IGEM:Stanford/2009/2009/07/10

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Entry title
Things to consider: FACS: would need to buy antibodies. Could use machine in Pediatrics or Grant building. Surface and intracellular staining? (esp for Foxp3? IL-17, too?) ELISA: could detect levels of IL-17 and IL-10 perhaps? Problematic because IL-10 could come from a contaminating Th1 population. Also, some subsets of Th17 cells may produce IL-10 depending on how they’re cultured. Might have to double this up with FACS—either CFSE or Foxp3/CD25/CD127 for Tregs. Qt-RT-PCR: could detect IL-17 and Foxp3 transcripts. A bit of a hairy procedure though. Western: probably not quantitative enough. Analysis of effects of RA: Reagents/Supplies: Media: RPMI, L-glu, FCS at least. Streptomycin/penicillin to ensure sterility. Antibodies: TGF-beta, IL-6, IL-2 at least, anti-IL-4, anti-IFN-gamma. If we can get IL-1beta, IL-21, and TNF for free, then we might use them, but these are not required. Stimulants: anti-CD3, anti-CD28, PMA/ionomycin Antibodies (pre-conjugated and on different channels) to Foxp3 (ab10563 100.0µg $340.00 http://www.abcam.com/index.html?pageconfig=reviews&intAbID=10563 would have to be conjugated to a flourochromed-secondary), IL-17 (intracellular) at least. If we have access to Ror-gamma-t we could use this too. If doing surface staining, then CD25, CD127, CD4 for Tregs, what about Th17? Maybe CCR6. Or do combination surface and intracellular? Sources for Protocols: http://www.natureprotocols.com/2007/11/05/isolation_of_treg_cells_and_tr.php for Treg isolation and Treg-Tresp coculture systems in mice
 * We would probably have to assay Th17/Treg balance on already-differentiated Th17 cells and Tregs in co-cultures. Whole blood PBMC or CD4+ T cells do not contain a high enough percentage of Th17 cells, and even when subjected to Th17 polarizing conditions, the fraction is so small that the error bar would be fairly high and our changes may not be truly indicative of the effects of RA/IL-6.
 * If using already-differentiated Th17 cells/Tregs would we subject naïve CD4 separately to Th17 and Treg polarizing conditions and then introduce stable populations into a coculture system? Or would sort whole blood for Th17/Treg (might be problematic on the Th17 side) and expand clones (might be problematic in terms of timing)?  Also, then we would have to consider the difference between nTregs and iTregs.
 * How to assess populations:
 * Would co-culture consist of just Th17/Treg? Irradiated APC as well? Would we have to add blocking antibodies to prevent induction of Th1/Th2 lineages?
 * Primarily analyzed effects of RA on naïve CD4 T cells under Th17 polarizing conditions. Reagents: negative selection kit (anti- CD8, CD11b, CD11c, CD25, CD45R (B220), NK1.1, and TER119) to enrich for CD4+CD25-; CD90.2/Thy1.2 beads for negatively selecting APCS (later irradiated); RPMI medium supplemented with FCS, L-glu, penicillin, stretopmycin, beta-mercaptoethanol, anti-CD3/anti-CD28, IL-2 for T cell cultures and stimulation; TGF-beta, IL-6, IL-2, TNF as exogenous cytokines for Th17 polarizing conditions in addition to OVA; Foxp3, CFSE and IL-17 for intracellular staining; all-trans retinoic acid in DMSO (Mucida et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science. 2007 Jul 13;317(5835):256-60.)
 * Analyzed effects of RA on naïve CD4 T cells under Th17 polarizing conditions. Reagents: MACS bead isolation kit for naïve CD4 T cells, anti-CD3/anti-CD28, IL-6, IL-21, TGF-beta, IL-1, all-trans retinoic acid for isolation and culturing; IL-17 and Foxp3 intracellular staining for assaying populations. (Xiao et al. Retinoic acid increases Foxp3+ regulatory T cells and inhibits development of Th17 cells by enhancing TGF-beta-driven Smad3 signaling and inhibiting IL-6 and IL-23 receptor expression. J Immunol. 2008 Aug 15;181(4):2277-84.)
 * Analyzed effects of RA (ARTA) on murine T cell differentiation under Th17 and Treg polarizing conditions. Reagents: MACS isolation kits (for T cells and APCs); RPMI+FCS+L-glu as media; Th17 polarizing conditions used IL-6, IL-2, anti-IL-4, anti-IFN-gamma, TGF-beta; ARTA dissolved in DMSO; intracellular cytokine measured by anti-IL-17-PE/anti-IL-17-APC and anti-FoxP3-FITC, Brefeldin A, PMA/ionomycin; also used ELISA for IL-17 in supernatant and qt-rt-PCR (Elias et al. Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway. Blood. 2008 Feb 1;111(3):1013-20.)
 * Whole blood (can we get any human samples from the Blood Bank?) or frozen PBMC or cultures of T cells
 * MACS selection kits if we don’t have access to cultures of naïve CD4 T cells or pre-differentiated cells (http://www.miltenyibiotec.com/en/PG_329_1082_CD4_CD25_CD127_dim_Regulatory_T_Cell_Isolation_Kit_II.aspx for Tregs--CD4+CD25+CD127-, product # 130-094-775; http://www.miltenyibiotec.com/en/PG_329_14_CD4_CD25_Regulatory_T_Cell_Isolation_Kit.aspx for Tregs and Tresp (CD4+CD25+ vs. CD4+CD25- product #130-091-301)
 * For culturing:
 * If intracellular staining/assessment by FACS
 * if ELISA: IL-17 (http://www.biocompare.com/ProductListings/27699/Human-IL-17-ELISA-EIA-Kits.html), TGF-beta (http://www.biocompare.com/ProductCategories/2538/Human-TGF-ELISA-Kits.html), IL-10 (http://www.biocompare.com/ProductListings/27692/Human-IL-10-ELISA-EIA-Kits.html). But this would be less indicative of the population ratios than FACS and would be problematic in terms of assessing Treg population because TGF-beta and IL-10 can come from different sources, especially contaminating populations of Teff.
 * if QT-RT-PCR: IL-17 (http://www.biocompare.com/ProductDetails/205600/Human-IL-17-Primer-Pair.html) and Foxp3 (FOXP3 (forward primer) 5'-GCCCTTGGACAAGGACCCGATG-3' FOXP3 (reverse primer) 5'-CATTTGCCAGCAGTGGGTAGGA-3' from Sgima-Genosys, http://cancerres.aacrjournals.org/cgi/content/full/68/8/3001)


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