MHC II

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(No Transcript)
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MHC II

• There are two alleles associated with MS
• DR15
• DQ6
• There are two protective alleles
• HLA-C554
• HLA-DRB111

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MHC II and T cell Interaction
T cell
Macrophage
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TGF-ß role in MS

• Does TGF-ß promotes pathogenic function of TH-17
  cells
• Or, immunoregulatory effects of TGF-ß play a role
  in TH-17 cells sensitivity and suppression

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TGF-ß

• Protein for cell (anti)proliferation,
  differentiation, and other functions in most
  cells
• Induces apoptosis
• Regulation of CD25 Regulatory T Cell
• Differentiation ofCD25 Regulatory Cell and TH-17
  cell
• Blocks activation of lymphocytes

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What evidence do they present in the introduction
that will lead them down a questioning path?

• 1. Without IL-23 there is no EAE, there are no
  CNS cells, does not appear to be TGF-b dependent
• 2. IL-23 drives the production of IL-7 by memory
  T cells
• 3. TGF-b leads to FoxP3 production which leads to
  regulatory T-cells
• 4. TGF-b seems to be an initial pathway, IL-23 is
  later
• 5. TGF-b levels are increased in time of remission

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Overall Objective

• Here, they look at responses of activated
  myelin-reactive T cells with treatments of IL-23
  or TGF-ß and IL-6

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Figure 1

• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

• Hypothesis
• If activated T-helper 17 cells receive a signal
  and lead to the production of IL-17, then both
  TGF-ß/IL-6 and IL-23 will lead to IL-17
  production, but only the IL-23 will differentiate
  into a pathogenic cell.

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• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

• Techniques you will need for Figure 1
• EAE Model (Experimental autoimmune
  encephalomyelitis)
• Mycobacterium tuberculosis H37Ra
• (killed and desiccated) PLP (139-151)
• 8-10 days

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IL-17
Demyelination!
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• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

• Techniques you will need for Figure 1
• Lymph node collection
• Cultured with IL-23 or TGF- ß and IL-6
• 4 days

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• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

• Techniques you will need for Figure 1
• Two different mice models SJL and C57BL/6
• SJL
• PLP (139-151)
• Relapsing-remitting clinical course
• C57BL/6
• MOG (35-55)
• Chronic-progressive clinical course

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• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

• Techniques you will need for Figure 1
• Flow cytometry (Fluorescence-activated Cell
  Sorter, FACS)

IL-17
CD4
Anti-IL-17
Anti-CD4
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• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

• FACS Contd

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• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

• Techniques you will need for Figure 1
• Thymidine Incorporation
• Proliferation

Fig2B
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• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

Fig1A
Fig1B
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Figure 1

• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

• Conclusion
• Both IL-23 and the combination of IL-6 and TGF-ß
  produce IL-17
• There is a large production of IL-17 with the
  combination of IL-6 and TGF-ß
• Only the IL-23 produced IL-17 causes infection
• Difference is due to increased IL-17 production
  rather than to increased expansion of
  PLP-specific cells

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• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

• Information you will need for Figure 1
• EAE model of MS

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• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

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• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

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Is their data believable?

• Activated TH-17 cells respond to IL-23 and to
  TGF- ß and IL-6/ TGF- ß and IL-6 abrogate
  pathogenic function of TH-17 cells

Fig1C
Table 1
S2A
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Figure 2
Gene expression profile of cytokine-stimulated T
cells

• Hypothesis
• If activated T-helper 17 cells can diverge down
  different pathways, then when we administer the
  treatment of TGF-ß and IL-6 we will differentiate
  down an alternative pathway from that of the
  IL-23 pathway leading to different gene
  expression.

