Treg-mediated immunosuppression involves activation of the Notch-HES1 axis by membrane-bound TGF-β (original) (raw)
Enhanced Notch1-HES1 activation in antigen-induced tolerance compared with inflammation. To investigate the possible role of Notch in the development of tolerance, we subjected BALB/c mice to a model of tolerance induced by antigen (OVA), involving repeated exposure to inhaled antigen as previously described (3, 22). CD4+ T cells isolated from tolerized mice and mice immunized for the development of airway inflammation were stimulated with OVA ex vivo, and the expression of Notch1 was investigated. Although the CD4+ T cells isolated from the inflammation group proliferated more in comparison with those isolated from the tolerance group, as described previously (3), the cells from the tolerance group displayed a higher level of Notch1 expression (Figure 1).
Upregulation of cell surface expression of Notch1 on CD4+ T cells from tolerized mice. Mice were exposed for 10 successive days to PBS (mice subsequently develop airway inflammation after OVA/alum immunization followed by OVA aerosol exposure) or to 1% OVA (to develop tolerance). Both groups were immunized with OVA/alum on days 21 and 27, and spleens were isolated (3). Spleens from 3 animals were pooled in each group, and CD4+ T cells were isolated and subjected to stimulation with OVA/APCs in vitro. The cells were stained for the expression of CD4, CD25, and Notch1. Since no staining was detected in the top left quadrants of the “Airway inflammation” and “Tolerance” panels, the total fluorescence value in the FL1 channel for the isotype control stain (top left and top right quadrants) was subtracted from the experimental stains to arrive at net percent-positive values of 5% and 15% for the airway inflammation and tolerance conditions, respectively. Expression of CD25 and Notch1 was determined on equal numbers of CD4+ T cells for each condition. Numbers in the dot plots denote percentages. Results shown are representative of 4 independent experiments.
Upregulation of Notch1 on target cells by cells expressing membrane-bound TGF-β. We established an assay system to directly assess Notch1 expression in response to cells expressing membrane-bound TGF-β (TGF-βm+) isolated from tolerized mice, which have regulatory function (3). In this assay, CD4+ T cells isolated from DO11.10 TCR transgenic mice, which are recognized by the clonotypic KJ1-26 antibody, were used as target cells, and the cells were incubated with either TGF-βm+ cells or cells devoid of membrane-bound TGF-β (TGF-βm–) (Figure 2, A and B). A small fraction of the cell mix in each group was stained with KJ1-26 and anti-Notch1, and cell extract was derived from the rest. DO11.10 CD4+ T cells exposed to regulatory TGF-βm+ cells showed 2-fold and 4- to 5-fold more Notch1 expression after 24 (data not shown) and 48 hours of coculture (Figure 2C), respectively. Expression of cleaved Notch1, and HES1, a downstream target of Notch1, was also detected in the extracts prepared from the cell mix containing TGF-βm+ and DO11 CD4+ T cells (Figure 2, D and E). It should be noted that the anti-Notch1 antibody used in this particular experiment recognizes cleaved Notch1 and NICD but not the intact protein.
Upregulation of Notch1 on target cells by TGF-βm+ but not by TGF-βm– cells. (A) Post-sort TGF-β flow cytometry profile of TGF-βm+ and TGF-βm– cells. TGF-βm+ cells were induced in mice using the tolerance protocol. On day 21, CD4+ T cells were prepared by negative selection, and cells expressing TGF-β on the cell surface (TGF-βm+) were separated from those devoid of cell surface TGF-β (TGF-βm–) using 2 rounds of sorting. The purity of the 2 populations was assessed by flow cytometry (~80% enrichment of TGF-βm+ cells). (B) Experimental strategy. TGF-βm+ or TGF-βm– cells were mixed with DO11.10 TCR transgenic CD4+ T cells (target cells) in a 1:1 ratio and stimulated in vitro with whole OVA protein and APCs. (C) Staining with anti-Notch1 and KJ1-26 antibody or matching isotype control of an aliquot of 105 total cells removed after 48 hours of incubation. Each dot plot contains approximately 1,000 events. Numbers in dot plots denote percentages. (D) Western blot analysis of the presence of cleaved Notch1 in total cell extracts (TCEs) prepared from the mixed cultures after 24 hours of incubation using antibody specific to cleaved Notch1. Expression of β-actin is shown as a marker for protein loading. (E) Analysis of HES1 expression by immunoblotting of nuclear extracts (NEs) prepared after 72 hours of culture. CREB-1 expression was examined to assess protein loading. Results shown are representative of 2 independent experiments.
