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The activation status of neuroantigen-specific T cells in the target organ determines the clinical outcome of autoimmune encephalomyelitis.

Kawakami N, Lassmann S, Li Z, Odoardi F, Ritter T, Ziemssen T, Klinkert WE, Ellwart JW, Bradl M, Krivacic K, Lassmann H, Ransohoff RM, Volk HD, Wekerle H, Linington C, Flügel A - J. Exp. Med. (2004)

Bottom Line: Using retrovirally transduced green fluorescent T cells, we now report that differential disease activity reflects different levels of autoreactive effector T cell activation in their target tissue.However, exclusively highly pathogenic T cells were significantly reactivated within the CNS.Low-level reactivation of weakly pathogenic T cells was not due to anergy because these cells could be activated by specific antigen in situ as well as after isolation ex vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroimmunology, Max-Planck Institute for Neurobiology, 82152 Martinsried, Germany.

ABSTRACT
The clinical picture of experimental autoimmune encephalomyelitis (EAE) is critically dependent on the nature of the target autoantigen and the genetic background of the experimental animals. Potentially lethal EAE is mediated by myelin basic protein (MBP)-specific T cells in Lewis rats, whereas transfer of S100beta- or myelin oligodendrocyte glycoprotein (MOG)-specific T cells causes intense inflammatory response in the central nervous system (CNS) with minimal disease. However, in Dark Agouti rats, the pathogenicity of MOG-specific T cells resembles the one of MBP-specific T cells in the Lewis rat. Using retrovirally transduced green fluorescent T cells, we now report that differential disease activity reflects different levels of autoreactive effector T cell activation in their target tissue. Irrespective of their pathogenicity, the migratory activity, gene expression patterns, and immigration of green fluorescent protein(+) T cells into the CNS were similar. However, exclusively highly pathogenic T cells were significantly reactivated within the CNS. Without local effector T cell activation, production of monocyte chemoattractants was insufficient to initiate and propagate a full inflammatory response. Low-level reactivation of weakly pathogenic T cells was not due to anergy because these cells could be activated by specific antigen in situ as well as after isolation ex vivo.

