Central nervous system destruction mediated by glutamic acid decarboxylase-specific CD4+ T cells

Abstract

High titers of autoantibodies against glutamic acid decarboxylase (GAD) 65 are commonly observed in patients suffering from type 1 diabetes as well as stiff-person syndrome (SPS), a disorder that affects the CNS, and a variant of SPS, progressive encephalomyelitis with rigidity and myoclonus. Although there is a considerable amount of data focusing on the role of GAD65-specific CD4(+) T cells in type 1 diabetes, little is known about their role in SPS. In this study, we show that mice possessing a monoclonal GAD65-specific CD4(+) T cell population (4B5, PA19.9G11, or PA17.9G7) develop a lethal encephalomyelitis-like disease in the absence of any other T cells or B cells. GAD65-reactive CD4(+) T cells were found throughout the CNS in direct concordance with GAD65 expression and activated microglia: proximal to the circumventricular organs at the interface between the brain parenchyma and the blood-brain barrier. In the presence of B cells, high titer anti-GAD65 autoantibodies were generated, but these had no effect on the incidence or severity of disease. In addition, GAD65-specific CD4(+) T cells isolated from the brain were activated and produced IFN-gamma. These findings suggest that GAD65-reactive CD4(+) T cells alone mediate a lethal encephalomyelitis-like disease that may serve as a useful model to study GAD65-mediated diseases of the CNS.

Figure 1

GAD65-reactive CD4⁺ T cells cause a lethal encephalomyelitis-like disease. A, Incidence of disease in NOD.scid mice transplanted with bone marrow transduced with retrovirus encoding the TCR listed. B, Disease severity as determined by disease score. An EAE-based scoring system was used to measure disease severity: 0 - normal; 1 – lethargic, ruffled fur, weight loss of at least 10%, flaccid tail; 2 – 1 plus abnormal righting reflex; 3 – impaired balance and/or ataxia, involuntary movements, weakness or stiffness of limbs, usually asymmetric; 4 – paralysis and/or persistent tremors, possible incontinence; 5 – moribund or death (32). The number of mice followed for incidence is as follows: PA21.14H4 (9), 4B5 (17), PA19.9G11 (8), and PA17.9G7 (11) while 5 mice from each group were monitored for disease score as outlined in the materials and methods section. C, Pituitary (upper panel) and thalamus (lower panel) sections of mice with a Grade 3 score (Grade 0 for PA21.14H4) were stained with either anti-mouse CD4 (red), anti-GAD65 (green) and DAPI (blue), or anti-Iba-1 (green) and DAPI (blue). The diagrams on the right side depict the anterior (Ant), intermedia (Int) and posterior (Post) regions of the pituitary gland (upper panel) and a sagittal section of a brain and the location of the thalamus (lower panel). Data is representative of 6 experiments with 8–17 mice per TCR group.

Figure 2

T cell reconstitution is equivalent between Rg mice expressing GAD65-specific and control TCRs. A, Representative flow cytometric plot illustrating GFP⁺TCR⁺ cells (top panel) and CD4⁺TCR⁺ cells (bottom panel) in the spleens of representative TCR NOD.scid Rg mice. The GFP⁺TCR⁺ plots were gated on live cells and the CD4⁺TCR⁺ plots were gated on live, GFP⁺ cells. B, Mean fluorescence of TCR expression of GFP⁺ cells in spleens of Rg mice represented in A. C, Number of GFP⁺TCR⁺CD4⁺ cells in the spleens of TCR Rg mice represented in A. Data is representative of 6 experiments with 4–11 mice per TCR group.

Figure 3

The presence of B cells and GAD65 autoantibodies in Rg mice has no effect on the disease mediated by GAD65-specific CD4⁺ T cells. A, Number of CD4⁺ T cells and CD19⁺ B cells in spleens of chimeric Rg mice receiving NOD.scid bone marrow transduced with retrovirus encoding the TCR and either NOD.Tcra^−/− or NOD.scid bone marrow transduced with retrovirus encoding YFP only. Numbers were determined by gating on live, GFP⁺ (T cells) or YFP⁺ (B cells) cells. B, Left panel depicts incidence of disease and right panel indicates disease severity of chimeric Rg mice receiving 2 × 10⁶ TCR retroviral-transduced bone marrow cells from NOD.scid and vector transduced bone marrow from either NOD.scid (T-cells alone) or NOD.Tcra^−/− (T cells + B cells) mice. The T cells alone group is depicted as closed symbols and the T cells plus B cells group is depicted as open symbols with a dashed line. The number of mice followed for incidence is as follows: PA21.14H4 (4), PA21.14H4 + B cells (7), 4B5 (7), 4B5 + B cells (15), PA19.9G11 (4), PA19.9G11 + B cells (10), PA17.9G7 (5) and PA17.9G7 + B cells (8) while 5 mice from each group were monitored for disease score as outlined. C, Pituitary gland sections from NOD.scid mice receiving 4B5- or PA19.9G11-transduced NOD.scid bone marrow plus NOD.Tcra^−/− YFP only transduced bone marrow, and an unmanipulated NOD.scid mouse as control. Sections were stained with anti-mouse CD4 (T cells), anti-mouse CD19 (B cells), anti-mouse GAD65 (GAD65) or anti-mouse Iba-1 (activated macrophages/microglial) (green) and counterstained with DAPI (blue). Mice in the 4B5 + B cells and PA19.9G11 + B cells groups displayed clinical signs of disease at the time of euthanasia. D, GAD65-specific autoantibody titres in serum from the chimeric Rg mice at various states of disease are depicted. Data is representative of 4 experiments with 4–15 mice per TCR group.

Figure 4

GAD65-specific CD4⁺ T cells isolated from the brain are activated and secreting IFN-γ. A, Reactivity of GFP⁺CD4⁺ splenic T cells (2.5 × 10⁴) purified by FACS to GAD65 whole protein was determined in the presence of 5 × 10⁵ irradiated APCs for 48 hr, pulsed with [³H]-thymidine and harvested 24 hrs later. Background counts were subtracted from the data depicted. B, Concentration of IL-2, IFN-γ and IL-17 in supernatants of cells depicted in A following incubation with 10μg/ml GAD65. C, Representative flow cytometric plots of CD4⁺ IFN-γ⁺ cells in the spleen, brain and pituitary of 4B5 and PA19.9G11 retrogenic mice displaying a disease score of 3 which was six weeks post transplant. Plots were gated on GFP⁺ cells. D, Percentage of GFP⁺CD4⁺IFN-γ⁺ cells in brains of 4B5 and PA19.9G11 Rg mice six weeks post transplant, displaying a disease score of 3. For A and B data is representative of 2 experiments with spleens and lymph nodes of 3–4 mice pooled for each TCR. For C and D data is representative of 4 experiments with 4 mice for each TCR.