Expression of Notch ligands by TGF-βm+ cells. Having previously shown specific immunosuppressive functions of TGF-βm+ but not TGF-βm– cells (3), and further demonstrated that only TGF-βm+ cells induce Notch activation in target cells (Figure 2E), we examined expression of the Notch ligands on the TGF-βm+ cells. Mice were exposed to aerosolized OVA to induce tolerance, and on day 21 after initiation of exposure to OVA, CD4+CD25+ T cells were isolated from the spleens of these mice. Simultaneously, CD4+CD25+ T cells were also isolated from the spleens of naive mice. We first assessed expression of TGF-βm+ on these cells by flow cytometry. While approximately 10% of the CD4+CD25+ cells freshly isolated from the tolerized mice were found to express membrane-bound TGF-β, no TGF-β expression was detected on cells isolated from naive mice, as reported by us and others previously (3, 6). When expression of Notch ligands on these cells was examined, all of these cells were found to express the 3 ligands Jagged-1, Delta-1, and Delta-4 at a very high level (Figure 3A). In contrast, when TGF-βm– cells were examined, expression of Jagged-1 and Delta-4 was not detected, and that of Delta-1 was greatly diminished compared with that in TGF-βm+ cells (several orders of magnitude based on mean fluorescence intensity; Figure 3B). The level of Delta-1 staining on TGF-βm– cells was comparable to that on CD4+ T cells from naive animals (i.e., was very low), which also lack expression of Jagged-1 and Delta-4 (data not shown).
High-level expression of Notch ligands on TGF-βm+, but not TGF-βm–, cells. (A) CD4+CD25+ cells were purified from the spleens of naive or tolerized mice. The isolated CD4+CD25+ cells were stained for the expression of surface TGF-β1 and either Jagged-1, Delta-1, or Delta-4. No TGF-βm+ cells were detected among the CD4+CD25+ cells isolated from naive mice, while 10% of the CD4+CD25+ cells isolated from tolerized mice expressed TGF-β on the cell surface. The dot plots shown were generated by gating on CD4+CD25+TGF-βm+ cells from tolerized mice after staining with isotype (goat IgG) control, anti–Jagged-1, anti–Delta-1, or anti–Delta-4 antibodies. (B) TGF-βm– T cells from tolerized animals were similarly stained and analyzed for the expression of the 3 Notch ligands. Similar numbers of gated events are shown in each panel.
Activation of the Notch1-HES1 axis by membrane-bound TGF-β. To determine whether cell surface–associated TGF-β has the distinct ability to activate the Notch1 pathway, we compared the ability of TGF-βm+ cells and soluble TGF-β (in doses from 1 pg/ml to 1 ng/ml) to induce cleavage of Notch and activate HES1 in target cells. DO11.10 CD4+ T cells were incubated with TGF-βm+ or TGF-βm– cells from tolerized mice or with naive CD4+ T cells (to maintain similar cell composition in the mix) plus different doses of soluble TGF-β (shown are results with 1 ng/ml of soluble TGF-β). Cell extracts were prepared from the cocultured cells, and expression of Foxp3 (shown previously by us to be expressed by TGF-βm+ but not by TGF-βm– cells), phospho-Smad3 (pSmad3; TGF-β–inducible factor), NICD, and HES1 was examined in the respective extracts. As noted by others, nuclear NICD is very difficult to detect because of its low abundance (21) and short half-life (23). Foxp3 expression was most prominent when TGF-βm+ cells were coincubated with target cells (Figure 4A). Some expression was detected when naive cells were included along with soluble TGF-β, which could be due to a small population of TGF-βm+ cells in naive mice as reported previously by us (3). Interestingly, while as expected, phosphorylated Smad3, an essential feature of TGF-β signaling, was detected in each case, NICD and HES1 were detected only when TGF-βm+ cells were present in the cell mix (Figure 4A). Since only high doses of soluble TGF-β were previously shown to induce Foxp3 expression in target CD4+CD25– T cells by RT-PCR techniques (24), we assessed the effect of different doses of soluble TGF-β on Foxp3 protein expression in CD4+CD25– T cells stimulated in culture using intracellular staining methods. As shown in Figure 4B, a dose-dependent increase in the frequency of Foxp3-expressing cells was clearly evident. To demonstrate Notch1 activation induced by membrane-bound TGF-β in target DO11 cells, cells were sorted after coculture (Figure 4C), and indeed HES1 expression was noted only in the sorted DO11 cells that were previously incubated with TGF-βm+ cells (Figure 4D).
