Beta cell MHC class I is a late requirement for diabetes

Abstract

Type 1 diabetes occurs as a result of an autoimmune attack on the insulin-producing beta cells. Although CD8 T cells have been implicated both early and late in this process, the requirement for direct interaction between these cells and MHC class I on the beta cells has not been demonstrated. By using nonobese diabetic mice lacking beta cell class I expression, we show that both initiation and progression of insulitis proceeds unperturbed. However, without beta cell class I expression, the vast majority of these mice do not develop hyperglycemia. These findings demonstrate that a direct interaction between CD8 T cells and beta cells is not required for initiation or early disease progression. The requirement for class I on beta cells is a relatively late checkpoint in the development of diabetes.

Fig. 1.

The β2M^a transgene was constructed as described (14). In brief, exons II and III of the β2M^a gene were flanked by 150-bp loxP sites inserted into the NdeI site of intron A and the BamHI site of intron C. Thin lines, introns and flanking DNA; boxes, exons; triangles, loxP sites; B, BamHI; Bg, BglI; E, EcoRI; N, NdeI; X, XbaI; and Xh, XhoI.

Fig. 2.

(a) HIP-Cre-mediated recombination occurs specifically in islets. DNA (6.25, 12.5, 25, 50, and 100 ng) was prepared from islets, spleen, thymus, mesenteric lymph nodes, and peripheral blood of male 100-day-old β2M^aβ2M^–/– HIP-Cre^– and HIP-Cre⁺ NOD mice. (i) Control PCR, which amplifies a 1.1-kbp product from the β2M^a transgene, a 950-bp product from the endogenous β2M^a gene, and a 2.5-kbp product from the β2M^–/– gene (not seen in all samples). (ii) PCR to detect the recombined β2M^a transgene, which amplifies an 800-bp product from the HIP-Cre-recombined gene and a 2.5-kbp product from the unrecombined gene (seen in the NOD control only). (b) Expression of H-2K^d is normal in all class I beta normal and class I beta bald NOD transgenic mice except the islets of class I beta bald transgenic mice, where expression is lost from the surface of most islet cells. Single-cell suspensions were made from islets, peripheral blood, spleen, mesenteric lymph nodes, and thymus of NOD class I beta normal, class I beta bald, and β2M^–/– sex-matched 70- to 160-day-old NOD mice. Cells were stained with biotinylated SF1.1.1 and streptavidin APC and analyzed by FACS. (c) HIP-cre-mediated recombination occurs specifically in islet beta cells. Islet cells were isolated from mouse pancreas and stained with SF1.1.1 (H-2K^d specific), then FACS-sorted into H-2K^d-positive and H-2K^d-negative pools (excluding dead cells). The sorted cells were then stained for insulin (red cells). Cells from NOD, class I beta normal, class I beta bald, and β2M^–/– NOD mice are shown. (Bar = 100 μm.)

Fig. 3.

(a) Degree of insulitis was not reduced in class I beta bald mice by histological analysis. Insulitis is scored in the following way: 0, no insulitis; 1, periductal insulitis; 2, circumferential insulitis; 3, intraislet infiltration; and 4, severe structural derangement. The mean scores of female β2M^aβ2m^+/– HIP-Cre⁺ (n = 7), β2M^–/– (n = 7), class I beta normal (n = 5), and class I beta bald (n = 13) NOD mice are shown. Black bars, 0; diagonal lines, 1; vertical lines, 2; gray, 3; and cross-hatched lines, 4. The proportion of islets exhibiting each level of insulitis severity was not significantly different between class I beta normal and class I beta bald NOD mice (P > 0.05). (b) Content of lymphocytic infiltrate of grade 3 islets from class I beta bald and class I beta normal NOD mice. Consecutive frozen sections taken from 250-day-old female mice were stained with the antibodies 53.6 (CD8 specific), GK1.5 (CD4 specific), and F4/80 (macrophage specific). Sections from β2M^+/–, class I beta normal, and class I beta bald NOD mice are shown. (Bar = 10 mm.)

Fig. 4.

Incidence of diabetes was significantly reduced in female class I beta bald NOD mice. Class I beta normal (▴, n = 27), and class I beta bald (▪, n = 39) NOD mice were followed for 250 days for development of diabetes. The incidence in class I beta bald NOD mice was significantly lower than littermate class I beta normal NOD mice (P < 0.020).