Mixed Bone Marrow Chimera Studies

Recipient C57BL/6J mice were irradiated with two doses of 550 rad, 3 h apart. Mice were fed with Uniprim diet (Envigo, Indianapolis, IN) for 2 weeks to prevent infections. The day following irradiation, bone marrow was isolated from donor mice and RBCs and T cells were depleted. Cell density was similarly adjusted from each strain, between 2.0 × 10⁷ to 4.0 × 10⁷ cells/ml. Bone marrow cells from WT C57Bl/6J or P2X7R^−/- and zDC-DTR mice were mixed 1:1 and 4.0 × 10⁶ to 8.0 × 10⁶ cells were injected intravenously per recipient mouse. Between 8 and 12 weeks following reconstitution, naïve CD45.1⁺ OT-II CD4⁺ T cells were injected i. v. into each chimera. The day after T cell transfer, chimeras were injected i. p. with either PBS or 1.25 µg Diphtheria Toxin (Sigma), to deplete cDCs. To maintain DT-induced DTR⁺ cell depletion, DT (1.25 µg/mouse) was injected every other day for the duration of experiments. Chimeras were immunized with 100 µg OVA_323-339 peptide in CFA at the tail base 24 h following the first DT injection. 7 days after immunization, inguinal LNs were collected and made into a single cell suspension. Single cell suspensions were restimulated with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of GolgiPlug for 4 h and then stained for flow cytometric analysis of IL-17A and IFNγ-producing CD45.1⁺ CD4⁺ T cells. Counting beads were used to determine absolute cell count.