Membrane-bound TGF-β, but not soluble TGF-β, activates the Notch1 pathway in target cells. TGF-βm+ or TGF-βm– CD4+ T cells were mixed with DO11.10 TCR transgenic CD4+ T cells in a 1:1 ratio and stimulated with OVA (whole protein)/APCs for 1 day. (A) Expression of Foxp3, pSmad3, HES1, and NICD was assessed in nuclear fractions of the cells by Western blot analysis using specific antibodies. CREB-1 expression is shown as a marker for protein loading. Densitometric reading of protein expression revealed pSmad3/CREB-1 ratios of 0.6, 0.4, and 0.7 and Foxp3/CREB-1 ratios of 0.05, 0.4, and 0.1 for TGF-βm–, TGF-βm+, and soluble TGF-β incubations with DO.11 T cells, respectively. (B) Induction of Foxp3 expression in CD4+CD25– T cells treated with different doses of soluble TGF-β for 72 hours in the presence of soluble anti-CD3 (2 μg/ml) and T cell–depleted splenocytes. Foxp3 was detected by intracellular staining techniques. Numbers in the dot plots denote percentages. (C) In separate experiments, after 3 days of coculture, the target KJ1-26+ DO11.10 TCR transgenic T cells were sorted from the modulatory KJ1-26– TGF-βm+ or TGF-βm– cells, and nuclear extracts were prepared from each fraction. (D) Analysis of HES1 expression by immunoblotting of nuclear extracts of KJ1-26– and KJ1-26+ FACS-sorted cells.
Inhibition of Notch1 activation compromises suppressive function of TGF-βm+ cells. To test the importance of Notch1 activation in the suppressor function of TGF-βm+ cells, we used an inhibitor of γ-secretase, the enzyme complex responsible for cleavage of Notch into its active intracellular transactivator NICD. We first carried out a control experiment to test the efficacy of the inhibitor, L-685,458, which has been previously used to block γ-secretase–mediated Notch signaling (25–27). We examined HES1 activation in the presence or absence of L-685,458 in CD4+ T cells stimulated with a combination of soluble anti-CD3 and APCs. As shown in Figure 5A, HES1 activation in stimulated CD4+ T cells was inhibited in the presence of L-685,458 but not in the presence of the vehicle (DMSO). Having confirmed the inhibitory effect of the γ-secretase inhibitor, we set up an in vitro cell proliferation assay (28) to test the importance of simultaneous TGF-β and Notch signaling in suppression by TGF-βm+Foxp3+ cells derived from tolerized mice. Unlike CD4+ T cells from tolerized mice, cells from mice immunized for inflammation readily proliferated in a recall response to OVA ex vivo in a dose-dependent fashion (Figure 5B). Their proliferation was, however, inhibited in the presence of cells from tolerized mice, which we previously showed involves cell-cell contact (3). However, use of L-685,458, at doses known to inhibit Notch activation (27), blocked the suppressive effect of TGF-βm+ cells (Figure 5B).
