Notch and presenilin regulate cellular expansion and cytokine secretion but cannot instruct Th1/Th2 fate acquisition - PubMed (original) (raw)

Notch and presenilin regulate cellular expansion and cytokine secretion but cannot instruct Th1/Th2 fate acquisition

Chin-Tong Ong et al. PLoS One. 2008.

Abstract

Recent reports suggested that Delta1, 4 and Jagged1, 2 possessed the ability to instruct CD4(+) T cell into selection of Th1 or Th2 fates, respectively, although the underlying mechanism endowing the cleaved Notch receptor with memory of ligand involved in its activation remains elusive. To examine this, we prepared artificial antigen-presenting cells expressing either DLL1 or Jag1. Although both ligands were efficient in inducing Notch2 cleavage and activation in CD4(+) T or reporter cells, the presence of Lunatic Fringe in CD4(+) T cells inhibited Jag1 activation of Notch1 receptor. Neither ligand could induce Th1 or Th2 fate choice independently of cytokines or redirect cytokine-driven Th1 or Th2 development. Instead, we find that Notch ligands only augment cytokine production during T cell differentiation in the presence of polarizing IL-12 and IL-4. Moreover, the differentiation choices of naïve CD4(+) T cells lacking gamma-secretase, RBP-J, or both in response to polarizing cytokines revealed that neither presenilin proteins nor RBP-J were required for cytokine-induced Th1/Th2 fate selection. However, presenilins facilitate cellular proliferation and cytokine secretion in an RBP-J (and thus, Notch) independent manner. The controversies surrounding the role of Notch and presenilins in Th1/Th2 polarization may reflect their role as genetic modifiers of T-helper cells differentiation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Functional Notch ligands on APCs do not affect APC-mediated T cell proliferation.

(A) Schematic of the experiment. Artificial APC lines were treated with mitomycin C (100 µg/ml for 1 h) and seeded either for co-culture reporter assay or for activating naïve CD4+ T cells isolated from DO11.10 mice. Irradiated BALB/c spleen cells were used as the natural APC control. Activated CD4+ T cells were assayed for the rate of proliferation, level of IFNγ and IL-4 production under the 4 polarizing conditions, and the presence of activated Notch1 and Lfng proteins. (B) The Notch ligands expressed on APC lines elicit Notch2 cleavage and RBP-J-dependent transcriptional activation of _TP1_-luciferase in co-culture system. Results are mean±S.D. of three independent experiments. (C) Functional Notch ligands on APC lines do not affect T cell proliferation. Naïve CD4+ T cells purified from DO11.10 mice were cultured with the different APC lines under various concentrations of OVA peptide, as indicated on the horizontal axis. The cultures were pulsed with [3H] thymidine at 48 h, harvested and analyzed at 60 h. Data represent c.p.m±s.d. from triplicate wells.

Figure 2

Figure 2. Notch ligands cannot instruct Th1/Th2 differentiation in the absence of inducing cytokines but only act selectively in co-stimulation.

(A) Notch ligands cannot induce a Th1 program under “drift” or “neutral” conditions, whereas only DLL-1 enhances IFN-γ production under Th1 polarizing conditions. Naïve CD4+ T cells from DO11.10 mice were purified to >99% purity by a two-step protocol, and primed with different APC lines and 0.3 µM OVA peptide in 4 polarizing conditions for 2 days. Activated T cells were expanded in fresh media for another 5 days, re-stimulated with 4 h of PMA/Ionomycin and stained for intracellular IFN-γ. B7: CHO-B7, DLL1: CHO-DLL1, J1: CHO-Jag1 and Spl: BALB/c spleen cells. Results are mean±s.d. from three independent experiments. (B) DLL1 and Jag1 ligands do not cause significant differences in the level of IL-4 production under “drift” and “neutral” conditions. They only marginally enhance IL-4 cytokine secretion under Th2 polarizing conditions. Activated T cells were re-stimulated on Day 7 with anti-CD3 antibody for 24 h and before supernatant collection and ELISA. Results are mean±s.d. of three independent experiments. The P value was determined by student two-tailed t test. (C) Only CHO-DLL1 APC line triggers Notch1 cleavage in activated CD4+ T cells. Naïve CD4+ T cells from DO10.11 mice were purified to >99% purity by the two-step protocol and primed with various APC lines and 0.3 µM OVA peptide in 3 different polarizing conditions. They were isolated 24 h later and probed with V1774 and actin antibodies. WB: western blot. (D) Detection of Lunatic Fringe in CD4+ T cells. Naïve CD4+ T cells purified from DO11.10 mice were activated with anti-CD3/CD28 antibodies under the 4 polarizing conditions for 24 h and harvested for western with anti-Lfng antibody. Lysate from newborn pup (P1) was used as positive control whereas negative control was lysate from NIH3T3 cells. WB: western blot.

Figure 3

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.

Figure 4

Figure 4. RBP-J and presenilins are not required for the production of intracellular IFN-γ in CD4+ T cells activated under Th1 polarizing conditions.

(A, B) Production of intracellular INF-γ is unaffected in different mutant genotypes in Th1 polarizing conditions when compared to Th2 polarizing conditions. Elevated levels of IFN-γ production are detected by ICS in RBP-J-deficient cells under Th1 polarizing conditions after re-stimulation with anti-CD3 but not with PMA/Ionomycin. Naïve CD4+ T cells from different genotypes were stimulated with anti-CD3/CD28 antibodies for 2 days in Th1 and Th2 conditions. Cells were then expanded into new media containing IL-2 cytokine. T cells were harvested and counted after 6 days of culture. An equal number of T cells were re-stimulated overnight with (A) anti-CD3 antibody, or (B) PMA/Ionomycin. Brefeldin A was added in the final 4 h of stimulation before intracellular staining. Each circle/diamond represents data of individual mouse. The P value was determined by two-tailed t test.

Figure 5

Figure 5. RBP-J-independent function of presenilin is required for optimal IFN-γ secretion from differentiated Th1 cells.

(A, B) Impaired IFNγ cytokine production by PS1/PS2-deficient T cells is not rescued by the compound loss of RBP-J. The experiments were conducted as described in Figure 4 except that the supernatant was harvested for ELISA analyses. Each data point indicates individual mouse and is presented as the relative level of IFN-γ secretion. This value is determined by calculating the percent of IFN-γ secretion from individual mouse over the maximum level attained in each separate experiment (see Materials and Methods for details). The P value was determined by two-tailed paired t test.

Figure 6

Figure 6. Reduced level of IL-4 secretion from PS1/PS2 and not RBP-J deficient Th2 cells.

(A, B) PS1/PS2-deficient T cells have significant reduction in IL-4 cytokine production when compared to RBP-J-deficient T cells independent of the mode of re-stimulation. The experiments were conducted as described in Figures 5. Data is presented as the relative level of IL-4, determined by comparing the IL-4 secretion from individual mouse over the maximum level attained in each separate experiment. The P value was determined by two-tailed paired t test.

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