Beta cells cannot directly prime diabetogenic CD8 T cells in nonobese diabetic mice

Abstract

Type 1 diabetes (T1D) is caused by the destruction of insulin-producing islet beta cells. CD8 T cells are prevalent in the islets of T1D patients and are the major effectors of beta cell destruction in nonobese diabetic (NOD) mice. In addition to their critical involvement in the late stages of diabetes, CD8 T cells are implicated in the initiation of disease. NOD mice, in which the beta2-microglobulin gene has been inactivated by gene targeting (NOD.beta2M-/-), have a deficiency in CD8 T cells and do not develop insulitis, which suggests that CD8 T cells are required for the initiation of T1D. However, neither in humans nor in NOD mice have the immunological requirements for diabetogenic CD8 T cells been precisely defined. In particular, it is not known in which cell type MHC class I expression is required for recruitment and activation of CD8 T cells. Here we have generated transgenic NOD mice, which lack MHC class I on mature professional antigen-presenting cells (pAPCs). These "class I APC-bald" mice developed periislet insulitis but not invasive intraislet insulitis, and they never became diabetic. Recruitment to the islet milieu does not therefore require cognate interaction between CD8 T cells and MHC class I on mature pAPCs. Conversely, such an interaction is critically essential to allow the crucial shift from periislet insulitis to invasive insulitis. Importantly, our findings demonstrate unequivocally that CD8 T cells cannot be primed to become diabetogenic by islet beta cells alone.

Fig. 1.

Loss of MHC class I is limited to the APC compartment in class I APC-bald NOD mice. Class I APC-bald NOD mice (n = 6), class I APC-normal NOD mice (n = 5), and NODβ₂M^−/− (n = 6) were analyzed for H-2K^d expression by FACS. Total conventional DCs (CD11c⁺), B lymphocytes (B220⁺ or CD19⁺), and macrophages (I-A^g7+ F4/80⁺ CD11b⁺) are shown, and the mean percentages (±SD) of H-2K^d-sufficient cells are indicated. All APC cell types from class I APC-bald NOD mice were largely deficient in MHC class I. In contrast, as expected, splenic T lymphocytes (CD4⁺, TCR⁺; Far Right) remain MHC class I-sufficient.

Fig. 2.

APCs of class I APC-bald NOD mice are unable to activate CD8 T cells in vivo. CFSE-labeled 8.3 TCR transgenic CD8 T cells were injected into either class I APC-normal mice (n = 5) or class I APC-bald NOD mice (n = 6). Six days after transfer, the percentage of proliferating 8.3 TCR⁺ CD8 T cells (R1) in the pancreatic lymph nodes of class I APC-normal mice (Upper) and class I APC-bald mice (Lower) was determined by flow cytometry. This result was representative of two independent experiments.

Fig. 3.

The islets of class I APC-bald NOD female mice have noninvasive insulitis lesions. (A and F) Hematoxylin and eosin stain of pancreata from >300-day-old, nondiabetic, class I APC-bald NOD mice showed infiltrates limited to the periislet region. Pancreatic sections were stained by immunofluorescence with anti-insulin antibody (red) and anti-CD8 antibody (green). Sections were additionally costained with anti-CD4 (B and G), anti-B220 (C and H), anti-CD11c (D and I), or anti-CD11b (E and J). The noninvasive infiltrate of pancreata from class I APC-bald NOD mice contained CD4 T cells, B cells, DCs, and macrophages, but it lacked CD8 T cells.

Fig. 4.

Insulitis severity. (A) A total of 208 islets of age-matched class I APC-normal NOD (n = 6), 159 islets of class I APC-bald NOD (n = 7), and 76 islets of NOD.β₂M^−/− (n = 3) were scored for the degree of insulitis as described in Materials and Methods. Bars: white, grade 0; horizontal lines, grade 1; gray, grade 2; vertical lines, grade 3; black, grade 4. (B) Class I APC-bald NOD mice did not develop invasive insulitis even when reconstituted with CD8 T cells (n = 8). Negative control NOD.scid mice (n = 2) and CD4⁺NOD.scid mice (n = 2) did not develop invasive insulitis, whereas positive control CD4⁺NOD.scid mice reconstituted with CD8 T cells (n = 4) did develop invasive insulitis. Splenocytes from class I APC-bald mice were capable of mediating invasive insulitis in NOD.scid recipient mice with class I-sufficient APCs. NOD.scid recipient mice developed invasive insulitis when reconstituted with either class I APC-bald (n = 3) or class I APC-normal (n = 5) splenocytes. (C) Immunofluorescent staining of invasive islet infiltrates in NOD.scid recipients of class I APC-normal splenocytes (i), class I APC-bald splenocytes (ii), and NOD CD4 and CD8 T cells (iii) demonstrates the presence of CD4 and CD8 T cells. Islet infiltrates of NOD.scid recipients of CD4 T cells alone were noninvasive (iv). All sections were stained with an anti-insulin antibody (red), an anti-CD8 antibody (green), and an anti-CD4 (blue).