T cell immunoregulatory abilities. We have followed natural killer (NK) T cell numbers in patients with IMD, both by flow cytometry using mAbs to the characteristic junctions found in the T cell receptors of this cell subtype, and by semiquantitative RT-PCR for the corresponding transcripts. Both before and after clinical onset, the representation of these cells in patients' PBMCs is reduced. We also report low numbers of resting CD4(+) CD25(+) T cells in IMD patients, a subset of T cells shown to have important immunoregulatory functions in abrogating autoimmunities in 3-day thymectomized experimental mice. Whereas a biased Th1 to Th2 cytokine profile has been suggested to underlie the pathogenesis of IMD in both species, we found defective production of IFN-gamma in our patients after in vitro stimulation of their PBMCs by phorbol-myristate acetate and ionomycin and both IFN-gamma and IL-4 deficiencies in V(alpha)24(+) NK T-enriched cells. These data suggest that multiple immunoregulatory T (Treg) cell defects underlie islet cell autoimmunity leading to IMD in humans and that these lesions may be part of a broad T cell defect.

Figure 1

NK T cell defect in IMD patients. (a) NK T cells in the peripheral blood were characterized by a three-color flow cytometry assay using mAbs to CD3 and to receptors bearing expressed Vα24 and Vβ11. The triple-positive population (CD3⁺Vα24⁺Vβ11⁺) showed marked reductions in both newly diagnosed (P < 0.0001) and long-term immune-mediated diabetic patients (P < 0.007) compared with controls. Nine multi-autoantibody–positive relatives of the 12 patients studied similarly had significantly reduced numbers of triple-positive cells (P < 0.0001). Whereas the type 2 diabetic patients also had lower levels than controls (P < 0.02), their deficiency was less marked than the IMD patients (P < 0.04). The horizontal lines represent means; n is the number of subjects in each group. (b) Comparisons are shown between NK T cells as defined by staining with CD3, Vα24, and mAb to the Vα24JαQ junction (6B11) to NK T cells defined by staining with CD3⁺Vα24⁺Vα11⁺ mAbs in patient and control groups. No differences were found in the NK T cell numbers as stained by the two different sets of mAbs. (c) A dot plot comparing the measurement of NK T cells using Vα24 and Vβ11 mAbs and Vα24 and the invariant JαQ junction mAb (6B11) in a representative normal control and a newly diagnosed IMD patient are shown. The patient has reduced doubly stained cells (upper-right quadrants) using either sets of mAbs. *P < 0.007, **P < 0.04.

Figure 2

PBMC and CD4^–CD8^– DN T cells from patients and normal controls were examined for Vα24JαQ expression by the Southern blot technique. (a and c) Vα24JαQ TCR transcripts are shown. (b and d) For the housekeeping gene HPRT expressions in the same subjects. (a) Expression of Vα24JαQ mRNA in the peripheral blood of IMD patients (lanes 1, 2, 4, 5, 6, 9, 10, and 11) are compared with normal controls (lanes 3 and 12). Lanes 7 and 8 compare the TCR expression in the DN population between controls and patients, respectively. Lane 13 is the negative control. (c) This figure part compares the expression of canonical Vα24JαQ transcripts in the patients with type 2 diabetes (lanes 1, 2, and 3) with a normal control (lane 4). Lane 5 is the negative control. The arrows indicate the position of the invariant Vα24JαQ and HPRT bands. (e) The relative expression intensities of the canonical Vα24JαQ TCR as normalized to HPRT gene expression in controls, IMD patients, and patients with type 2 diabetes are shown. The mRNA levels were all determined by RT-PCR followed by quantification of radiolabel by phosphorimaging. Shown in e are the mean levels of Vα24JαQ calibrated to the amount of HPRT gene expression in the sample. The bars indicate means plus 1 SE. Significant differences from the normal control group are *P < 0.05 and ** P < 0.01.

Figure 3

Deficiency of CD4⁺CD25⁺ T cells in IMD patients. Resting PBMCs were stained with mAbs to CD4 and CD25 in a two-color flow cytometry assay and analyzed by flow cytometry. In newly diagnosed IMD as well as long-standing type 1 diabetic patients, the mean percentage of these immunoregulatory cells of CD4⁺ T cells was reduced to 2.6 ± 0.23 (P < 0.001) and 3.7 ± 0.69 (P < 0.002), respectively, with 6.9 ± 0.4 and 6.3 ± 0.48 (P = NS) in the normal control and type 2 diabetic groups, respectively. The horizontal line represents the mean of that group.

Figure 4

Defective Th1 cytokine production in IMD patients at various stages of the disease. PBMCs from various groups were stimulated with PMA + I and were analyzed for the cell surface expression of CD3 and intracellular IFN-γ and IL-4 cytokines. Each dot represents one subject. (a) This figure depicts IFN-γ production in the patient groups. The percentage of T cells secreting IFN-γ was significantly reduced in newly diagnosed (P < 0.001) and long-standing IMD patients (P < 0.03) compared with the normal controls. CD3⁺ T cells were further characterized into CD4⁺ (b) and CD8⁺ (c) subsets for the cytokine production. CD4⁺ T cells showed significantly reduced IFN-γ production in both newly diagnosed (P < 0.003) and long-standing IMD patients (P < 0.005). Three out of seven ICA⁺ relatives so tested also had reduced numbers of T cells secreting IFN-γ (P < 0.01), while the other four were in the normal range. (d) Percentage of IL-4–secreting T cells was not reduced in IMD patient groups compared with controls. The horizontal bars represent the mean of that group.