Notch ligand Delta-like 4 promotes regulatory T cell identity in pulmonary viral infection

Abstract

Regulatory T (T_reg) cells establish tolerance, prevent inflammation at mucosal surfaces, and regulate immunopathology during infectious responses. Recent studies have shown that Delta-like ligand 4 (Dll4) was up-regulated on antigen presenting cells after RSV infection and its inhibition leads to exaggerated immunopathology. The present studies outline the role of Dll4 in T_reg cell differentiation, stability and function in RSV infection. Here we found that Dll4 was expressed on CD11b⁺ pulmonary DC in the lung and draining lymph nodes in wild type BALB/c mice after RSV infection. Dll4 neutralization exacerbated RSV-induced disease pathology, mucus production, group 2 innate lymphoid cell (ILC2) infiltration, IL-5 and IL-13 production, as well as IL-17A+ CD4 T cells. Dll4 inhibition decreased the abundance of CD62L^hiCD44^loFoxp3⁺ central T_reg cells (cT_R) in draining lymph nodes. The RSV-induced disease was accompanied by an increase in Th17-like effector phenotype in Foxp3⁺ T_reg cells and a decrease in Granzyme B expression after Dll4 blockade. Finally, Dll4-exposed induced T_reg (iT_reg) cells maintained CD62L^hiCD44^lo cT_R cell phenotype, had increased Foxp3 expression, became more suppressive, and were resistant to Th17 skewing in vitro. These results suggest that Dll4 activation during differentiation sustained T_reg cell phenotype and function to control RSV infection.

Introduction

Notch is a highly conserved signaling pathway that contributes to cell differentiation and function. Engagement of Notch receptors (Notch 1-4) and Notch ligands (Delta-like ligand 1, 3, 4, Jagged 1, 2) initiates cleavage of the Notch intracellular domain (NICD). NICD binds to recombination binding protein-J (RBP-Jκ) and activates the transcription of Notch target genes in association with a co-activator of the Mastermind like family (MAML 1-3). Notch signaling regulates various stages of T cells lineage development and differentiation, including early stages of thymocyte development, as well as CD4 T helper (Th) differentiation (1, 2). In the priming stage of Th cell differentiation, Notch ligand Dll4 sensitized antigen responses and augmented T cells activation (3), while separate studies demonstrated that Dll4 and Notch could activate production of IFNγ and the Th1 transcription factor T-bet (4–6). During Th2 differentiation, Jagged1 and intracellular Notch promoted Gata3 and Il4 expression (4, 7, 8), while Dll4 suppressed Il4, Il5, and Il13 (9, 10). Furthermore, Dll4 and Notch was reported to promote Th17 and Th9 differentiation by enhancing Il17a, Rorc and Il9 expression (11, 12). Together, Notch can serve as an amplifier for persistent Th1, Th2, and Th17 cell differentiation (5), suggesting that it is not a skewing signal, but rather enhances co-activation. However the role of Dll4 and Notch signaling in T_reg cells remains unresolved. Jagged2 and specific receptors, Notch1 and Notch3, promoted the T_reg cell master transcription factor—Foxp3 expression and T_reg cell survival (13–17), and RBP-Jκ was reported to directly bind the Foxp3 promoter and regulate Foxp3 transcription (17). In contrast, inactivating Notch signaling after Foxp3 is expressed enhanced T_reg cell numbers and promoted tolerance (18). Blockade of Notch receptors and Notch ligands expanded Foxp3+ T cell populations in vivo in experimental autoimmune encephalomyelitis (EAE), Graft-versus-host disease (GvHD), and Type 1 diabetes (T1D) (19–22). However, the role of Notch ligands in T_reg cell development and their resistance to inflammation during infection has not been well-defined.

Notch ligands can be induced on antigen presenting cells by pathogen-associated molecular patterns (PAMPs) (4, 7). Pathogens themselves can also induce Notch ligands. Studies showed that Respiratory syncytial virus (RSV) induced Dll4 expression on dendritic cells in vitro (9, 23), and Dll4 blockade exacerbated RSV-induced Th2 airway pathogenesis (9). Since T_reg cells are required to limit pulmonary inflammation and pathogenic Th2 responses during RSV infections (24–26), we hypothesized that initial exposure of Dll4 may modulate peripherally-induced T_reg (iT_reg) cell differentiation, homeostasis and stability to control the intensity of the immune response and lung pathology during RSV infection. In the present study, we report that Dll4 sustained CD62L^hiCD44^lo central T_reg cells and solidified iT_reg cell identity during infection. This study defines novel roles of Dll4 in iT_reg cell subset regulation and iT_reg cell stability.