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Intrathecal injection of antigen activates weakly pathogenic TGFP cells and aggravates clinical disease. Intrathecal injection of 20 μg of specific antigen (A, S100β; B, MOG, closed squares and black bars) or 20 μg of control antigen (OVA, open triangles) was performed at day 4 (A, S100β-EAE) or day 5 (B, LE-MOG-EAE) after T cell transfer. Control animals, which did not receive intrathecal injection, are shown (black circles). Weight loss was monitored daily, at day 4 (A) or 5 (B) in closer time intervals (4/5a: 1 h before; 4/5b: 4 h after; and 4/5c: 8 h after intrathecal S100β/MOG injection). Animals that had received 20 μg of specific antigen intrathecally showed enhanced weight loss starting 8 h after antigen injection. Animals of TLE-MOG-GFP cell–induced EAE developed clinical symptoms reaching maximal scores of three (B, black bars). Mean value and standard deviation of five independent experiments (S100β) and three independent experiments (MOG) are shown, including eight animals/treatment group. (C) TS100β-GFP (orange histograms) or TLE-MOG-GFP cells (yellow histograms) were isolated from spinal cords of control antigen (shaded histograms, 20 μg OVA) or specific antigen (overlay histograms, 20 μg S100β or MOG, respectively)–treated animals 4 h after intrathecal injection, and were analyzed cytofluorometrically for the expression of IL-2R and OX-40 antigen (OX-40). TGFP cells from specific antigen-treated but not OVA-treated animals up-regulated OX-40 antigen and IL-2R. (D) Intracellular IFNγ staining of TS100β-GFP (orange histograms) or TLE-MOG-GFP cells (yellow histograms) after intrathecal treatment with control antigen (shaded histograms, 20 μg OVA) or specific antigen (overlay histograms, 20 μg S100β or MOG, respectively). 4 h after intrathecal antigen injection TGFP cells in the CNS (top left histogram, TS100β-GFP cells, 24% of cells were IFNγ+; middle left histogram, TLE-MOG-GFP cells, 10% of cells were IFNγ+) but not in the spleen (top right histogram, TS100β-GFP cells, 2% of cells were IFNγ+; middle right histogram, TLE-MOG-GFP cells, 1% of cells were IFNγ+) up-regulated IFNγ production. Upon stimulation with PMA/ionomycin (unshaded overlay histograms), a high percentage of TLE-MOG-GFP cells isolated from CNS (bottom left histogram), and spleen (bottom right histogram) produced IFNγ. Representative results of two independent sets of experiments/antigen are shown.
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fig6: Intrathecal injection of antigen activates weakly pathogenic TGFP cells and aggravates clinical disease. Intrathecal injection of 20 μg of specific antigen (A, S100β; B, MOG, closed squares and black bars) or 20 μg of control antigen (OVA, open triangles) was performed at day 4 (A, S100β-EAE) or day 5 (B, LE-MOG-EAE) after T cell transfer. Control animals, which did not receive intrathecal injection, are shown (black circles). Weight loss was monitored daily, at day 4 (A) or 5 (B) in closer time intervals (4/5a: 1 h before; 4/5b: 4 h after; and 4/5c: 8 h after intrathecal S100β/MOG injection). Animals that had received 20 μg of specific antigen intrathecally showed enhanced weight loss starting 8 h after antigen injection. Animals of TLE-MOG-GFP cell–induced EAE developed clinical symptoms reaching maximal scores of three (B, black bars). Mean value and standard deviation of five independent experiments (S100β) and three independent experiments (MOG) are shown, including eight animals/treatment group. (C) TS100β-GFP (orange histograms) or TLE-MOG-GFP cells (yellow histograms) were isolated from spinal cords of control antigen (shaded histograms, 20 μg OVA) or specific antigen (overlay histograms, 20 μg S100β or MOG, respectively)–treated animals 4 h after intrathecal injection, and were analyzed cytofluorometrically for the expression of IL-2R and OX-40 antigen (OX-40). TGFP cells from specific antigen-treated but not OVA-treated animals up-regulated OX-40 antigen and IL-2R. (D) Intracellular IFNγ staining of TS100β-GFP (orange histograms) or TLE-MOG-GFP cells (yellow histograms) after intrathecal treatment with control antigen (shaded histograms, 20 μg OVA) or specific antigen (overlay histograms, 20 μg S100β or MOG, respectively). 4 h after intrathecal antigen injection TGFP cells in the CNS (top left histogram, TS100β-GFP cells, 24% of cells were IFNγ+; middle left histogram, TLE-MOG-GFP cells, 10% of cells were IFNγ+) but not in the spleen (top right histogram, TS100β-GFP cells, 2% of cells were IFNγ+; middle right histogram, TLE-MOG-GFP cells, 1% of cells were IFNγ+) up-regulated IFNγ production. Upon stimulation with PMA/ionomycin (unshaded overlay histograms), a high percentage of TLE-MOG-GFP cells isolated from CNS (bottom left histogram), and spleen (bottom right histogram) produced IFNγ. Representative results of two independent sets of experiments/antigen are shown.

Mentions: The failure of the CNS to activate infiltrating S100β and MOG-specific T cells in the Lewis rat could be explained if T cells with these specificities were rendered functionally unresponsive. Therefore, we isolated TS100β−GFP, TMBP-GFP, TLE-MOG-GFP, and TDA-MOG-GFP cells from the spleen 4 d after transfer and immediately confronted them with their cognate antigen in vitro (Table III). Irrespective of their antigen specificity or ability to induce clinical EAE, all four TCLs retained their ability to proliferate in response to their cognate antigen in vitro (Table III). Furthermore, intracellular IFNγ staining revealed that TS100β−GFP and TLE-MOG-GFP cells isolated from spleen and CNS can be induced to express IFNγ after stimulation with PMA/ionomycin in vitro (Fig. 5, G and H, and Fig. 6 D). Therefore, the inability of certain T cell specificities to induce clinical EAE in the Lewis rat cannot be attributed to anergy.


