Inherent ER stress in pancreatic islet β cells causes self-recognition by autoreactive T cells in type 1 diabetes

Abstract

Type 1 diabetes (T1D) is an autoimmune disease characterized by pancreatic β cell destruction induced by islet reactive T cells that have escaped central tolerance. Many physiological and environmental triggers associated with T1D result in β cell endoplasmic reticulum (ER) stress and dysfunction, increasing the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that β cell ER stress induced by environmental and physiological conditions generates abnormally-modified proteins for the T1D autoimmune response. To test this hypothesis we exposed the murine CD4(+) diabetogenic BDC2.5 T cell clone to murine islets in which ER stress had been induced chemically (Thapsigargin). The BDC2.5 T cell IFNγ response to these cells was significantly increased compared to non-treated islets. This β cell ER stress increased activity of the calcium (Ca(2+))-dependent PTM enzyme tissue transglutaminase 2 (Tgase2), which was necessary for full stress-dependent immunogenicity. Indeed, BDC2.5 T cells responded more strongly to their antigen after its modification by Tgase2. Finally, exposure of non-antigenic murine insulinomas to chemical ER stress in vitro or physiological ER stress in vivo caused increased ER stress and Tgase2 activity, culminating in higher BDC2.5 responses. Thus, β cell ER stress induced by chemical and physiological triggers leads to β cell immunogenicity through Ca(2+)-dependent PTM. These findings elucidate a mechanism of how β cell proteins are modified and become immunogenic, and reveal a novel opportunity for preventing β cell recognition by autoreactive T cells.

Keywords: Autoimmunity; ER stress; Post-translational modification; Type 1 diabetes; β cell.

Fig. 1. Primary islets from many mouse strains elicit effector responses from BDC2.5 T cell clones

(A) Schematic of the BDC2.5 T cell assay protocol. Antigen presenting cell (APC). (B) The immunogenicity of NOD.scid islet cells was measured by BDC2.5 T cell assay. Data are mean IFNγ secretion ± SEM. ** p < 0.01. (C) The immunogenicity of NOD.scid and C57BL/6 (left) or BALB/c (right) islet cells was measured by BDC2.5 T cell assay. Data are mean IFNγ secretion ± SEM.

Fig. 2. β cell ER stress increases recognition by diabetogenic T cells

(A) Primary NOD.scid islets were incubated with 5 µM Thaps or control (Ctrl.) for 1 hr, washed extensively, and dispersed. Cell lysates were analyzed for the phosphorylation of UPR proteins PERK and eIF2α. Data are representative of 3 independent experiments. Densitometry data are phosphorylation levels normalized by β-actin and relative to that in control (Ctrl.) treated cells. (B) The immunogenicity of NOD.scid islet cells treated with 5 µM Thaps or control for 1 hr was measured by BDC2.5 T cell assay. Either APC or T cells were omitted from assay to demonstrate the necessity of both cell types for full IFNγ secretion. Data are mean IFNγ secretion ± SEM. *** p < 0.001.

Fig. 3. ER stress-induced immunogenicity is Ca²⁺ dependent

(A) Dispersed NOD.scid islet cells were labeled with Fluo-4 and analyzed by live imaging with an Olympus Fluoview FV1000 microscope for 350 sec at room temperature. At 70 sec, the cells were exposed to 5 µM Thaps or controls. Scale bars, 50 µm. Arrows indicate cells with greater Fluo-4 intensity after exposure to Thaps. Line graphs represent the intensity of Fluo-4 in each sample over time. Data are representative of 3 independent experiments. Quantified data are change in Fluo-4 intensity from baseline to peak, with mean ± SEM. ** p < 0.01. (B) The immunogenicity of primary NOD.scid islet cells treated with 2.5 µM Bapta or control (Ctrl.) prior to incubation with 5 µM Thaps or control was measured by BDC2.5 T cell assay. Data are relative IFNγ secretion compared to 5 µM Thaps, with mean ± SEM. *** p < 0.001

Fig. 4. β cell ER stress increases the activity of Tgase2

Primary NOD.scid islets were incubated with 5 µM Thaps or control (Ctrl.) for 1 hr. The cells were lysed and Tgase2 activity was measured by microtiter plate assay. Data are relative Tgase2 activity compared to control (Ctrl.) islet cells, with mean ± SEM. ** p < 0.01.

Fig. 5. Modification of CHgA by Tgase2 increases immunogenicity

(A) Alignment of the sequences of WE14 and CHgA_351–370. (B) Synthetic CHgA_351–370 was incubated with purified Tgase2 at 37°C for 3 hr. To confirm the dependence of these reactions on Ca²⁺, the reactions were repeated without CaCl₂ (0 mM Ca²⁺) or with increased EDTA (10 mM EDTA). The reaction products were analyzed by SDS-PAGE. Lane 1: CHgA_351–370 alone; Lane 2: Tgase2 alone; Lane 3: CHgA_351–370 incubated with Tgase2; Lane 4: CHgA_351–370 incubated with Tgase2 in 0 mM Ca²⁺; Lane 5, CHgA_351–370 incubated with Tgase2 in 10 mM EDTA. (C) The immunogenicity of CHgA_351–370 incubated with Tgase2 was measured by BDC2.5 T cell assay. Data are mean IFNγ secretion ± SEM. * p < 0.05.

