Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue

Abstract

Activation of autoreactive T cells can lead to autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM). The initiation and maintenance of IDDM by dendritic cells (DC), the most potent professional antigen-presenting cells, were investigated in transgenic mice expressing the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) under the control of the rat insulin promoter (RIP-GP mice). We show that after adoptive transfer of DC constitutively expressing the immunodominant cytotoxic T lymphocyte (CTL) epitope of the LCMV-GP, RIP-GP mice developed autoimmune diabetes. Kinetic and functional studies of DC-activated CTL revealed that development of IDDM was dependent on dose and timing of antigenic stimulation. Strikingly, repeated CTL activation by DC led to severe destructive mononuclear infiltration of the pancreatic islets but also to de novo formation of islet-associated organized lymphoid structures in the pancreatic parenchyma. In addition, repetitive DC immunization induced IDDM with lymphoid neogenesis also in perforin-deficient RIP-GP mice, illustrating that CD8(+) T cell-dependent inflammatory mechanisms independent of perforin could induce IDDM. Thus, DC presenting self-antigens not only are potent inducers of autoreactive T cells, but also help to maintain a peripheral immune response locally; therefore, the induction of autoimmunity against previously ignored autoantigens represents a potential hazard, particularly in DC-based antitumor therapies.

Figure 1

Blood glucose levels in RIP-GP mice after single or repetitive intravenous immunization with H8-DC. Mice were immunized with either a single dose of (A) 10⁶ DC (n = 8) on day 0 or (B) 10⁵ DC (n = 4) on day 0, or repetitive doses of (C) 10⁵ DC (n = 8) on days 0, 6, and 12 or (D) 10⁴ DC (n = 8) on days 0, 2, 4, and 6. Arrows, Day of DC injection. Values of four representative mice per group are shown.

Figure 2

Expansion of GP33-specific TCR transgenic T cells by H8-DC. 5 × 10⁵ spleen cells from TCR transgenic mice were adoptively transferred into C57BL/6 mice on day −1. Mice were immunized intravenously once with (A) 10⁵ H8-DC on day 0 or repetitively with (B) 10⁵ H8-DC on days 0, 2, 4, and 6. The expansion of the TCR transgenic T cells in blood, spleen, and hepatic lymph nodes (hep. Ln) was followed by three-color FACS^® analysis and is depicted as percentage of Vα2⁺ Vβ8.1⁺ (tg-TCR ⁺) cells in the CD8⁺ compartment (mean of three animals per time point). Arrows, Day of injection of H8-DC. Data from one of two experiments are shown.

Figure 3

Immunohistological analysis of pancreatic islets and submandibular salivary glands in RIP-GP mice after H8-DC immunization. 10⁵ H8-DC were adoptively transferred on days 0, 6, and 12, and pancreata were analyzed on day 7 (A), day 14 (B), and day 21 (C) for the presence of insulin-producing islet cells, CD8⁺ and CD4⁺ T cells, B220⁺ B cells, and CD11c⁺ DC. Pancreas sections from untreated RIP-GP mice were also stained but were essentially negative except for insulin. (D) Control staining of submandibular salivary gland of a diabetic RIP-GP mouse on day 14 with the respective markers. Original magnification: ×100.

Figure 4

Organization of newly formed lymphoid tissues in RIP-GP mice after repetitive priming with H8-DC. Pancreata were analyzed by immunohistochemistry on day 21 by staining for the indicated markers. (A) Newly formed organized lymphoid tissue was found periductally in the pancreatic parenchyma (arrow); a pancreatic lymph node located outside the pancreatic parenchyma is also shown (arrowhead). Distribution of infiltrating (B) CD4⁺ T cells and (C) B cells in islets and newly formed lymphoid tissues. (D) Magnification of the boxed area in A showing an insulin-positive islet (arrow) and periductal, de novo–formed lymphoid tissue (arrowhead) in close vicinity to an artery (*). Distinct spatial distribution of (E) CD8⁺ T cells, (F) B cells, and (G) follicular DC (FDC) in the newly formed lymphoid tissue. (H) Magnification of the boxed area in G showing the 4C11⁺ follicular DC network. (I) Staining for PNAd. The region corresponding to the boxed area in G was photographed showing PNAd⁺ blood vessels in the newly formed lymphoid tissue. Original magnifications: A–C, ×24; D–G, ×63; H and I, ×250.