Extended Data Figure 5. Development and characterization of mice with IEC-specific Cd1d1 deletion.

a, Schematic map of the targeting strategy for generation of Cd1d1^fl/fl mice. A LoxP (L83) site was inserted at 10310 and a FNFL (Frt-Neo-Frt-LoxP) cassette at 12190 to flank exons 2, 3, and 4 (about 1.9 kb) of the Cd1d1 gene to generate the ‘floxed/neo’ Cd1d1 allele. A gene-targeting vector was constructed by retrieving one 5 kb long homology arm (5′ to L83), one 1.9 kb sequence containing exon 2/3/4, FNFL cassette, and one 2 kb short homology arm (end of FNFL to 3′). The FNFL cassette conferred G418 resistance during gene targeting in PTL1 (129B6 hybrid) embryonic stem (ES) cells. The targeted Cd1d1 allele was PCR amplified and sequenced to confirm the targeted C57BL/6 allele based on the C57BL/6-specific mutation in the Cd1d2 allele. Three targeted ES cells with targeted C57BL/6 allele were injected into C57BL/6 blastocysts to generate chimaeric mice. Male chimaeras were bred to bACTFlpe females or EIIa-Cre females to transmit the floxed Cd1d1 allele (Cd1d1^L/+) (with neo cassette removed by Fple recombinase) through the germ line. Mice carrying the floxed Cd1d1 allele were crossed to tissue-specific VillinCre-ER^T2 and exons 2, 3 and 4 and Frt-LoxP were removed by Cre recombinase in intestinal epithelial cells. b–e, Impaired CD1d- but not MHC-class I-restricted antigen presentation in Cd1d1^ΔIEC mice. b, Cd1d1^ΔIEC mice exhibit normal numbers of invariant NKT cells in liver, spleen and colonic lamina propria as determined by flow cytometry using α-GalCer/CD1d tetramers (n = 4 mice per group). c, Representative histograms of CD1d cell surface expression on colonic IECs of the indicated mouse strains before (left, n =5 mice per group) and 6 h after (right, n =5 mice per group) rectal oxazolone challenge. d, CD1d-mediated antigen presentation by colonic IECs of the indicated mouse strains (n = 5 mice per group). Presentation of the model glycolipid antigen α-GalCer to invariant NKT-cell hybridomas 24.7 and DN32.D3 and of endogenous antigens (autoreactivity) to the non-invariant NKT-cell hybridoma 14S.6 is shown. e, Presentation of H2-K^b-restricted SIINFEKL presentation by the indicated IECs (n = 5 mice per group) to the SIINFEKL-responsive hybridoma RF33.70. Results representative of three independent experiments are shown b–e, Mean ±s.e.m. of triplicate cultures are shown. Student’s t-test was applied. f, Intestinal epithelial CD1d is protective in oxazolone colitis. Representative macroscopic colon images of Cd1d1^ΔIEC and wild-type littermates upon rectal challenge with oxazolone (Ox., n =5 mice per group) or vehicle (ethanol (EtOH), n =4 mice per group).