The activation status of neuroantigen-specific T cells in the target organ determines the clinical outcome of autoimmune encephalomyelitis.

Kawakami N, Lassmann S, Li Z, Odoardi F, Ritter T, Ziemssen T, Klinkert WE, Ellwart JW, Bradl M, Krivacic K, Lassmann H, Ransohoff RM, Volk HD, Wekerle H, Linington C, Flügel A - J. Exp. Med. (2004)

Intrathecal injection of antigen activates weakly pathogenic TGFP cells and aggravates clinical disease. Intrathecal injection of 20 μg of specific antigen (A, S100β; B, MOG, closed squares and black bars) or 20 μg of control antigen (OVA, open triangles) was performed at day 4 (A, S100β-EAE) or day 5 (B, LE-MOG-EAE) after T cell transfer. Control animals, which did not receive intrathecal injection, are shown (black circles). Weight loss was monitored daily, at day 4 (A) or 5 (B) in closer time intervals (4/5a: 1 h before; 4/5b: 4 h after; and 4/5c: 8 h after intrathecal S100β/MOG injection). Animals that had received 20 μg of specific antigen intrathecally showed enhanced weight loss starting 8 h after antigen injection. Animals of TLE-MOG-GFP cell–induced EAE developed clinical symptoms reaching maximal scores of three (B, black bars). Mean value and standard deviation of five independent experiments (S100β) and three independent experiments (MOG) are shown, including eight animals/treatment group. (C) TS100β-GFP (orange histograms) or TLE-MOG-GFP cells (yellow histograms) were isolated from spinal cords of control antigen (shaded histograms, 20 μg OVA) or specific antigen (overlay histograms, 20 μg S100β or MOG, respectively)–treated animals 4 h after intrathecal injection, and were analyzed cytofluorometrically for the expression of IL-2R and OX-40 antigen (OX-40). TGFP cells from specific antigen-treated but not OVA-treated animals up-regulated OX-40 antigen and IL-2R. (D) Intracellular IFNγ staining of TS100β-GFP (orange histograms) or TLE-MOG-GFP cells (yellow histograms) after intrathecal treatment with control antigen (shaded histograms, 20 μg OVA) or specific antigen (overlay histograms, 20 μg S100β or MOG, respectively). 4 h after intrathecal antigen injection TGFP cells in the CNS (top left histogram, TS100β-GFP cells, 24% of cells were IFNγ+; middle left histogram, TLE-MOG-GFP cells, 10% of cells were IFNγ+) but not in the spleen (top right histogram, TS100β-GFP cells, 2% of cells were IFNγ+; middle right histogram, TLE-MOG-GFP cells, 1% of cells were IFNγ+) up-regulated IFNγ production. Upon stimulation with PMA/ionomycin (unshaded overlay histograms), a high percentage of TLE-MOG-GFP cells isolated from CNS (bottom left histogram), and spleen (bottom right histogram) produced IFNγ. Representative results of two independent sets of experiments/antigen are shown.
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Related In: Results  -  Collection

