miR-146a deficiency leads to increased TRAF6 expression in T cells stimulated by alloantigen. (A-C) CD4⁺/CD8⁺ T cells (C57BL/6) were isolated 48 hours after stimulation with allogeneic BMDC (BALB/c) in vitro. (A) RNA was isolated from purified T cells, and the expression of Traf6 was analyzed by microarray. (B) RNA was isolated from purified T cells and reverse transcribed into cDNA, followed by qRT-PCR. Traf6 expression of T cells isolated from miR-146a^−/− or miR-146a^+/+ mice is shown. (C) Purified T cells were lysed and analyzed for TRAF6 protein levels by western blot (n = 4 per group). (D) T cells were isolated from the spleens and LNs of allo-HCT recipients on day 9, lysed, and analyzed for TRAF6 expression. Transferred and reisolated T cells were from miR-146a^−/− (n = 7) or miR-146a^+/+ mice (n = 8) as indicated. (E) The amount of IκB is shown for T cells treated as described in C (n = 7 per group). (F) A proposed simplified mechanism for how the TRAF6/NF-κB/TNF axis is regulated by miR-146a in alloreactive T cells. Dividing lines indicate that lanes were run on the same gel but were noncontiguous. ***P < .0001; **P < .01; *P < .05.