Figure 2. RORγt is a transcriptional repressor of Btla.
(A) Expression of RORγt and CD127 in freshly isolated lymphocytes gated on TCRβ− live cells from the iLN (left) and PP (right) of Rorcgfp/+ mice (representative of 8 mice).
(B, C) MFI for RORγt-GFP expression in iLN γδ T cells and PP ILCs (B), and for BTLA expression in RORγt+ γδT cells and ILCs (C).
(D) Expression of BTLA and GFP in cells transduced with pMSCV-IRES-GFP-RORγt retrovirus (RV-RORγt) or with empty retrovirus (RV) (representative of two transduction experiments and six different passages after transduction).
(E) mRNA levels (relative to L32) of Rorc and Btla in FACS-sorted RORγt-GFP−, RORγt-GFPLow and RORγt-GFPHigh cells that were transduced with RV-RORγt (each symbol represents a different passage of the indicated FACS-sorted cell populations). In (B–C) each symbol represents a mouse and in (B) lines are medians.
(F) VISTA plot of sequence similarity (>70%, 100 bp, pink) between the 5kb promoter regions of the human and mouse BTLA coding genes and graphical representation of the conserved RORγt binding sites and their positions relative to the transcription start (indicated by arrow).
(G) PCR analysis using primers specific for the RORγt binding sites −49 and −369 following a ChIP assay with anti-RORγ or control IgG in RV-RORγt transduced cells (representative of two experiments).
(H) Btla promoter reporter activity in Jurkat cells co-transfected with wild type or mutated promoter, and the indicated amounts of RORγt expressing plasmid (mean±sem of two experiments with two replicates each).
(I) Btla promoter reporter activity in Jurkat cells co-transfected with wild-type promoter in the presence or absence of wild-type (wt) or Activation Function domain 2 (AF2)-mutant or DNA Binding Domain (DBD)-mutant RORγt (mean±sem of two experiments with two replicates each). See also Figure S2.