C57bl 6ncr mice

Wild type (WT) C57BL/6NCr mice were originally purchased from Charles River, Frederick, MD, and used as breeders. Systemic and CRAMP^flox/flox mice were generated as detailed in Supplemental Figure 1. All mice used in this study were produced and maintained in the same room in the animal facility at the National Cancer Institute at Frederick, Frederick, MD. For colitis experiments, mice were transferred to another room on the same floor just before the experiment and kept there until the end. Six to 12 week-old male mice were used throughout the study. The experimental protocols of this study were approved by the Frederick National Laboratory for Cancer Research Animal Care and Use Committee, Frederick, MD, and all experiments were performed in accordance with relevant guidelines and regulations.
To generate littermates, CRAMP heterozygotes (CRAMP^+/−) were generated by crossing CRAMP^−/− mice to WT mice, and several cages of CRAMP^+/− breeding pairs were set up. Pups were genotyped and weaned at 4 weeks. WT or CRAMP^−/− pups were single housed for up to 13 weeks and feces were collected.

The EGFRvIII-specific CAR vector has been previously described (12 (link),34 (link)). Briefly, second generation CARs were defined by the intracellular signaling components CD28 and CD3z, and third generation CARs were defined by CD28, 4–1BB, and CD3z. To produce CARs deficient in Lck signaling, two amino acid substitutions were introduced in the PYAP motif in the CD28 signaling domain of the CAR transgene through site-directed mutagenesis (30 (link)). CARs were generated by retroviral transduction (35 (link)). Briefly, retroviral supernatant was produced by cotransfection of HEK 293T cells using Lipofectamine 2000 Transfection Reagent (Invitrogen) with MSGV1-EGFRvIII CAR retroviral vectors and pCL-Eco helper plasmid (Imgenex). On the same day, splenocytes were freshly harvested from C57BL/6NCr mice (Charles River Laboratories) and cultured in R10 mouse T-cell media supplemented with 50units/mL IL-2 and 2.5μg/mL Concanavalin A. After 48 hours, splenic T cells were transduced with retroviral supernatant on non-tissue culture 24-well plates previously coated with 0.5 mL of RetroNectin (Clontech) at a concentration of 25 μg/mL in PBS. Cells were plated at a density of 1×10⁶/mL in viral supernatant supplemented with 50units/mL IL-2. Cells were split every 24 hours for two days.

Experiments were conducted using nine week-old female C57BL/6NCrmice (Charles River). Animals were housed in sterile micro isolator cages in an air-conditioned room with 12 hour light/dark cycles. Animals (n = 6 per group) were maintained on 25 IU vitamin D/kg, 100 IU vitamin D/kg or 10,000 IU vitamin D/kg throughout the study (Research Diets Inc, New Brunswick, NJ) [20 (link)] . All other diet components were identical.

Animal experiments were approved by the Institutional Animal Care and Use Committees of the NCI and performed in accordance with the National Institutes of Health (NIH) guidelines. C57BL/6NCR mice were purchased from Charles River Laboratory. Thy1.1 and Ly5.1 Pmel-1 TCR-transgenic (Pmel, C57BL/6 .Cg-/Cy Tg [TcraTcrb] 8Rest/J) mice were maintained in house under specific pathogen-free conditions, as previously described (Overwijk et al., 2003 (link)). All animals used in our experiments were between 6–8 weeks of age.

Procedures were approved by the Institutional Animal Care and Use Committee at Roswell Park Comprehensive Cancer Center. Retired female C567BL6/Ncr mice (6–8 month old), and female and male 6–8 week old C57BL6/Ncr mice were purchased from Charles River and were housed in pathogen-free facilities. 6–8 week female and male C57BL/6J mice were purchased from Jackson Laboratories for chimeric experiments and housed in pathogen-free facilities. Female and male breeding pairs of B6.129-Ido1^tm1Alm/J mice (Ido1^−/−), B6.FVB-1700016L21Rjk^{Tg(Itgax-DTR/EGFP)57Lan}/j mice (CD11c-DTR), and B6.129S2-Cd28^tm1Mak/J (CD28^−/−) mice were purchased from Jackson Laboratories. Female and male breeding pairs of CD28-AYAA knock in (KI) mice were derived as previously reported (Boomer and Green, 2010 (link)). Upon receipt all animals were house and bred in the Division of Laboratory Animal Resources (RPCI, Buffalo, NY) in a pathogen-free barrier facility. Age matched 6–8 week old femurs and tibias from B6.129S4-Cd80^tm1ShrCd86^tm2Shr/J (CD80/CD86^−/−) crossed with C57BL/6 ^{Tg(TcraTcrb)1100Mjb}/J mice were kindly provided by Drs. Stephen Schoenberger and Joey Lee (La Jolla Institute for Allergy and Immunology). Age matched 6–8 week old femurs and tibias from C57BL/6^{Tg(UBC-GFP)30Scha}/J were generously provided by Dr. Michael Nemeth and Jennifer Peresie (RPCCC).

Areg^Fl/Fl mice were generated using mouse B6 ES cells obtained through the EUCOMM (European Conditional Mouse Mutagenesis Program) consortium. Areg^Fl/Fl allele was bred to Foxp3^YFP-cre and CD4-cre mice, and mice were screened for maintenance of the C57BL/6N Nnt allele. C57BL/6NCr mice were purchased from Charles River. For details of Areg^Fl/Fl and other mice used in this study, see the Supplemental Experimental Procedures. Generation and treatments of mice were performed under protocol 08-10-023 approved by the Sloan Kettering Institute (SKI) Institutional Animal Care and Use Committee. All mouse strains were maintained in the SKI animal facility in accordance with institutional guidelines.