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The cellular location of self-antigen determines the positive and negative selection of autoreactive B cells.

Ferry H, Jones M, Vaux DJ, Roberts IS, Cornall RJ - J. Exp. Med. (2003)

Bottom Line: To test for differences in the B cell response to intracellular and extracellular self-antigens, we sequestered a tolerogenic cell surface antigen intracellularly by addition of a two amino acid endoplasmic reticulum (ER) retention signal.In contrast to cell surface antigen, which causes the deletion of autoreactive B cells, the intracellularly sequestered self-antigen failed to induce B cell tolerance and was instead autoimmunogenic.The intracellular antigen positively selected antigen-binding B cells to differentiate into B1 cells and induced large numbers of IgM autoantibody-secreting plasma cells in a T-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.

ABSTRACT
Systemic autoimmune disease is frequently characterized by the production of autoantibodies against widely expressed intracellular self-antigens, whereas B cell tolerance to ubiquitous and highly expressed extracellular antigens is strictly enforced. To test for differences in the B cell response to intracellular and extracellular self-antigens, we sequestered a tolerogenic cell surface antigen intracellularly by addition of a two amino acid endoplasmic reticulum (ER) retention signal. In contrast to cell surface antigen, which causes the deletion of autoreactive B cells, the intracellularly sequestered self-antigen failed to induce B cell tolerance and was instead autoimmunogenic. The intracellular antigen positively selected antigen-binding B cells to differentiate into B1 cells and induced large numbers of IgM autoantibody-secreting plasma cells in a T-independent manner. By analyzing the impact of differences in subcellular distribution independently from other variables, such as B cell receptor affinity, antigen type, or tissue distribution, we have established that intracellular localization of autoantigen predisposes for autoantibody production. These findings help explain why intracellular antigens are targeted in systemic autoimmune diseases.

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Generation of a transgene expressing a ubiquitous intracellular membrane-bound lysozyme. Derived from the construct used to express mHEL on the cell surface (KLK) (1), the sole modification is the addition of a dilysine ER reteintion motif to the cytoplasmic tail (black box). In other respects, the constructs are identical, including the lysozyme domain and the H-2Kb transmembrane region. Restriction sites: X, XhoI; N, NotI; S, SalI; C, ClaI.
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fig1: Generation of a transgene expressing a ubiquitous intracellular membrane-bound lysozyme. Derived from the construct used to express mHEL on the cell surface (KLK) (1), the sole modification is the addition of a dilysine ER reteintion motif to the cytoplasmic tail (black box). In other respects, the constructs are identical, including the lysozyme domain and the H-2Kb transmembrane region. Restriction sites: X, XhoI; N, NotI; S, SalI; C, ClaI.

Mentions: To study the mechanisms involved in maintaining tolerance to intracellular self-antigens, we modified the tolerogenic cell surface mHEL construct by adding a dilysine ER retention motif to the COOH-terminal cytoplasmic tail (mHEL-KK; Fig. 1) (29). Proteins carrying dilysine or other dibasic motifs bind to cytosolic COP I proteins, which causes their continuous and avid retrieval from the golgi to the ER (30). Apart from the cytoplasmic tail, the mHEL and mHEL-KK constructs were identical and shared the same ubiquitous class I promoter, extracytoplasmic lysozyme domain, and transmembrane segment. Three lines of transgenic mice carrying the ER-restricted HEL were produced using fertilized eggs from (CBA×C57BL/6)F1 mice and backcrossed six generations to C57BL/6. The transgenic lines, designated mHEL-KK1, mHEL-KK2, and mHEL-KK3, were healthy and bred normally. Histological examination of fixed and permeabilized spleen and thymus showed expression in many cells with a typical ER distribution on confocal microscopy (Fig. 2, A and B) . Flow cytometric analysis for cell surface and intracellular antigen in spleen and BM cells with anti-HEL antibodies confirmed that expression was intracellular (Fig. 2 C). In each organ sample preparation there were a minority of cells that were positive for surface-exposed HEL, ranging from 0.015% lymphocytes in BM to 0.4% in spleen samples (Fig. 2 D), but these were membrane-permeable necrotic or late stage apoptotic cells as determined by staining with propidium iodide (not depicted). In transgenic mice expressing mHEL on the cell surface, sHEL is present in serum at levels in excess of 200 ng/ml due to proteolytic cleavage from the intact protein (unpublished data). In the mHEL-KK lines, sHEL was present in a range of 2–10 ng/ml, which would be compatible with release from cellular debris or rare surface expression (Fig. 2 E).