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Gene expression profile of cytokine-stimulated T
cells

• Techniques you will need for Figure 2
• RT-PCR Refresh
• Measuring mRNA levels

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Figure 2
Gene expression profile of cytokine-stimulated T
cells
Fig2A
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Figure 2
Gene expression profile of cytokine-stimulated T
cells
Fig2B
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Figure 2
TGF- ß and IL-6 abrogate pathogenic function of
TH-17 cells

• Conclusion
• IL-17f is up-regulated in the presence of IL-23,
  TGF- ß and IL-6
• IL-22 is up-regulated in the presence of IL-23
• IL23r is up-regulated in the presence of IL-23

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Figure 2
TGF- ß and IL-6 abrogate pathogenic function of
TH-17 cells

• Conclusion
• IL-17f is up-regulated in the presence of IL-23,
  TGF- ß and IL-6
• IL-22 is up-regulated in the presence of IL-23
• IL23r is up-regulated in the presence of IL-23

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Figure 3
TGF- ß and IL-6-stimulated cell do not establish
inflammation

• Hypothesis
• If there is differential gene expression in the
  activated T-helper 17 cells that lead to
  pathogenesis, then only those cells that are
  administered with IL-23 will establish an
  inflammatory response and lead to the entry of
  those cells into the central nervous system.

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Gene expression profile of cytokine-stimulated T
cells

• Techniques you will need for Figure 3
• How is this experiment done differently? (3A)
• Donor cells to a Recipient

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Gene expression profile of cytokine-stimulated T
cells

• Techniques you will need for Figure 3
• What else? (3A)
• Isolated from CNS and Spleen

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Figure 3
TGF- ß and IL-6-stimulated cell do not establish
inflammation
Fig3A
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Figure 3
Fig3B
Fig3C
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Gene expression profile of cytokine-stimulated T
cells

• Techniques you will need for Figure 3
• What is happening here? Looks familiar? (3D)
• Proliferation
• So what?
• Taggable

• Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU)

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Gene expression profile of cytokine-stimulated T
cells
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Figure 3
Fig3D
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Figure 3
TGF- ß and IL-6-stimulated cell do not establish
inflammation
Fig3E
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Figure 3
TGF- ß and IL-6-stimulated cell do not establish
inflammation

• Conclusions

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Figure 4
TGF- ß and IL-6 reduce chemokine production by
TH-17 cells

• Hypothesis
• If TGF-ß and IL-6 are not leading T-helper 17
  cells to interleukin inflammatory responses, then
  when we administer this combination to the TH-17
  cells we will observe reduced levels of chemokine
  production.

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Figure 4
TGF- ß and IL-6 reduce chemokine production by
TH-17 cells
Fig4A
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Figure 4
TGF- ß and IL-6 reduce chemokine production by
TH-17 cells
Fig4B
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Figure 4
TGF- ß and IL-6 reduce chemokine production by
TH-17 cells

• Conclusions

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Figure 5
IL-10 is upregulated by TGF- ß and IL-6 but not
by IL-23

• Hypothesis
• If IL-10 and TGF-ß/IL-6 are leading to protective
  effects against EAE, then when we administer
  TGF-ß/IL-6, and not IL-23, to T-helper 17 cells
  we will see IL-10 production.

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Gene expression profile of cytokine-stimulated T
cells

• Techniques you will need for Figure 5
• Elisa? Yes! What else?(5A)
• Magnetic-activated cell sorting (MACS)
• Cool

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Gene expression profile of cytokine-stimulated T
cells
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Figure 5
IL-10 is upregulated by TGF- ß and IL-6 but not
by IL-23
Fig5A
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Figure 5
IL-10 is upregulated by TGF- ß and IL-6 but not
by IL-23
Fig5B
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Figure 5
IL-10 is upregulated by TGF- ß and IL-6 but not
by IL-23
Fig5C
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Figure 5
IL-10 is upregulated by TGF- ß and IL-6 but not
by IL-23
Fig5D
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Figure 5
IL-10 is upregulated by TGF- ß and IL-6 but not
by IL-23
Fig5E
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Figure 5
IL-10 is upregulated by TGF- ß and IL-6 but not
by IL-23

• Conclusions

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Figure 6
Bystander suppression mediated via IL-10

• Hypothesis
• If IL-10 is leading to pathogenic T-helper cell
  deactivation through the TGF-ß/IL-6 pathway, then
  when we administer TGF-ß/IL-6 and allow the cells
  time to produce IL-10, we see decrease in EAE
  score that is not exhibited when IL-10 is
  inhibited.

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Figure 6
Bystander suppression mediated via IL-10
Fig6B
Fig6A
Fig6C
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Figure 6
Bystander suppression mediated via IL-10

• Conclusions