A Defect in Deletion of NucleosomeSpecific Autoimmune T Cells in Lupus Prone Thymus: Role of Thymic

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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
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Systemic lupus erythematosus

• Chronic, potentially debilitating or fatal
  autoimmune disease in which the immune system
  attacks the bodys cells and tissue, resulting in
  inflammation and tissue damage
• Treatment Immunosupressors currently there is
  no cure.

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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.

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Background

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

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Materials/Methods
SBF
B10

• SWR

SNF1
SBF1
MHC Matched
Lupus Prone
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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

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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

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Flow Cytometry

• 3 color staining used FITC, Biotin, PE
• Anti CD4, CD8, and CD69 pan TCR (H57597) TCRs
  V4, V8, V17, and TCRs V2, V3.2, V8, V11.1/11.2
  Antibodies

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Calculating Percent Specific Apoptosis

• ( experimental apoptosis - spontaneous
  apoptosis)/(100 -the percentage of spontaneous
  apoptosis) x 100

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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

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Presentation of autoepitopes by thymic APC

• APCs DCs, Macrophages, Epithelial cocultured
  with T cell hybridomas
• Specific for H471-94

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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 (H57597),
  biotinylated anti-CD69 or anti- CD25
• Flow cytometry

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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

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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

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Results
SWR and SBF1
SNF1

• Required concentration of H471-94 needed to
  induce apoptosis depends on amount of expression
  of Tg TCR

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Results

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

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Results

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

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Results

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

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Results

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

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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

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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.

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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.

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Questions?