Fig. 6. ER stress in NIT-1 insulinomas increases immunogenicity in a Ca²⁺-dependent manner

(A) NIT-1 insulinoma cells were incubated with 5 µM Thaps or control (Ctrl.) for 1 hr and washed extensively. Cell lysates were analyzed for the phosphorylation of UPR proteins PERK and eIF2α. Data are representative of 3 independent experiments. Densitometry data are phosphorylation levels normalized by β-actin and relative to that in control (Ctrl.) treated cells. (B) The immunogenicity of NIT-1 cells treated with 5 µM Thaps or control was measured by BDC2.5 T cell assay. Either APC or T cells were omitted from assay to demonstrate the necessity of both cell types for full IFNγ secretion. Data are mean IFNγ secretion ± SEM. *** p < 0.001. (C) NIT-1 cells were labeled with Fluo-4 and analyzed by live imaging with an Olympus Fluoview FV1000 microscope for 350 sec at room temperature. At 70 sec, the cells were exposed to 5 µM Thaps or controls. Scale bars, 50 µm. Arrows indicate cells with greater Fluo-4 intensity after exposure to Thaps. Line graphs represent intensity of Fluo-4 in each sample over time. Data are representative of 3 independent experiments. Quantified data are change in Fluo-4 intensity from baseline to peak, with mean ± SEM. ** p < 0.01. (D) The immunogenicity of NIT-1 cells treated with 2.5 µM Bapta or control (Ctrl.) prior to incubation with 5 µM Thaps or control was measured by BDC2.5 T cell assay. Data are relative IFNγ secretion compared to 5 µM Thaps, with mean ± SEM. ** p < 0.01 (E) NIT-1 cells were incubated with 5 µM Thaps or control (Ctrl.) for 1 hr. The cells were lysed and Tgase2 activity was measured by microtiter plate assay. Data are relative Tgase2 activity compared to control (Ctrl.) NIT-1 cells, with mean ± SEM. * p < 0.05.

Fig. 7. Tgase2 activity contributes to β cell immunogenicity

(A) NIT-1 cells were transduced with control shRNA (Ctrl. shRNA) or Tgase2-targeting shRNA (Tgase2 shRNA). Tgase2 expression was measured by qRT-PCR. Data are relative Tgase2 expression compared to cells transduced with control shRNA, with mean ± SEM. ** p < 0.01. (B) The immunogenicity of NIT-1 cells transduced with control shRNA (Ctrl. shRNA) or Tgase2-targeting shRNA (Tgase2 shRNA) prior to incubation with 5 µM Thaps or control (Ctrl.) for 1 hr was measured by BDC2.5 T cell assay. Data are relative IFNγ secretion compared to NIT-1 cells transduced with Ctrl. shRNA and incubated with 5 µM Thaps, with mean ± SEM. * p < 0.05.

Fig. 8. Physiological cues increase β cell ER stress and immunogenicity

Non-immunogenic NIT-1 cells (2–5×10⁶) were transplanted under the kidney capsule of NOD.scid mice. (A) Serum insulin levels in control mice (Ctrl.) or mice transplanted with NIT-1 cells (NIT-1) were measured by ELISA. Medians are indicated with a line. Data are representative of 4 independent experiments. * p < 0.05, ** p < 0.01. (B) The onset of hypoglycemia in control mice (No Transplant) or mice transplanted with NIT-1 cells (NIT-1 Transplant) was monitored over time. p < 0.001 (C) At the onset of hypoglycemia, the mice were sacrificed and the NIT-1 cells were explanted. Cell lysates of cultured NIT-1 cells (NIT-1) or explanted NIT-1 cells (Explant) were analyzed for the phosphorylation of UPR proteins PERK and eIF2α. Data are representative of 3 independent experiments. Densitometry data are phosphorylation levels normalized by β-actin and relative to that in cultured NIT-1 cells. (D) Cultured NIT-1 cells or explanted NIT-1 cells were lysed, and Tgase2 activity was measured by ex vivo Tgase2 activity assay. Data are mean Tgase2 activity, with mean ± SEM. *** p < 0.001. (E) The immunogenicity of cultured NIT-1 cells or explanted NIT-1 cells was measured by BDC2.5 T cell assay. Data are mean IFNγ secretion ± SEM. * p < 0.05.

Fig. 9. Model

(A) Under resting conditions, CHgA is translated, folded in the ER, and packaged in secretory granules. Cytosolic Ca²⁺ remains low, and Tgase2 activity remains low. (B) β cell exposure to physiological or environmental triggers of ER stress leads to increased ER burden. (C) Ca²⁺ stores are released from the ER, increasing cytosolic Ca²⁺, and (D) activating Ca²⁺-dependent PTM enzymes such as Tgase2. (E) Activated Tgase2 translocates to the ER to modify CHgA, (F) which is packaged into secretory granules. If presented to auto-reactive T cells by APC, modified CHgA breaks peripheral tolerance and facilitates immune recognition of β cells.