Materials and Methods

Mice

6-8 week old female BALB/cJ and C57BL/6J mice were purchased from Jackson Laboratory. Female CD45.1 (B6-Ly5.1/Cr) mice were purchased from Charles River. Foxp3^eGFP mice (B6.Cg-Foxp3^tm2(EGFP)Tch/J, stock number 006772) were bought from Jackson Laboratory and bred in house. CD4-specific DNMAML mice (Cd4-Cre × R26^DNMAMLf) and CD4-specific Rbpj knockout mice (Cd4-Cre × Rbpj^f/f) are generated as described (27–29). All mice were housed in the University Laboratory Animal Facility under animal protocols approved by the Animal Use Committee in University of Michigan.

RSV infection and In vivo neutralization of Dll4

RSV Line 19 was clinical isolate originally from a sick infant in University of Michigan Health System to mimic human infection (30). BALB/cJ mice were anesthetized and infected intratracheally (i.t) with 1 × 10⁵ pfu of Line 19 RSV, as previously described (9). For Dll4 blockade in vivo, 2.5 mg of purified polyclonal anti-Dll4 antibody or control IgG were injected intraperitoneally (i.p) two hours before RSV infection at day 0. Same dose of control or anti-Dll4 antibody were given on day 2, 4, and 6.

Histopathology

Left lobe of lung was fixed with 4% formaldehyde and embedded in paraffin, and 5 μm of section were stained with Periodic acid-Schiff (PAS) stain to detect mucus.

RNA Isolation and Quantitative PCR

RNA were extracted with TRIzol (Invitrogen) by following manufacturers protocol, and one μg of total RNA were reverse transcribed to cDNA to determine gene expression using Taqman gene expression primer/probe sets. Dll1, Dll4, Jag1, and Jag2 were detect by SYBR as described (31). Delta4 primers: 5’-AGGTGCCACTTCGGTTACACAG-3’ and 5’-CAATCACACACTCGTTCCTCTCTTC-3’. Muc5ac and Gob5 expression were assessed by custom primers as described (32). Detection was performed in ABI 7500 Real-time PCR system. Gene expression was calculated using the ΔΔCt method and normalized with Gapdh as input control.

Primary cells Isolation and Cytokine production assay

Mice lungs were chopped. Lung and mediastinal lymph node were enzymatically digested using 1 mg/mL Collagenase A (Roche) and 25 U/ml DNaseI (Sigma-Aldrich) in RPMI 1640 with 10% fetal calf serum for 45 min at 37°C. Tissue were further dispersed through 18 gauge needle/10 mL syringe, and filtered through 100-μm nylon mesh twice. 5 × 10⁵ cells from mediastinal lymph node cells were plated in 96-well and re-stimulated with 10⁵ pfu RSV Line 19 for 48 hours. IFN-γ, IL-4, IL-5, IL-13, IL-17A, IL-10, IL-9 level in supernatant were measured with Bio-plex™ cytokine assay (Bio-Rad).

Extracellular and Intracellular Flow cytometry analysis

Single-cell suspension of lung and lymph node were stimulated with 100 ng/mL Phorbol-12-myristate 13-acetate (PMA), 750 ng/mL Ionomycin, 0.5 μL/mL GolgiStop (BD), 0.5 μL/mL GolgiPlug (BD) for 5 hours if mentioned. After excluding dead cells with LIVE/DEAD Fixable Yellow stain (Invitrogen), cells were pre-incubated with anti-FcγR III/II (Biolegend) for 15 minutes and labeled with the following antibody from Biolegend, unless otherwise specified: anti-B220 (RA3-6B2), CD3 (145-2C11), CD4 (GK1.5), CD8α (53-6.7), CD11b (M1/70), CD11c (N418), CD25 (PC61), CD44 (IM7), CD45 (30-F11), CD62L (MEL-14), CD69 (H1.2F3), CD103 (2E7), CD127 (SB/199), CCR7 (4B12), Dll1 (HMD1-3), Dll4 (HMD4-1), Gr-1 (RB6-8C5), I-A/I-E (M5/114.15.2), ST2 (DIH9). For Innate lymphoid cells staining, Lineage markers were anti-CD3, CD11b, B220, Gr-1, TER119. After 30 minutes of incubation at 4°C, cells were washed and proceed to intracellular staining.

