HLA class I hyper-expression unmasks beta cells but not alpha cells to the immune system in pre-diabetes

Abstract

Human leukocyte antigens of class-I (HLA-I) molecules are hyper-expressed in insulin-containing islets (ICI) of type 1 diabetic (T1D) donors. This study investigated the HLA-I expression in autoantibody positive (AAB+) donors and defined its intra-islet and intracellular localization as well as proximity to infiltrating CD8 T cells with high-resolution confocal microscopy. We found HLA-I hyper-expression had already occurred prior to clinical diagnosis of T1D in islets of AAB+ donors. Interestingly, throughout all stages of disease, HLA-I was mostly expressed by alpha cells. Hyper-expression in AAB+ and T1D donors was associated with intra-cellular accumulation in the Golgi. Proximity analysis showed a moderate but significant correlation between HLA-I and infiltrating CD8 T cells only in ICI of T1D donors, but not in AAB+ donors. These observations not only demonstrate a very early, islet-intrinsic immune-independent increase of HLA-I during diabetes pathogenesis, but also point towards a role for alpha cells in T1D.

Keywords: Alpha cells; Beta cells; HLA class I; Type 1 diabetes; nPOD.

Fig. 1.

HLA class I is hyper-expressed in the islets of T1D as well as in AAB+ donors. FFPE sections of human pancreata were stained with anti-HLA-I (magenta), anti-insulin (green) and anti-glucagon (red) and Hoechst (blue). (a) Representative images of an islet from a non-diabetic (ND) donor (nPOD 6034), an autoantibody positive (AAB+) donor (nPOD 6197), an insulin-containing islet (ICI) of a T1D donor (nPOD 6198) and an insulin-deficient islet (IDI) of a T1D donor (nPOD 6198) are shown. Images were acquired using a confocal microscope LSM 780 with a 63× 1.4na objective. HLA-I was quantified using Zen software. (b) The percentage of total islet area positive for HLA-I was measured in at least 20 islets per section from non-diabetic donors (n=10), single autoantibody positive (n=5), double autoantibody positive (n=5) and type 1 diabetic donors (n=12). (c) Percentage of total islet area positive for HLA-I presented by case. Each data point represents an islet. An arbitrary threshold of 20% was set above which HLA-I was considered hyper-expressed. *** p=0.0004 ****p<0.0001.

Fig. 2.

HLA class I is mainly expressed on alpha cells irrespective of disease status. (a) Percentage of colocalization of HLA-I and glucagon (alpha cells- circle) and HLA-I and insulin (beta cells - square) quantified using the Mander’s overlap coefficient using Zen software. Each data point represents an islet. (b) Mean percentage of HLA-I positive area per cell type. ND: non-diabetic; AAB+: autoantibody positive; T1D: type 1 diabetes; ICI: insulin-containing islet; IDI: insulin-deficient islet. **** p<0.0001.

Fig. 3.

HLA class I colocalizes with Golgi marker GM130 in the islets of AAB+ and T1D donors. Percentage of colocalization of HLA-I and GM130 quantified using Mander’s overlap coefficient in the islets from non-diabetic donor (nPOD 6034), autoantibody positive donors (nPOD 6181 and 6197) and a type 1 diabetic donor (nPOD 6212). Colocalization analysis was performed in Zen software. ND: Non-diabetic; AAB+: autoantibody positive; T1D: type 1 diabetes. *p<0.05 **p<0.01.

Fig. 4.

Proximity analysis of HLA class I in the surroundings of CD8 T cells. Pancreatic sections from organ donors were stained for HLA-I, CD8, insulin and glucagon. CD8 T cells were detected in the pancreatic tissue sections and a proximity analysis was performed to detect the HLA-I expression in the surroundings of CD8 T cells. (a) Quantification of HLA-I using ImagePro premier in the radials surrounding the CD8 T cells, 100 being the closest to the CD8 cell and 400 the furthest presented by case. Each data point represents the level of HLA-I in a radial circle. (b) Schematic representation of CD8 T cell and the radial circles from 100 to 400 used for the proximity analysis. (c) Quantification of HLA-I presented as the sum of the radials from 100 to 400 and presented by group. Each data point represents an islet. Each data point represents the level of HLA-I in a radial circle (d) Correlation between the number of CD8 T cells per field of view and the total expression of HLA-I in the surroundings of those CD8 T cells. Each data point represents a field of view. ND: non-diabetic; AAB+: autoantibody positive; T1D: type 1 diabetes; ICI: insulin-containing islet; IDI: insulin-deficient islet.