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fig6: Intrathecal injection of antigen activates weakly pathogenic TGFP cells and aggravates clinical disease. Intrathecal injection of 20 μg of specific antigen (A, S100β; B, MOG, closed squares and black bars) or 20 μg of control antigen (OVA, open triangles) was performed at day 4 (A, S100β-EAE) or day 5 (B, LE-MOG-EAE) after T cell transfer. Control animals, which did not receive intrathecal injection, are shown (black circles). Weight loss was monitored daily, at day 4 (A) or 5 (B) in closer time intervals (4/5a: 1 h before; 4/5b: 4 h after; and 4/5c: 8 h after intrathecal S100β/MOG injection). Animals that had received 20 μg of specific antigen intrathecally showed enhanced weight loss starting 8 h after antigen injection. Animals of TLE-MOG-GFP cell–induced EAE developed clinical symptoms reaching maximal scores of three (B, black bars). Mean value and standard deviation of five independent experiments (S100β) and three independent experiments (MOG) are shown, including eight animals/treatment group. (C) TS100β-GFP (orange histograms) or TLE-MOG-GFP cells (yellow histograms) were isolated from spinal cords of control antigen (shaded histograms, 20 μg OVA) or specific antigen (overlay histograms, 20 μg S100β or MOG, respectively)–treated animals 4 h after intrathecal injection, and were analyzed cytofluorometrically for the expression of IL-2R and OX-40 antigen (OX-40). TGFP cells from specific antigen-treated but not OVA-treated animals up-regulated OX-40 antigen and IL-2R. (D) Intracellular IFNγ staining of TS100β-GFP (orange histograms) or TLE-MOG-GFP cells (yellow histograms) after intrathecal treatment with control antigen (shaded histograms, 20 μg OVA) or specific antigen (overlay histograms, 20 μg S100β or MOG, respectively). 4 h after intrathecal antigen injection TGFP cells in the CNS (top left histogram, TS100β-GFP cells, 24% of cells were IFNγ+; middle left histogram, TLE-MOG-GFP cells, 10% of cells were IFNγ+) but not in the spleen (top right histogram, TS100β-GFP cells, 2% of cells were IFNγ+; middle right histogram, TLE-MOG-GFP cells, 1% of cells were IFNγ+) up-regulated IFNγ production. Upon stimulation with PMA/ionomycin (unshaded overlay histograms), a high percentage of TLE-MOG-GFP cells isolated from CNS (bottom left histogram), and spleen (bottom right histogram) produced IFNγ. Representative results of two independent sets of experiments/antigen are shown.
Mentions: The failure of the CNS to activate infiltrating S100β and MOG-specific T cells in the Lewis rat could be explained if T cells with these specificities were rendered functionally unresponsive. Therefore, we isolated TS100β−GFP, TMBP-GFP, TLE-MOG-GFP, and TDA-MOG-GFP cells from the spleen 4 d after transfer and immediately confronted them with their cognate antigen in vitro (Table III). Irrespective of their antigen specificity or ability to induce clinical EAE, all four TCLs retained their ability to proliferate in response to their cognate antigen in vitro (Table III). Furthermore, intracellular IFNγ staining revealed that TS100β−GFP and TLE-MOG-GFP cells isolated from spleen and CNS can be induced to express IFNγ after stimulation with PMA/ionomycin in vitro (Fig. 5, G and H, and Fig. 6 D). Therefore, the inability of certain T cell specificities to induce clinical EAE in the Lewis rat cannot be attributed to anergy.

Bottom Line: Using retrovirally transduced green fluorescent T cells, we now report that differential disease activity reflects different levels of autoreactive effector T cell activation in their target tissue.However, exclusively highly pathogenic T cells were significantly reactivated within the CNS.Low-level reactivation of weakly pathogenic T cells was not due to anergy because these cells could be activated by specific antigen in situ as well as after isolation ex vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroimmunology, Max-Planck Institute for Neurobiology, 82152 Martinsried, Germany.

ABSTRACT
The clinical picture of experimental autoimmune encephalomyelitis (EAE) is critically dependent on the nature of the target autoantigen and the genetic background of the experimental animals. Potentially lethal EAE is mediated by myelin basic protein (MBP)-specific T cells in Lewis rats, whereas transfer of S100beta- or myelin oligodendrocyte glycoprotein (MOG)-specific T cells causes intense inflammatory response in the central nervous system (CNS) with minimal disease. However, in Dark Agouti rats, the pathogenicity of MOG-specific T cells resembles the one of MBP-specific T cells in the Lewis rat. Using retrovirally transduced green fluorescent T cells, we now report that differential disease activity reflects different levels of autoreactive effector T cell activation in their target tissue. Irrespective of their pathogenicity, the migratory activity, gene expression patterns, and immigration of green fluorescent protein(+) T cells into the CNS were similar. However, exclusively highly pathogenic T cells were significantly reactivated within the CNS. Without local effector T cell activation, production of monocyte chemoattractants was insufficient to initiate and propagate a full inflammatory response. Low-level reactivation of weakly pathogenic T cells was not due to anergy because these cells could be activated by specific antigen in situ as well as after isolation ex vivo.

Show MeSH
Related in: MedlinePlus