5 min at room temperature cells were surface stained for cd4 efluor 450 cd25 pe lap perp at 4 u00b0 c for 30 min in pbs followed by viability dye efluor 506 stain at 4 u00b0 c for 30 min in pbs fixation and foxp3 alexa fluor 647 stain was performed using the transcriptional factor staining buffer set other cells were surface stained with cd4 efluor 450 cd49b apc and lag3 pe tr1 staining at 4 u00b0 c for 30 min in pbs followed by viability dye efluor 506 controls included isotype control single positive and unstained cells all kits and antibodies were purchased form ebiosciences san diego calif flow cytometry was performed on a lsr ii system bd bioscience san jose calif and data were analyzed with fcsexpress software de novo software los angeles calif mice were fed with ctb c2 250 mg twice per day for 2 days and sacrificed 5 hours after the last gavage tissue was collected frozen cryo sectioned and stained as described 19 the following antibodies were used rabbit anti fviii light chain 1 200 santa cruz biotechnology dallas tex biotin anti mouse cd11c 1 200 bd biosciences san jose calif alexa fluor 488 donkey anti rabbit.