Distinct T Cell Activation Program: BHLHB2 Transcription Factor Study
Explore the role of the BHLHB2 transcription factor in T cell activation, examining how co-stimulation via CD28 affects T cell phenotype and differentiation. Discover insights from shRNA targeting BHLHB2 mRNA and its impact on primary cells. Details on epitope-tagged BHLHB2 expression and the competitive advantages observed with BHLHB2 over-expression in Jurkat cells.
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Presentation Transcript
Bluelab meeting BHLHB2 in T cell activation Jonathan Esensten 19 January 2010
Hypothesis: co-stimulation via CD28 during activation induces a distinct T cell phenotype and differentiation program that is controlled by one or more transcription factors.
Top mRNA hit: BHLHB2
shRNAs were designed to target 3 locations in the BHLHB2 mRNA: 2455 1088 502 3 Bhlhb2 mRNA 5 An additional scrambled (SCRM) hairpin sequence was cloned into pSICO-R vector (with constitutive GFP). Vector was packaged into lenti virus.
Unfortunately, this shRNA construct does not appear to work in primary cells.
[. . .] Also: Senior author of Sun et al emailed us saying that the autoimmune disease that they reported disappeared when then moved to an SPF mouse facility.
CD4+ cells from BHLHB2 +/+ or -/- littermates were purified from splenocytes and activated under the indicated conditions for 24 hours before harvest IFN (relative) IL-2 (relative) 80% of WT 40% of WT anti-CD3 - + + - + + anti-CD3 - + + - + + anti-CD28 - - + - - + anti-CD28 - - + - - + BHLHB2 +/+ BHLHB2 -/- BHLHB2 +/+ BHLHB2 -/- Cells courtesy of Ann Marini and Robert Lipsky
Epitope-tagged BHLHB2 expression vector for ChIP AMP CMV TATA box Rep origin 1 5' LTR (RU5) HIV env/gp8 HIV Intergrase (Flap) pCSP NEP FugW.GAD.TCR164 091009 Exon-1 11672 bp hUBC-1 Exon-2 GAD164 alpha BamHI 3' LTR P2A WPRE GAD165 beta EGFP structure of the inserts eGFP 2A hBHLHB2 6xHIS A2 EcoRI eGFP 2A hBHLHB2 B1
BHLHB2 over-expression gives Jurkat cells a competitive advantage. . .at lease initially
Jurkat cells were sorted for GFP+ fraction 3 days after infection pre-sort uninfected post-sort 5.4% 6xHIS-tagged 1.5% untagged GFP
Day 12 post-sort 6xHIS-tagged untagged uninfected 0.1% 99.2% 97.6%
Poor activation using anti-CD3 and anti-CD28 with large numbers of cells 24hrs, anti-CD3 + aCD28 unstimulated 6xHIS-tagged untagged CD69 CD25
PMA and ionomycin stimulation 24hrs, PMA + ionomycin unstimulated 6xHIS-tagged untagged CD69 CD25
qPCR on Jurkat cells infected with pFUGW-eGFP-2A-BHLHB2, ativated with P+I, JE 1-8-09 1000000 expression relative to unstimulated 100000 10000 1000 100 10 1 CD25 un CD25 act IL-2 un IL-2 act BHLHB2 unBHLHB2 act
fragmented DNA from chromatin prep 800 500 400 average: ~300 bp 300 200 100 A2-activated, 2 ug A2-activated, 8 ug A2-unstim, 2 ug A2-unstim, 8 ug
First results. . . ChIP of IL-2 locus and control loci in Jurkat cells infected with BHLHB2-6xHIS, JE 1-15-10 0.045 0.04 enrichement relative to input 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0 neg control pos control IL-2 exon 3 IL-2 promoter
Could the primers be the problem? IL-2 promoter: product: 376 bp IL-2 intron 3: product: 307 bp Chr. 8 origin negative controlmyc1: product: 249 bp Rev-erba (DEC1 positive control)product: 401 BP
Troubleshooting the ChIP asssay - primers product is too large - epitope is blocked during fixation - antibody not binding to protein A - non-specific binding to beads requires more washing
Next steps Mouse work - Confirm IL-2 production defect in the naive CD4+ cells from BHLHB2 KO mice - Basic activation and proliferation assays - Investigate possible Treg defects
Thank you Jeff Bluestone David Gumbiner Weihong Liu Amy Putnam Todd Eagar Donors B-G Dimitri de Kouchkovsky Art Weiss Shirley Zhu Chris Barker Jennifer Gregg the entire Bluestone and Anderson labs