Reversal of suppression by TGF-βm+ cells by inhibition of Notch1 signaling. (A) The γ-secretase inhibitor L-685,458 inhibits Notch1-induced HES1 activation in stimulated CD4+ T cells. Naive CD4+ T cells were stimulated with soluble anti-CD3 (2 μg/ml) plus APCs for 16 hours in the presence or absence of L-685,458 or 0.1% DMSO (vehicle). Nuclear extracts were prepared, and presence of HES1 was determined by immunoblotting. CREB-1 expression was assessed as a control for protein loading. (B) The γ-secretase inhibitor reverses the suppressive functions of TGF-βm+ cells. Mice were first exposed to PBS (inflammation group; Inf.) or 1% OVA (tolerance group; Tol.) daily for 10 days and were then immunized with OVA/alum on days 21 and 27. Splenic CD4+ T cells isolated on day 34 were stimulated in vitro with different concentrations of OVA (1–200 μg/ml) and mitomycin C–treated, T cell–depleted APCs at equivalent cell numbers (105 cells each per well). In mixed cultures containing CD4+ T cells from both groups, twice the number of cells from the tolerance group was added. The γ-secretase inhibitor L-685,458 was added at a concentration of 1 or 10 μM. After 72 hours of incubation, cells were pulsed with [3H]thymidine to assess cell proliferation. The proliferative response of CD4+ T cells from naive mice is shown as a negative control. *P < 0.05, mixed cultures with inhibitor treatment versus cultures without treatment. Each data point represents the mean ± SEM of triplicate wells. Shown is a representative experiment of 3.
TGF-βm+ cells induce direct interaction between cleaved Notch1 and pSmad3 in target cells. The ability of an inhibitor of γ-secretase to block the suppressive function of TGF-βm+ cells prompted us to seek biochemical evidence that membrane-bound TGF-β does cross-talk with the Notch1 pathway in target cells. In these experiments, we used cell extracts prepared from target DO11.10 cells that were briefly incubated with TGF-βm+ cells, TGF-βm– cells, naive CD4+ T cells, or soluble TGF-β (2 ng/ml). Cell extracts were prepared, and anti-pSmad3 antibody, coupled to agarose beads, was used to immunoprecipitate pSmad3 from the cell lysates. An isotype was used as a negative control for immunoprecipitation with TGF-βm+ cells. As shown in Figure 6, anti-pSmad3 antibody, but not the control antibody, resulted in coimmunoprecipitation of cleaved Notch1 only when DO11 T cells were incubated with TGF-βm+ cells. However, this was not because pSmad3 was present only in this particular cell lysate. As expected, pSmad3 was induced in the DO11 T cells when they were cocultured with TGF-βm– cells, which, we have shown previously, secrete soluble TGF-β (3), or when the cells were directly treated with soluble TGF-β (Figure 6). No pSmad3 was detected when DO11 cells were incubated with naive CD4+ T cells (negative control). As previously suggested (3), the biochemical data provided additional evidence that membrane-bound TGF-β has specific properties that cannot be substituted by soluble TGF-β, at least at doses that are otherwise inhibitory for CD4+ T cell activation as we and others have previously shown (29–31).
Membrane-bound TGF-β, but not soluble TGF-β, induces interaction between cleaved Notch1 and pSmad3. DO11.10 CD4+ T cells were cocultured with TGF-βm+ cells, TGF-βm– cells, naive CD4+ T cells, or soluble TGF-β (2 ng/ml) for 24 hours, and cell extracts were prepared. The extracts were subjected to immunoprecipitation using anti-pSmad3 or isotype control (with TGF-βm+ cells). The immunoprecipitates were analyzed by immunoblotting with antibody against cleaved Notch1, and the blot was stripped and reprobed with anti-pSmad3. While all lysates contained similar levels of Smad3, which is constitutively expressed in cells (data not shown), Smad3 was phosphorylated in the presence of TGF-βm+ cells, TGF-βm– cells, or soluble TGF-β, but not naive cells.
