Initiation of autoimmune diabetes by developmentally regulated presentation of islet cell antigens in the pancreatic lymph nodes

Abstract

Little is known about the events triggering lymphocyte invasion of the pancreatic islets in prelude to autoimmune diabetes. For example, where islet-reactive T cells first encounter antigen has not been identified. We addressed this issue using BDC2.5 T cell receptor transgenic mice, which express a receptor recognizing a natural islet beta cell antigen. In BDC2.5 animals, activated T cells were found only in the islets and the lymph nodes draining them, and there was a close temporal correlation between lymph node T cell activation and islet infiltration. When naive BDC2.5 T cells were transferred into nontransgenic recipients, proliferating cells were observed only in pancreatic lymph nodes, and this occurred significantly before insulitis was detectable. Surprisingly, proliferation was not seen in 10-day-old recipients. This age-dependent dichotomy was reproduced in a second transfer system based on an unrelated antigen artificially expressed on beta cells. We conclude that beta cell antigens are transported specifically to pancreatic lymph nodes, where they trigger reactive T cells to invade the islets. Systemic or extrapancreatic T cell priming, indicative of activation via molecular mimicry or superantigens, was not seen. Compromised presentation of beta cell antigens in the pancreatic lymph nodes of juvenile animals may be the root of a first "checkpoint" in diabetes progression.

Figure 1

Activated lymphocytes in BDC2.5 mice. (A) CD69 expression on CD4⁺Vβ4⁺ T cells from spleen (SP), ILNs, MLNs, PLNs, and IILs from a young BDC2.5 mouse. The percentage of positive cells included within the indicated region is shown for each histogram. (B) Representative biparametric CD69/CD44 and CD69/ CD62L histograms of gated CD4⁺ T cells from the same sources. (C) Underrepresentation of nontransgenic Vα2⁺ TCRs among CD69⁺CD4⁺ lymphocytes in PLNs compared with ILNs from BDC2.5 mice. Dashed line, an approximation of the percentage of Vα2⁺ cells within CD4⁺ T cells in the whole lymphoid system of the analyzed mouse (estimated from pooled data of spleen and LN T cells).

Figure 2

Temporal correlation between the activation of T cells in PLNs and islet infiltration in young BDC2.5 mice. Percentage of infiltrated islets was plotted against the percentage of CD69⁺ among CD4⁺ T cells in PLNs in BDC2.5 mice of different ages. Each dot represents an individual mouse.

Figure 3

Proliferation of CFSE-labeled transferred BDC2.5 spleen cells occurs selectively in PLNs. (A) The transfer protocol: splenocytes from BDC2.5 mice are labeled with CFSE in vitro, then transferred to Cα⁰/NOD recipients. Proliferation of the transferred CD4⁺Vβ4⁺ T cells is visualized, a few days after transfer, as progressive decrease of labeling intensity in individual cells. (B) CFSE label 3 d after transfer to Cα⁰/ NOD recipients in PLNs, ILNs, axillary LNs (ALN), cervical LNs (CLN), MLNs, RLNs, iliac LNs (ILILN), parathymic LNs (PATLN), and spleen. (C) Proliferation in PLNs occurs in Cα⁰/NOD, RAG⁰/NOD, and in normal NOD recipients.

Figure 4

Proliferation of transferred BDC2.5 cells in the PLNs occurs before islet cell infiltration. In a representative experiment, a cohort of Cα⁰/NOD mice were injected with 5 × 10⁷ CFSE-labeled BDC2.5 spleen cells and subsequently analyzed for CFSE dilution in PLNs and ILNs at various times after transfer. The “proliferation index” shown for each histogram is obtained by dividing the fraction of proliferating cells by the fraction of nonproliferating cells (gates as shown). Insulitis values on right: number of infiltrated islets over the total number of islets scored.

Figure 5

A comparison between adult and 10-d-old BDC2.5 mice as donors and recipients in the transfer model. (A) 5 × 10⁷ T cells from 10-d-old or adult BDC2.5 donors were transferred to adult Cα⁰/NOD recipients. The graph is a summary of several independent experiments, showing the proliferation index of CD4⁺Vβ4⁺ T cells in PLNs at different time points, calculated as for Fig. 4; each dot represents an individual mouse. (B) BDC2.5 donor T cells were transferred to adult or 10-d-old Cα⁰/NOD mice, as indicated (proliferation index as above).

Figure 6

Proliferation of OVA-specific CD8⁺ T cells in adult and 10-d-old RIP-mOVA mice. Recipients were injected with 2 × 10⁷ splenocytes from OT-I transgenic mice, and loss of CFSE by proliferation was evaluated in gated CD8⁺ T cells in ILNs, RLNs, and PLNs (see above) 67 h after transfer.