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A Defect in Deletion of Nucleosome-Specific Autoimmune T Cells in Lupus – Prone Thymus: Role of Thymic Dendritic Cells

A Defect in Deletion of Nucleosome-Specific Autoimmune T Cells in Lupus – Prone Thymus: Role of Thymic Dendritic Cells. Marissa A. Michaels, Hee-Kap Kang, Arunan Kaliyaperumal, Ebenezar Satyaraj, Yan Shi, and Syamal K. Datta

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A Defect in Deletion of Nucleosome-Specific Autoimmune T Cells in Lupus – Prone Thymus: Role of Thymic Dendritic Cells

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  1. A Defect in Deletion of Nucleosome-Specific Autoimmune T Cells in Lupus – Prone Thymus: Role of Thymic Dendritic Cells Marissa A. Michaels, Hee-Kap Kang, Arunan Kaliyaperumal, Ebenezar Satyaraj, Yan Shi, and Syamal K. Datta Division of Rheumatology, Departments of Medicine and Microbiology-Immunology, Northwestern University Feinberg School of Medicine Presented by: Mark Cruz and Sandra Peery

  2. Systemic lupus erythematosus • Chronic, potentially debilitating or fatal autoimmune disease in which the immune system attacks the body’s cells and tissue, resulting in inflammation and tissue damage • Treatment: Immunosupressors; currently there is no cure.

  3. Experimental Goal • To study central and peripheral regulation of nucleosome – specific T cells in normal and lupus-prone mice. To determine if positive or negative selection is different for nucleosomes or other similar major endogenous autoantigens in lupus prone mice.

  4. Background • Elusive diagnosis • Most commonly harms the kidneys (lupus nephritis), heart, joints (rheumatological), skin, lungs, blood vessels and brain/nervous system.

  5. Materials/Methods SBF B10 SWR SNF1 SBF1 MHC Matched Lupus Prone

  6. Important Terms • Dendritic Cells: Large, motile, weakly phatocytic professional APCs. Essential in presentation of antigen to CD4+ T cells • Immunomagnetic Beads: superparamagnetic, monosized polymer particles coated with antibodies

  7. The Nucleosome • Structural unit of DNA • Consists of chormatin surrounded by Histone proteins • Two copies of each Histone protein (H2A, H2B, H3, and H4) • H2B59-73 [Control] • H471-94 [Major product of apoptosis]

  8. Flow Cytometry • 3 color staining used [FITC, Biotin, PE] • Anti CD4, CD8, and CD69; pan TCR (H57–597); TCRs V4, V8, V17, and TCRs V2, V3.2, V8, V11.1/11.2 Antibodies

  9. Calculating Percent Specific Apoptosis • (% experimental apoptosis - % spontaneous apoptosis)/(100 -the percentage of spontaneous apoptosis) x 100

  10. Presentation of autoepitopes by thymic APC • Thymic DC prepared by collagenase-DNase digestion + two rounds of purification using CD11c-immunomagnetic beads • DC purity confirmed by flow cytometry for CD11c and MHC II • Non DC APC [Macrophages, epithelial cells] gathered form Miltenyi column flow combined with anti-Thy 1.2 and C’

  11. Presentation of autoepitopes by thymic APC • APC’s [DC’s, Macrophages, Epithelial] cocultured with T cell hybridomas • Specific for H471-94

  12. TCR down regulation assay • Splenic CD4+ T cells co cultured with irradiated B + Macrophage cells with different concentrations of the control or test peptide for 18 hours • Later stained with anti anti-TCR-FITC (H57–597), biotinylated anti-CD69 or anti- CD25 • Flow cytometry

  13. Results • Number of Thymocytes in Different Mice Strains • SNF1 Mice = Lupus Prone • Larger numbers of thymocytes • SWR and SBF1 Mice = Normal • Smaller numbers of thymocytes

  14. Results • Number of Thymocytes that Underwent Apoptosis in the Different Mice Strains • SNF1 Mice = Lupus Prone • Smaller numbers of non-viable thymocytes • SWR and SBF1 Mice = Normal • Larger numbers of non-viable thymocytes

  15. Results SWR and SBF1 SNF1 • Required concentration of H471-94 needed to induce apoptosis depends on amount of expression of Tg TCR

  16. Results • Apoptosis when induced by H471-94 and anti-CD3 antibody

  17. Results • The degree of IL-2 produced by hybridomas when stimulated by the epitopes on the thymic dendritic cells in the different mice strains.

  18. Results • Percent Incidence of Mice with lupus nephritis at certain ages in different mice strains.

  19. Results • Percent suppression of IFN gamma in different mice strains and cell types.

  20. Summary • The thymus of the SNF1 mice deleted fewer thymocytes • The thymic dendritic cells from the lupus prone mice had lower expression of the Tg TCR • SNF1 thymocytes had lower expression of the Tg TCR • Defect in deletion in lupus prone mice was not due to any unusual resistance of the thymocytes • Some strains of the lupus prone mice were more prone to develop lupus nephritis

  21. Conclusions • Peptide epitopes from nucleosomes are presented to developing thymocytes. • The amount of nucleosome epitope displayed on the dendritic cells determine if the auto immune T cells are or are not deleted. • Defect in deletion is not in the thymic lymphoid cells as all strains were equally susceptible to other means of deletion. • Defensive mechanisms are possible even in lupus prone backgrounds.

  22. What it all means • The researchers found that there is a defect in the central tolerance that is selective for nucleosome autoantigens and is caused by inefficient presentation of the relevant epitopes by thymic dendritic cells.

  23. Questions?

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