MHC II

α1

α2

β1

β2

membrane

Peptide-binding cleft• There are two alleles associated

with MS• DR15 • DQ6

• There are two protective alleles • HLA-C554 • HLA-DRB1*11

MHC II and T cell Interaction

Macrophage

T cell

TGF-β role in MS

• Does TGF-β promotes pathogenic function of TH-17 cells

• Or, immunoregulatory effects of TGF-β play a role in TH-17 cells sensitivity and suppression

TGF-β

• 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

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

Overall Objective

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

Figure 1

• Hypothesis– If activated T-helper 17 cells receive a signal and

lead to the production of IL-17, then both TGF-β/IL-6 and IL-23 will lead to IL-17 production, but only the IL-23 will differentiate into a pathogenic cell.

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

Activated TH-17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β 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

IL-17

Demyelination!

Activated TH-17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β 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- β and IL-6- 4 days

Activated TH-17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β 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

Techniques you will need for Figure 1

Flow cytometry (Fluorescence-activated Cell Sorter, FACS)

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

IL-17

Anti-IL-17

CD4

Anti-CD4

FACS Cont’d

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

IL-17 CD4

Techniques you will need for Figure 1

Thymidine Incorporation

- Proliferation

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

Fig2B

Fig1A

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

Fig1B

Figure 1

• Conclusion– Both IL-23 and the combination of IL-6 and TGF-β

produce IL-17– There is a large production of IL-17 with the

combination of IL-6 and TGF-β – 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

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

Information you will need for Figure 1

EAE model of MS

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

Disease Grade Clinical Symptoms 0 Healthy 1 Flaccid tail 2 Impaired righting reflex and hindlimb weakness 3 Partial hindlimb paralysis 4 Complete hindlimb paralysis 5 Hindllimb paralysis with partial forelimb paralysis 6 Moribund

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

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

Is their data believable?

S2A

Fig1C

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

Table 1

Disease Grade Clinical Symptoms 0 Healthy 1 Flaccid tail 2 Impaired righting reflex and hindlimb weakness 3 Partial hindlimb paralysis 4 Complete hindlimb paralysis 5 Hindllimb paralysis with partial forelimb paralysis 6 Moribund

Figure 2

• Hypothesis– If activated T-helper 17 cells can diverge down

different pathways, then when we administer the treatment of TGF-β and IL-6 we will differentiate down an alternative pathway from that of the IL-23 pathway leading to different gene expression.

Gene expression profile of cytokine-stimulated T cells

Techniques you will need for Figure 2

RT-PCR Refresh

- Measuring mRNA levels

Gene expression profile of cytokine-stimulated T cells

Figure 2Gene expression profile of cytokine-stimulated T cells

Fig2A

Figure 2Gene expression profile of cytokine-stimulated T cells

Fig2B

Figure 2

• Conclusion– IL-17f is up-regulated in the presence of IL-23,

TGF- β and IL-6– IL-22 is up-regulated in the presence of IL-23– IL23r is up-regulated in the presence of IL-23

TGF- β and IL-6 abrogate pathogenic function of TH-17 cells

Figure 2

• Conclusion– IL-17f is up-regulated in the presence of IL-23,

TGF- β and IL-6– IL-22 is up-regulated in the presence of IL-23– IL23r is up-regulated in the presence of IL-23

TGF- β and IL-6 abrogate pathogenic function of TH-17 cells

Figure 3

• 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.

TGF- β and IL-6-stimulated cell do not establish inflammation

Techniques you will need for Figure 3

How is this experiment done differently? (3A)

- Donor cells to a Recipient

Gene expression profile of cytokine-stimulated T cells

Techniques you will need for Figure 3

What else? (3A)

- Isolated from CNS and Spleen

Gene expression profile of cytokine-stimulated T cells

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

Fig3A

Figure 3

Fig3B Fig3C

Techniques you will need for Figure 3

What is happening here? Looks familiar? (3D)

- Proliferation

-So what?-Taggable

Gene expression profile of cytokine-stimulated T cells

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

Gene expression profile of cytokine-stimulated T cells

Figure 3Fig3D

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

Fig3E

Figure 3

• Conclusions

TGF- β and IL-6-stimulated cell do not establish inflammation

Figure 4

• Hypothesis– If TGF-β 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.

TGF- β and IL-6 reduce chemokine production by TH-17 cells

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

Fig4A

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

Fig4B

Figure 4

• Conclusions

TGF- β and IL-6 reduce chemokine production by TH-17 cells

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

• Hypothesis– If IL-10 and TGF-β/IL-6 are leading to protective

effects against EAE, then when we administer TGF-β/IL-6, and not IL-23, to T-helper 17 cells we will see IL-10 production.

Techniques you will need for Figure 5

Elisa? Yes! What else?(5A)

Magnetic-activated cell sorting (MACS) -Cool

Gene expression profile of cytokine-stimulated T cells

Gene expression profile of cytokine-stimulated T cells

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

Fig5A

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

Fig5B

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

Fig5C

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

Fig5D

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

Fig5E

Figure 5

• Conclusions

IL-10 is upregulated by TGF- β and IL-6 but not by IL-23

Figure 6Bystander suppression mediated via IL-10

• Hypothesis– If IL-10 is leading to pathogenic T-helper cell

deactivation through the TGF-β/IL-6 pathway, then when we administer TGF-β/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.

Figure 6Bystander suppression mediated via IL-10

Fig6AFig6B

Fig6C

Figure 6

• Conclusions

Bystander suppression mediated via IL-10