CREM-deficient DC provide enhanced Ag-specific T cell proliferation. a, BMDCs of C57BL/6 animals were treated with the TLR4 agonist LPS, the TLR7 agonist R848, and the TLR9 agonist CpG for 8 h. RNA was extracted and real-time PCR for CREM was performed and equalized to GAPDH and ribosomal protein L values (n = 3 experiments). b, BMDCs of C57BL/6 animals were stimulated with the TLR4 agonist LPS for 24 h and nuclear proteins were extracted. CREM protein expression was detected by Western blot (n = 2 experiments). c, BMDCs of Crem−/− or Crem+/+ animals were incubated together with T cells from allogenic BALB/c mice. Three days later, T cells were labeled with thymidine and its incorporation was measured 24 h later (n = 5 experiments, p = 0.0065, paired t test). d, BMDCs of DO11.10 Crem−/− or DO11.10 Crem+/+ animals were either left untreated or were pulsed with OVA peptide (1 μM) for 2 h and extensively washed with PBS. BMDCs were cocultured with CFSE-labeled (1 μM) T lymphocytes of OVA-transgenic DO11.10 CREM heterozygote (HZ) mice in ratios ranging from 1:10 to 1:100. FACS analysis of T cells was performed after 3–5 days. Number represents percentage of proliferated cells in each quadrant (n = 4 experiments).