Glatiramer acetate-specific T cells in the brain express T helper 2/3 cytokines and brain-derived neurotrophic factor in situ

Abstract

The ability of a remedy to modulate the pathological process in the target organ is crucial for its therapeutic activity. Glatiramer acetate (GA, Copaxone, Copolymer 1), a drug approved for the treatment of multiple sclerosis, induces regulatory T helper 2/3 cells that penetrate the CNS. Here we investigated whether these GA-specific T cells can function as suppressor cells with therapeutic potential in the target organ by in situ expression of T helper 2/3 cytokines and neurotrophic factors. GA-specific cells and their in situ expression were detected on the level of whole-brain tissue by using a two-stage double-labeling system: (i) labeling of the GA-specific T cells, followed by their adoptive transfer, and (ii) detection of the secreted factors in the brain by immunohistological methods. GA-specific T cells in the CNS demonstrated intense expression of the brain-derived neurotrophic factor and of two antiinflammatory cytokines, IL-10 and transforming growth factor beta. No expression of the inflammatory cytokine IFN-gamma was observed. This pattern of expression was manifested in brains of normal and experimental autoimmune encephalomyelitis-induced mice to which GA-specific cells were adoptively transferred, but not in control mice. Furthermore, infiltration of GA-induced cells to the brain resulted in bystander expression of IL-10 and transforming growth factor beta by resident astrocytes and microglia. The ability of infiltrating GA-specific cells to express antiinflammatory cytokines and neurotrophic factor in the organ in which the pathological processes occur correlates directly with the therapeutic activity of GA in experimental autoimmune encephalomyelitis/multiple sclerosis.

Fig. 1.

Immunohistochemical analysis of BDNF expression by GA-specific cells in the brain. Activated labeled GA-specific cells were injected into the peritoneum of either EAE-induced (A–C, G–J, and M–O) or normal (D–F) mice. After 7 days, the mice were perfused and brain sections (20 μm) were stained immunocytochemically for BDNF expression. (A–C) Area surrounding the lateral ventricle. (D–F) Choroid plexus in the lateral ventricle. (G–I) Perivascular infiltration in the cortex. (J) Overlap image of enlarged Hoechst-labeled, GA-specific cells and their BDNF secretion in the cortex. (M–O) Enlarged images of single GA-specific PK-labeled cells in the cortex. Controls demonstrate BDNF expression in corresponding brain regions of an EAE-induced mouse (K) and a normal mouse (L). (Scale bar: A–F, K, and L, 100 μm; G–I, 20 μm; J and M–O, 10 μm.)

Fig. 2.

Immunohistochemical analysis of IL-10 expression by GA-specific cells in brains of EAE-induced mice. (A–F) Perivascular infiltration in the thalamus. (G–I) Enlarged images of single cells in the thalamus. (L–N) Single cells in the cortex. (O–R) Nonoverlapping distribution of IL-10-expressing, GA-labeled cells and additional population of cells expressing IL-10 and GFAP in tissue surrounding the lateral ventricle. Controls demonstrate IL-10 expression in corresponding brain regions of an EAE-induced mouse (J) and a normal mouse (K). (Scale bar: A–C, 50 μm; D–F, 20 μm; G–I and L–N, 10 μm; J, K, and O–R, 100 μm.)

Fig. 3.

Immunohistochemical analysis of TGF-β expression by GA-specific cells in brains of EAE-induced mice. (A–D) Perivascular infiltration in the cortex. (G–I) 3D reconstruction of PK-labeled cells in the thalamus by confocal scanning microscope. (J–M) Nonoverlapping distribution of TGF-β-expressing GA-labeled cells and additional population of cells expressing TGF-β and GFAP in tissue surrounding a blood vessel. Controls demonstrate TGF-β in corresponding brain region of an EAE-induced mouse (E) and a normal mouse (F) in the cortex. (Scale bar: A–C, 50 μm; D, 20 μm; E, F, and M–P, 100 μm; G–L, 10 μm.)

Fig. 4.

Lack of expression of IFN-γ by GA-specific cells in the brain. Cells in the cortex of an EAE-induced mouse (A–C) and a normal mouse (D–F). Expression of IFN-γ in corresponding brain sections of controls: an EAE-induced mouse (G and H) and a normal mouse (I). (Scale bar: A–F and H,40 μm; G and I, 100 μm.)