In vivo evidence for the contribution of human histocompatibility leukocyte antigen (HLA)-DQ molecules to the development of diabetes

Abstract

Although DQA1*0301/DQB1*0302 is the human histocompatibility leukocyte antigen (HLA) class II gene most commonly associated with human type 1 diabetes, direct in vivo experimental evidence for its diabetogenic role is lacking. Therefore, we generated C57BL/6 transgenic mice that bear this molecule and do not express mouse major histocompatibility complex (MHC) class II molecules (DQ8(+)/mII(-)). They did not develop insulitis or spontaneous diabetes. However, when DQ8(+)/mII(-) mice were bred with C57BL/6 mice expressing costimulatory molecule B7-1 on beta cells (which normally do not develop diabetes), 81% of the DQ8(+)/mII(-)/B7-1(+) mice developed spontaneous diabetes. The diabetes was accompanied by severe insulitis composed of both T cells (CD4(+) and CD8(+)) and B cells. T cells from the diabetic mice secreted large amounts of interferon gamma, but not interleukin 4, in response to DQ8(+) islets and the putative islet autoantigens, insulin and glutamic acid decarboxylase (GAD). Diabetes could also be adoptively transferred to irradiated nondiabetic DQ8(+)/mII(-)/B7-1(+) mice. In striking contrast, none of the transgenic mice in which the diabetes protective allele (DQA1*0103/DQB1*0601, DQ6 for short) was substituted for mouse MHC class II molecules but remained for the expression of B7-1 on pancreatic beta cells (DQ6(+)/mII(-)/B7-1(+)) developed diabetes. Only 7% of DQ(-)/mII(-)/B7-1(+) mice developed diabetes at an older age, and none of the DQ(-)/mII(+)/B7-1(+) mice or DQ8(+)/mII(+)/B7-1(+) mice developed diabetes. In conclusion, substitution of HLA-DQA1*0301/DQB1*0302, but not HLA-DQA1*0103/DQB1*0601, for murine MHC class II provokes autoimmune diabetes in non-diabetes-prone rat insulin promoter (RIP).B7-1 C57BL/6 mice. Our data provide direct in vivo evidence for the diabetogenic effect of this human MHC class II molecule and a unique "humanized" animal model of spontaneous diabetes.

Figure 1

Development of diabetes spontaneously in HLA-DQ transgenic mice. (A) Spontaneous diabetes in DQ8 experiments. Six groups of mice were used in the study as indicated. Number of mice per group was as follows: n = 21 (10 female, 11 male) DQ8⁺/mII⁻/B7⁺; n = 15 (8 female, 7 male) DQ8⁻/mII⁻/B7⁺; n = 16 (11 female, 5 male) DQ8⁺/mII⁻/B7⁻; n = 14 (8 female, 6 male) DQ8⁻/mII⁻/B7⁻; n = 17 (6 female, 11 male) DQ8⁺/mII⁺/B7⁺; and n = 12 (9 female, 3 male) DQ8⁺/mII⁻/B7⁻. (B) Spontaneous diabetes in DQ6 experiments. Four groups of mice were used in the study as indicated. Number of mice per group was as follows: n = 16 (9 female, 7 male) DQ6⁺/mII⁻/B7⁺; n = 12 (5 female, 7 male) DQ6⁻/mII⁻/B7⁺; n = 9 (4 female, 5 male) DQ6⁺/mII⁺/B7⁺; and n = 15 (6 female, 9 male) DQ6⁺/mII⁻/B7⁻. Mice were housed under specific pathogen-free conditions. Diabetes was determined by monitoring of urinary glucose and confirmed by blood glucose (>250 mg/dl).

Figure 2

Adoptively transferred diabetes. 10 × 10⁷ diabetic DQ8⁺/mII⁻/B7⁺ splenocytes (depleted red blood cells) were transferred intravenously into irradiated recipients (as indicated). Diabetes was determined as above. Number of mice per group was as follows: n = 9 (4 female, 5 male) DQ8⁺/mII⁻/B7⁺; n = 8 (4 female, 4 male) DQ⁻/mII⁻/B7⁺; n = 3 (male) DQ8⁺/mII⁻/B7⁻; n = 3 (male) DQ8⁻/mII⁻/B7⁻; and n = 3 (male) DQ8⁺/mII⁻/B7⁺* (these recipients were adoptively transferred with purified CD8⁺ splenocytes only from diabetic DQ8⁺/mII⁻/B7⁺ mice).

Figure 4

ICAM-1 staining of pancreatic sections of nondiabetic (a) and diabetic (b) DQ8⁺/mII⁻/B7⁺ mice.

Figure 3

Immunohistochemis-try staining of pancreatic sections of diabetic DQ8⁺/mII⁻/B7⁺ mice (top panels, a–c) and diabetes-free DQ6⁺/mII⁻/B7⁺ mice (bottom panels, d–f). Islet infiltrates were stained positive for CD4⁺ T cells (a), B220⁺ B cells (b), and CD8⁺ T cells (c).

Figure 5

In vitro proliferation (A) and IFN-γ production (B) of diabetic splenocytes to islets and islet β cell autoantigens. 2 × 10⁵/well of splenocytes (depleted red blood cells) were cultured in medium (Click's containing 5% heat-inactivated FCS) alone or with antigens (as indicated). Cytokine production was measured in the supernatants of the cultures (3 d) by ELISA.

Figure 6

Staining of frozen sections of salivary gland from diabetic DQ8⁺/mII⁻/B7⁺ mice (top panels, a and b) and normal DQ6⁺/mII⁻/B7⁺ mice (bottom panels, c and d). Lymphocyte infiltrates were stained positive for CD4 (a and c) and CD8 (b and d) T cells.