Perforin-independent beta-cell destruction by diabetogenic CD8(+) T lymphocytes in transgenic nonobese diabetic mice

Abstract

Autoimmune diabetes in nonobese diabetic (NOD) mice results from destruction of pancreatic beta cells by T lymphocytes. It is believed that CD8(+) cytotoxic T lymphocytes (CTLs) effect the initial beta-cell insult in diabetes, but the mechanisms remain unclear. Studies of NOD.lpr mice have suggested that disease initiation is a Fas-dependent process, yet perforin-deficient NOD mice rarely develop diabetes despite expressing Fas. Here, we have investigated the role of perforin and Fas in the ability of beta cell-reactive CD8(+) T cells bearing a T-cell receptor (8.3-TCR) that is representative of TCRs used by CD8(+) CTLs propagated from the earliest insulitic lesions of NOD mice, and that targets an immunodominant peptide/H-2Kd complex on beta cells, to effect beta-cell damage in vitro and in vivo. In vitro, 8.3-CTLs killed antigenic peptide-pulsed non-beta-cell targets via both perforin and Fas, but they killed NOD beta cells via Fas exclusively. Perforin-deficient 8.3-TCR-transgenic NOD mice expressing an oligoclonal or monoclonal T-cell repertoire developed diabetes even more frequently than their perforin-competent littermates. These results demonstrate that diabetogenic CD8(+) CTLs representative of CTLs putatively involved in the initiation of autoimmune diabetes kill beta cells in a Fas-dependent and perforin-independent manner.

Figure 1

Incidence of autoimmune diabetes in NOD/Lt, NOD.PO^+/–, and NOD.PO^–/– mice. Data corresponds to 41 NOD.PO^–/– females, 31 NOD.PO^–/– males, 21 NOD.PO^+/– females, seven NOD.PO^+/– males, 114 NOD/Lt females, and 59 NOD/Lt males. See the text for statistics.

Figure 2

Flow cytometric profiles of thymocytes and splenocytes from 8.3-NOD.PO^+/– and 8.3-NOD.PO^–/– mice. CD4, CD8, and Vβ8.1/8.2 profiles of thymocytes (a) and splenic cells (b) from 8.3-NOD.PO^+/– and 8.3-NOD.PO^–/– mice (n = 4–6 mice per group, 8–12 weeks old). Upper panels show CD4 vs. CD8 contour plots of cells stained with anti-CD8/PE, anti–Vβ8.1/8.2-FITC, and anti-CD4/biotin plus Streptavidin-PerCP. The lower panels show the Vβ8.1/8.2 fluorescence histograms of each T-cell subset after electronic gating. Numbers indicate the average percentage of cells (upper panels) or number of Vβ8.1/8.2⁺ cells (lower panels) in each subset. DN, double-negative cells. DP, double-positive cells.

Figure 3

Functional activity of 8.3-CD8⁺ T cells from 8.3-NOD.PO^–/– mice. (a) Proliferation of CD8⁺ T cells from NOD/Lt, 8.3-NOD.PO^+/–, and 8.3-NOD.PO^–/– mice in response to NOD islet cells. (b) Cytokine secretion by splenic CD8⁺ T cells from NOD/Lt, 8.3-NOD.PO^+/–, and 8.3-NOD.PO^–/– mice in response to stimulation with NOD islet cells. (c) Differentiation of 8.3-CTLp from 8.3-NOD.PO^+/– and 8.3-NOD.PO^–/– mice into CTL. Splenic CD8⁺ T cells were incubated with NRP-pulsed NOD splenocytes for seven days and then used as effectors in ⁵¹Cr release assays employing NRP- or tum-pulsed RMA-SK^d cells as targets (at a 1:10 target/effector ratio). Bars show the SEMs. (d) Cytotoxic activity of islet-derived CD8⁺ T-cell clones from 8.3-NOD.PO^–/– mice at a 1:3 target/effector ratio.

Figure 4

Cytotoxic activity of 8.3-CTLs from 8.3-NOD.PO^+/– and 8.3-NOD.PO^–/– mice against peptide-pulsed L1210-Fas⁺ and L1210-Fas^– cells. 8.3-CTL lines (a) and clones (b) were generated as in Figure 3 and tested at a 1:10 (for lines) or 1:3 (for clones) target/effector ratio . Bars show the SEMs.

Figure 5

β-cell cytotoxicity of 8.3-CTLs from 8.3-NOD.PO^+/– and 8.3-NOD.PO^–/– mice. NRP-differentiated 8.3-CTLs were tested in cytotoxicity assays against islet cells from NOD/Lt, C57BL/6, and NOD.lpr/lpr mice (at a 1:10 target/effector ratio).

Figure 6

Spontaneous diabetes in 8.3-NOD.PO^+/– and 8.3-NOD.PO^–/– mice. Data corresponds to 17 8.3-NOD.PO^+/– females, 24 8.3-NOD.PO^+/– males, 44 8.3-NOD.PO^–/– females, and 27 8.3-NOD.PO^–/– males. See the text for statistics.

Figure 7

Spontaneous diabetes in RAG-2^–/– 8.3-NOD.PO^+/+ vs. RAG-2^–/– 8.3-NOD.PO^–/– mice. Data corresponds to five RAG-2^–/– 8.3-NOD.PO^–/– females, six RAG-2^–/– 8.3-NOD.PO^–/– males, 38 RAG-2^–/– 8.3-NOD.PO^+/+ females, and 34 RAG-2^–/– 8.3-NOD.PO^+/+ males. See text for statistics.