MHC II α1α1 α2α2 β1β1 β2β2 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 T H -17 cells Or, immunoregulatory effects of TGF-β play a role in T H -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 T H -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 T H -17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β and IL-6 abrogate pathogenic function of T H -17 cells

Techniques you will need for Figure 1 EAE Model (Experimental autoimmune encephalomyelitis) - Mycobacterium tuberculosis H37Ra (killed and desiccated) + PLP ( ) days

Activated T H -17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β and IL-6 abrogate pathogenic function of T H -17 cells Techniques you will need for Figure 1 Lymph node collection -Cultured with IL-23 or TGF- β and IL days

Activated T H -17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β and IL-6 abrogate pathogenic function of T H -17 cells Techniques you will need for Figure 1 Two different mice models: SJL and C57BL/6 -SJL -PLP ( ) -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 T H -17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β and IL-6 abrogate pathogenic function of T H -17 cells IL-17 Anti-IL-17 CD4 Anti-CD4

FACS Cont’d Activated T H -17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β and IL-6 abrogate pathogenic function of T H -17 cells IL-17 CD4

Techniques you will need for Figure 1 Thymidine Incorporation - Proliferation Activated T H -17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β and IL-6 abrogate pathogenic function of T H -17 cells Fig2B

Fig1A Activated T H -17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β and IL-6 abrogate pathogenic function of T H -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 T H -17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β and IL-6 abrogate pathogenic function of T H -17 cells

Information you will need for Figure 1 EAE model of MS Activated T H -17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β and IL-6 abrogate pathogenic function of T H -17 cells

Is their data believable? S2A Fig1C Activated T H -17 cells respond to IL-23 and to TGF- β and IL-6/ TGF- β and IL-6 abrogate pathogenic function of T H -17 cells Table 1

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 2 Gene expression profile of cytokine-stimulated T cells Fig2A

Figure 2 Gene 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 T H -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 T H -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 3 TGF- 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 3 Fig3D

Figure 3 TGF- 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 T H -17 cells

Figure 4 TGF- β and IL-6 reduce chemokine production by T H -17 cells Fig4A

Figure 4 TGF- β and IL-6 reduce chemokine production by T H -17 cells Fig4B

Figure 4 Conclusions TGF- β and IL-6 reduce chemokine production by T H -17 cells

Figure 5 IL-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

Figure 5 IL-10 is upregulated by TGF- β and IL-6 but not by IL-23 Fig5A

Figure 5 IL-10 is upregulated by TGF- β and IL-6 but not by IL-23 Fig5B

Figure 5 IL-10 is upregulated by TGF- β and IL-6 but not by IL-23 Fig5C

Figure 5 IL-10 is upregulated by TGF- β and IL-6 but not by IL-23 Fig5D

Figure 5 IL-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 6 Bystander 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 6 Bystander suppression mediated via IL-10 Fig6A Fig6B Fig6C

Figure 6 Conclusions Bystander suppression mediated via IL-10