Blocking Notch1 activation in vivo prevents the ability of TGF-βm+ cells to suppress allergic airways disease. To test the importance of Notch1 activation in the suppressor function of TGF-βm+ cells in vivo, an anti-Notch1 antibody was used to block Notch1 activation. This antibody (distinct from the one used to detect cleaved Notch1 or NICD) was previously shown to antagonize Notch1 functions during thymocyte development (32). We first tested the inhibitory function of this antibody toward HES1 induction in target cells by TGF-βm+ cells. As shown in Figure 7, the antibody blocked HES1 activation induced by TGF-βm+ cells.
Inhibition of HES1 expression in the presence of anti-Notch1 antibody. TGF-βm+ cells were mixed with DO11 T cells in the presence or absence of anti-Notch1 antibody (1 μg/ml or 10 μg/ml) or control antibody, and HES1 expression was determined in nuclear extracts of the cells. Results are representative of 2 independent experiments.
We have previously shown that adoptive transfer of TGF-βm+ cells from tolerized mice to naive mice suppresses development of airway inflammation in the recipient mice (3). Using this approach, the relevance of the Notch1 pathway in suppression in vivo was examined using the anti-Notch1 antibody (Figure 8). The ability of TGF-βm+ cells to inhibit all of the hallmarks of allergic airways disease, ranging from antigen-specific IgE levels in the blood to Th2-type cytokine secretion and eosinophilic airway inflammation (Figure 8, group 3), was completely reversed in the presence of anti-Notch1 but not control antibody (Figure 8, group 5). Interestingly, while the OVA-specific IgE profile shows the dominant inhibitory effect of TGF-β1 on Th2 effects, the OVA-specific IgA levels reflect a dominant effect of Th2 cytokines such as IL-5. While TGF-β1 promotes a low frequency of IgA-secreting B cells (33, 34), Th2 cytokines, particularly IL-5, amplify this response severalfold (35, 36). The low levels of IgA in the bronchoalveolar lavage (BAL) fluid of mice that received TGF-βm+ cells with or without isotype control (groups 3 and 6) are further proof of the ability of TGF-βm+ cells to inhibit Th2-mediated inflammation. Tolerance was similarly inhibited when the anti-Notch1 antibody was used in conjunction with antigen exposure during the 10-day exposure period (data not shown). Collectively, these data indicate that membrane-bound TGF-β, induced in CD4+ T cells by antigen inhalation (3), requires functional Notch1 to exert its immunosuppressive functions. Although the precise targets of the anti-Notch1 antibody that prevented the immunosuppressive effects of TGF-βm+ cells cannot be ascertained from this experiment, a Th cell is a likely target.
Neutralization of Notch1 prevents active suppression by adoptively transferred TGF-βm+ cells. (A) Experimental setup. Anti-Notch1 antibody (50 μg/mouse) or matching isotype control was administrated i.p. into recipient animals 1 hour before OVA/alum injection (days 21 and 27) or aerosol challenge (day 35). Control mice were immunized with OVA/alum (airway inflammation) or subjected to the tolerance protocol and challenged with aerosolized OVA. Twenty-four hours after the last OVA challenge, mice were processed for different endpoints. (B) Shown are analysis of IgE in sera and IgA, and cytokine levels (IL-5, IL-13, IL-10 [undetectable] and TGF-β1) and cell differentials in BALF obtained from 6 groups of animals. Total TGF-β1 levels were measured after acid treatment of BALF. Active TGF-β1, measured in the absence of acid treatment, was detected at approximately 100 pg/ml only in the BALF of mice that received TGF-βm+ cells (lanes 3 and 6). *P < 0.05, TGF-βm+ recipients that also received anti-Notch1 antibody versus mice that received TGF-βm+ cells alone. (C–H) Tissue histology is also shown. Lung infiltrates were of +5 grade in all TGF-βm– cell transfers (F) or in control OVA/OVA–immunized mice (C), between +4 and +5 in mice that received TGF-βm+ cells and anti-Notch1 antibody treatment (G), compared with grades between +1 and +2 in mice that received TGF-βm+ cells alone (E) or isotype control (H), or in tolerized mice (D). There were 3 mice per group, and the results are representative of 2 independent experiments. Ag, antigen. Magnification, ×40.