Notch and Presenilin Regulate Cellular Expansion and Cytokine Secretion but Cannot Instruct Th1/Th2 Fate Acquisition
Figure 3
RBP-J independent function of presenilin is required for optimal T cell expansion.
(A) Efficient deletion of Presenilin1 and RBP-J alleles by CD4-cre transgene. Naïve CD4+ T cells isolated from conditional knockout littermates (n≥7 animals per genotype) showed no detectable level of RBP-J and presenilin1 proteins. WB: western blot. (B) Removal of PS1/PS2 proteins abolished Notch1 signaling in activated naïve CD4+ T cells. Activated Notch1 is detected in activated control CD4+ T cells under both Th1 and Th2 polarizing conditions but not detected in T cells that have targeted ablation of PS1/PS2 alleles. Non transfected HEK293T cells were used as negative control, while cells transfected with PCS2+N1ΔE was used as a positive control. CD4+ T cells were isolated with anti-CD4 magnetic beads on MACS column to >95% purity from the spleens of 2 months old littermates. This protocol allows co-purification of natural APCs that provide the Notch ligands (compare with Figure 2C where no Notch1 activation was observed when a two-step purification method was used). T cells were activated with anti-CD3/CD28 in Th1 or Th2 polarizing conditions for 24 h. T cells were then FACS sorted for CD4+ population and probed with V1744 antibody. WB: western blot. (C) Proliferation capacity was measured by 3[H]-thymidine incorporation. Reduced proliferation was observed in PSdko and PSRtko cells under both Th1 and Th2 polarizing conditions. Rko cells were not significantly different than controls. Results are presented as mean±S.D. of five wells and representative of at least three independent experiments. (D, E) Reduction in the final number of viable PSdko and PSRtko T cells 6 days after activation. Naïve CD4+ T cells from different genotypes were stimulated with anti-CD3/CD28 antibodies for 2 days in Th1 and Th2 conditions before they were expanded in fresh media containing IL-2 cytokine. After 6 days of culture, T cells were harvested and viable cells were counted. Each circle/diamond indicates data of individual mouse. In Figures 1–4, circle (○) indicates data point in which CD4+ T cells were expanded by regimen 2 (Supplemental Table 4B), whereas diamond (◊) indicates result in which T cells were expanded according to regimen 3 (Supplemental Table 4C). The P value was determined by student two-tailed t test. (F, G) The expression of T-bet and GATA-3 is unaffected by the removal of RBP-J and/or presenilins. T cells activated in Th1 or Th2 conditions were harvested and probed with T-bet or GATA-3 antibodies after 6 days in culture. Results are representative of three independent experiments. WB: western blot.