For intracellular staining, cells were fixed and permeabilized with Transcription factors staining buffer set (eBioscience). Cells were labeled with antibody from eBioscience: Foxp3 (FJK-16s), IL-17A (eBio17B7), IL-13 (eBio13A), GATA3 (TWAJ), RORγt (AFKJS-9), GzmB (NGZB) for 30 minutes at room temperature. Flow cytometry data were acquired from LSR II (BD) or Novocyte (ACEA) flow cytometer and were analyzed with FlowJo software (TreeStar).

Naïve CD4 T Cell Isolation and Stimulation

CD4⁺CD25⁻CD62L^hiCD44^lo naïve T cells were enriched from spleen using the naïve CD4 T cells isolation kit (Miltenyi Biotec) with more than 92% purity. Naïve T cells were then plated and cultured in 24-well plates. 10⁶ / 0.5 mL of naïve T cells were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant Dll4 (1.65 μg/mL or the dose mentioned; R&D); to skew toward in vitro-induced T_reg cells (iT_reg), human TGFβ1 (2 ng/mL; R&D System) and mouse IL-2 (10 ng/mL; R&D System) were added at the same time; to re-stimulate toward in vitro-induced Th17 cells (Th17), mouse IL-6 (10 ng/mL; R&D System), human TGFβ1 (2 ng/mL; R&D System), anti-IFNγ neutralizing antibody (10 μg/mL; eBioscience), anti-IL-4 neutralizing antibody (10 μg/mL; eBioscience), and anti-IL-12/23 p40 neutralizing antibody (10 μg/mL; eBioscience) were added at 5 × 10⁵/ 0.2 mL of viable iT_reg culture or 10⁵/ 0.2 mL sorted eGFP⁺ iT_reg if mentioned.

Cell sorting and In vitro suppression assay

Single cell sorting was performed on FACSAria II (BD). DAPI⁻CD4⁺eGFP⁺ viable T_reg were sorted with more than 93% efficiency. Suppression assay was performed as described with small modification (33). In brief, naïve T cells isolated from CD45.1 mice were labeled with Cell Trace Violet (CTV) (Invitrogen). 2.5 × 10⁴ of labeled CD45.1+ naïve T cells were co-cultured with serial-diluted eGFP⁺ T_reg in 96 well round bottom plate. 0.625 μL of Dynabeads^® mouse T activator CD3/CD28 (Invitrogen) were added to 0.2 mL of culture. After 72 hours, cells were harvested, and CD45.1⁺ responder cell proliferation were accessed by CTV dilution.

Statistical Analysis

Data were analyzed by Prism6 (GraphPad). Data presented are mean values ± SEM. Comparison of two groups was performed in unpaired, two-tailed, Student’s t-test. Comparison of three or more groups was analyzed by ANOVA with Tukey’s post-tests. Significance was indicated at the level of *:p<0.05, **: p<0.005, ***: p<0.0005

Results

Up-regulation of Dll4 during RSV infection on CD11b+ pulmonary DC

Previously published data have shown that Dll4 expression was up-regulated on dendritic cells post RSV infection in vitro (9, 23). To further characterize the expression of Dll4 during RSV infection in vivo, BALB/cJ mice were intratracheally (i.t.) infected with 1 × 10⁵ pfu of RSV before harvesting of lung tissues at 2 and 6 days post infection (dpi). Delta-like ligand 4 (Dll4) transcripts were up-regulated significantly at 6 dpi, while transcripts for the other Notch ligands Dll1, Jagged (Jag) 1 and Jag2, were not increased in the lung (Fig 1A). After gating on MHC^hi CD11c⁺ dendritic cells (Fig 1B), Dll4 expression level was increased consistently on MHCII^hi CD11c⁺ dendritic cells after 6 dpi (Fig 1C). To further understand which type of DC expressed Dll4 after RSV infection, two populations of pulmonary dendritic cells (DC) were identified to determine Dll4 expression: MHCII^hi CD11c⁺ CD11b⁺ CD103⁻ DC and MHCII^hi CD11c⁺ CD103⁺ CD11b⁻ DC. CD11b⁺ DC expressed Dll4, whereas few of the CD103⁺ DC expressed Dll4 during RSV infection (Fig 1D). When we examined the number of DC in lung and draining lymph node at 6 dpi, increasing numbers of Dll4⁺ CD11b⁺ DC in both lung and mediastinal lymph node (mLN) were detected as the infection progressed. In contrast, the number of Dll4⁺ CD103⁺ DC was significantly lower and variable in lung or mLN after infection (Fig 1E). These data indicated that Dll4 was expressed on pulmonary DC, especially CD11b⁺ DC, after RSV infection in vivo.