The cellular location of self-antigen determines the positive and negative selection of autoreactive B cells.

Ferry H, Jones M, Vaux DJ, Roberts IS, Cornall RJ - J. Exp. Med. (2003)

Generation of a transgene expressing a ubiquitous intracellular membrane-bound lysozyme. Derived from the construct used to express mHEL on the cell surface (KLK) (1), the sole modification is the addition of a dilysine ER reteintion motif to the cytoplasmic tail (black box). In other respects, the constructs are identical, including the lysozyme domain and the H-2Kb transmembrane region. Restriction sites: X, XhoI; N, NotI; S, SalI; C, ClaI.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194246&req=5

fig1: Generation of a transgene expressing a ubiquitous intracellular membrane-bound lysozyme. Derived from the construct used to express mHEL on the cell surface (KLK) (1), the sole modification is the addition of a dilysine ER reteintion motif to the cytoplasmic tail (black box). In other respects, the constructs are identical, including the lysozyme domain and the H-2Kb transmembrane region. Restriction sites: X, XhoI; N, NotI; S, SalI; C, ClaI.
Mentions: To study the mechanisms involved in maintaining tolerance to intracellular self-antigens, we modified the tolerogenic cell surface mHEL construct by adding a dilysine ER retention motif to the COOH-terminal cytoplasmic tail (mHEL-KK; Fig. 1) (29). Proteins carrying dilysine or other dibasic motifs bind to cytosolic COP I proteins, which causes their continuous and avid retrieval from the golgi to the ER (30). Apart from the cytoplasmic tail, the mHEL and mHEL-KK constructs were identical and shared the same ubiquitous class I promoter, extracytoplasmic lysozyme domain, and transmembrane segment. Three lines of transgenic mice carrying the ER-restricted HEL were produced using fertilized eggs from (CBA×C57BL/6)F1 mice and backcrossed six generations to C57BL/6. The transgenic lines, designated mHEL-KK1, mHEL-KK2, and mHEL-KK3, were healthy and bred normally. Histological examination of fixed and permeabilized spleen and thymus showed expression in many cells with a typical ER distribution on confocal microscopy (Fig. 2, A and B) . Flow cytometric analysis for cell surface and intracellular antigen in spleen and BM cells with anti-HEL antibodies confirmed that expression was intracellular (Fig. 2 C). In each organ sample preparation there were a minority of cells that were positive for surface-exposed HEL, ranging from 0.015% lymphocytes in BM to 0.4% in spleen samples (Fig. 2 D), but these were membrane-permeable necrotic or late stage apoptotic cells as determined by staining with propidium iodide (not depicted). In transgenic mice expressing mHEL on the cell surface, sHEL is present in serum at levels in excess of 200 ng/ml due to proteolytic cleavage from the intact protein (unpublished data). In the mHEL-KK lines, sHEL was present in a range of 2–10 ng/ml, which would be compatible with release from cellular debris or rare surface expression (Fig. 2 E).

Bottom Line: To test for differences in the B cell response to intracellular and extracellular self-antigens, we sequestered a tolerogenic cell surface antigen intracellularly by addition of a two amino acid endoplasmic reticulum (ER) retention signal.In contrast to cell surface antigen, which causes the deletion of autoreactive B cells, the intracellularly sequestered self-antigen failed to induce B cell tolerance and was instead autoimmunogenic.The intracellular antigen positively selected antigen-binding B cells to differentiate into B1 cells and induced large numbers of IgM autoantibody-secreting plasma cells in a T-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.

ABSTRACT
Systemic autoimmune disease is frequently characterized by the production of autoantibodies against widely expressed intracellular self-antigens, whereas B cell tolerance to ubiquitous and highly expressed extracellular antigens is strictly enforced. To test for differences in the B cell response to intracellular and extracellular self-antigens, we sequestered a tolerogenic cell surface antigen intracellularly by addition of a two amino acid endoplasmic reticulum (ER) retention signal. In contrast to cell surface antigen, which causes the deletion of autoreactive B cells, the intracellularly sequestered self-antigen failed to induce B cell tolerance and was instead autoimmunogenic. The intracellular antigen positively selected antigen-binding B cells to differentiate into B1 cells and induced large numbers of IgM autoantibody-secreting plasma cells in a T-independent manner. By analyzing the impact of differences in subcellular distribution independently from other variables, such as B cell receptor affinity, antigen type, or tissue distribution, we have established that intracellular localization of autoantigen predisposes for autoantibody production. These findings help explain why intracellular antigens are targeted in systemic autoimmune diseases.

Show MeSH
Related in: MedlinePlus