Autoreactive human T-cell receptor initiates insulitis and impaired glucose tolerance in HLA DR4 transgenic mice

Abstract

A human T-cell receptor (TcR) derived from an autoreactive T-cell specific for GAD65, from a subject at high risk for autoimmune diabetes, was introduced into HLA-DR4 transgenic mice. The source of TcR was a CD4(+) T(H)1(+) T-cell clone which responded to an immunodominant epitope of the human islet protein GAD65, an epitope shared with both GAD65 and GAD67 in the mouse. The resulting HLA-DR4/GAD-TcR transgenic mice on a Rag2(o/o)/I-Ab(o/o)/B6 background exhibited a CD4(+) infiltrate into pancreatic islets that correlated with a loss of insulin in infiltrated islets. These mice also exhibited a subclinical impaired tolerance to exogenously fed glucose as assayed by an intraperitoneal glucose tolerance test. T cells containing the GAD65/67 (555-567) responsive TcR undergo strong negative selection as evidenced by a 10-fold lower thymocyte cellularity compared to non-TcR transgenic mice, and clonotype peripheral T cells represented approximately 1% of CD4(+) T cells in Rag2 sufficient mice. Upon in vitro stimulation, GAD65/67 555-567 responsive T cells secrete interferon-gamma, minimal interleukin (IL)-2 and tumor necrosis factor-alpha, and no IL-4, IL-5, IL-10, or IL-17, consistent with a T(H)1 profile. These data demonstrate that CD4(+) T cells specific for a naturally processed epitope within GAD can specifically home to pancreatic islets and lead to impaired islet beta-cell function in diabetes-associated HLA-DR4 transgenic mice on the relatively non-autoimmune C57BL/6 background. The relatively slow progression and patchy insulitis are reminiscent of the chronic pre-clinical phase similar to a majority of human at-risk subjects, and models these indolent features of human T1D.

Figure 1

Tissue cellularity and GAD TcR expression in DR4/164 GAD65 555–567 specific TcR transgenic mice. Cellularity for Thymus (A), Spleen (B), and Lymph nodes(C) represent the average counts (n=3 mice each) for Non TcR transgenic (Non TcR tg) and TcR transgenic mice (TcR tg) at 8 weeks of age. CD4 vs. CD8 profiles in thymus of DR4 and DR4/164 GAD TcR mice (D). Expression of human TcR Vβ5.1 transgene on CD4⁺ lymph node cells from the DR4/164 mouse is shown (E) along with clonotypic expression of the Vα12.1/Vβ5.1 TcR on CD4-gated lymph node cells (F). CD4⁺ human Vβ5.1⁺ transgenic T cells express an increase in the use of endogenous mouse TcR Vα compared to Vβ5.1⁻ T cells (G).

Figure 2

Intracellular FoxP3 expression on CD4⁺ T cells in GAD65 555–567 TcR transgenic and non-transgenic DR4 mice. FoxP3 expression in lymph node cells from DR4/164 TcR transgenic mice showing a correlation of FoxP3 expression with CD25 expression on CD4⁺ gated cells (A). Average percentages from 3 mice (8 wks of age) of CD4⁺ FoxP3⁺ expressing cells of all CD4⁺ cells (B). Percentages are shown for CD4⁺/Vβ5.1⁺/FoxP3⁺ (Tg Vβ⁺) and CD4⁺/Vβ5.1⁻/FoxP3⁺ (TgVβ⁻) for DR4/164 GAD-TcR mice and for non-TcR transgenic mice (Non-tg).

Figure 3

Antigen-specific dose-response and cytokine analysis of in vitro stimulated 164 GAD TcR T cells. Splenocytes from Dw14/164/Rag2^o/o mice (2e5) were stimulated with increasing amount of GAD65 555–567 or control IGRP 247–258 peptide for 96 hrs. 3H-thymidine was added at 72 hrs and incorporation was assayed by scintillation counting (A). Cytokines (pg/ml) from supernatants from proliferation assays (at 100 µg/ml antigen) taken at 72 hours (B). Experiment was repeated 3 times with similar results.

Figure 4

TcR transgenic CD4⁺ cells in DR4/164/Rag2^o/o mice exhibit an activated phenotype. CD4⁺ lymph node T cells, but not CD8⁺ cells from DR4/164/Rag2^o/o mice (10 weeks of age) are CD44^Hi and CD62L⁻.

Figure 5

Islet cell infiltrate in DR4/164/Rag2^o/o TcR transgenic mice. H & E staining on formalin fixed paraffin embedded pancreatic tissue from a 28 week old female DR4/164/Rag2^o/o mouse (A). Islets were scored as 0-no infiltrate, 1-less than 33%, 2-less than 66%, or 3-greater than 66%. A summary of islet infiltrate scores for 12 female and 6 male DR4/164/Rag2^o/o mice is shown in (B) and (C) respectively. Islets were scored as 0-no infiltrate (white bars), 1-less than 33% (light grey bars), 2-less than 66% (dark grey bars), or 3-greater than 66% (black bars).

Figure 6

Detection of CD4⁺ cells and loss of insulin staining in infiltrated islets from DR4/164/Rag2^o/o mice. Immunofluorescence staining for insulin (blue) and CD4 (green) in an infiltrated islet from a 28 week old female DR4/164/Rag2^o/o mouse (A). Immunohistochemistry staining for insulin (brown) in infiltrated islets from a female DR4/164/Rag2^o/o mouse (B).

Figure 7

Impaired response to exogenous glucose in DR4/164/Rag2^o/o mice. DR4/164/Rag2^o/o (164, filled circles) and DR4/Rag2^o/o (Non TcR tg, open circles) mice were fasted for 6 hours prior to glucose injection and blood glucose monitoring (see methods). 4 mice 30 weeks of age were used in each group.