Figure 1. Dll4 expresses on CD11b+ pulmonary DC during RSV infection.

A. Abundance of Dll1, Dll4, Jag1, and Jag2 mRNA in RSV-infected lung at the indicated time points relative to uninfected naïve lung

B. Representative flow cytometric analysis showing the gating strategy of viable MHC-II ^hi CD11c⁺ dendritic cells (DC) in RSV-infected lung at 6 dpi

C. Representative flow cytometric analysis showing the mean fluorescence intensity of Dll4 on Dll4⁺ MHC-II ^hi CD11c⁺ DC from RSV-infected lung

D. Percent of Dll4⁺ in CD11b⁺ or CD103⁺ DC (left, gating strategy) from infected lung

E. Numbers of Dll4⁺ CD11b+ DC or Dll4⁺ CD103⁺ DC in the lungs (left) or draining mediastinal lymph nodes (mLN) (right) of naïve or RSV-infected mice.

Data represent mean ± s.e.m. Data were from one experiment representative of two experiments with 4 to 5 mice per time point, with samples from each mouse processed and analyzed separately. * P<0.05; ** P<0.005; *** P<0.0005; NS: no significance (unpaired two-tailed t-test)

Inhibition of Dll4 exacerbated RSV-induced immunopathology in vivo

Next, we explored the role of Dll4 in controlling RSV immunopathology by blocking Dll4 in vivo during RSV infection. We have previously demonstrated that our polyclonal anti-Dll4 antibody is specific for Dll4 and not other Notch ligands (9). The polyclonal anti-Dll4 antibody decreased the abundance of Notch target gene Hes1 transcripts in activated T cells (Th0) in co-culture with fixed Dll4-expressing stromal cells (Sup Fig 1A), indicating that anti-Dll4 antibody blocked Dll4-mediated Notch activation in vitro. We next aimed at blocking Dll4 in vivo by injecting 2.5 mg of purified anti-Dll4 antibody intraperitoneally (i.p). Mice were then infected intratracheally (i.t) with RSV 2 hours after antibody injection at day 0, followed by i.p injection of anti-Dll4 every other day. After 8 days, the abundance of Hey1—Notch target gene transcripts was decreased in the lung (Sup Fig 1B) and sorted CD4 T cells (Sup Fig 1C) of anti-Dll4-treated mice, suggesting that systemic administration of anti-Dll4 antibodies inhibited Notch signaling activation in CD4 T cells in vivo. Dll4 blockade increased peribronchial infiltration by mononuclear cell clusters and drove mucus production in the airway as assessed in periodic-acid Schiff (PAS)-stained lung sections (Fig 2A). As an indication of RSV-induced pathology, expression of the mucus-associated gene gob5 (mClca3) was substantially increased (Fig 2B), but the viral protein mRNA expression measured with RSV-F and RSV-G were not significantly different at either 6 dpi or 8 dpi (Fig 2C and data not shown). While Dll4 inhibition increased total CD4 T as described (Fig 2D) (9), data also showed that more CD4 T cells acquired a CD44^hiCD62L^lo effector phenotype (Fig 2E). Another activation marker CD25 was not changed in CD4 T cells at 8 dpi (data not shown). Thus, Dll4 inhibition significantly altered lung immunopathology during RSV infection. Besides CD4 T cells, RSV infection can also expand group 2 innate lymphoid cells (ILC2) between 4 to 6 dpi in lung to be mucogenic and immunopathogenic (34). Here we investigate if Notch ligand Dll4 affects ILC2 homeostasis. Strikingly, Dll4 inhibition increased the number of Lin-CD45⁺CD127⁺CD90.2⁺ST2⁺CD25⁺ ILC2 in lung (Fig 2F) and mLN (Fig 2G) at 6 dpi. Thus, Dll4 blockade increased ILC2 in the lungs during RSV infection.

Figure 2. Blockade of Dll4 drives mucus production, accumulation of activated CD4 T cells, group 2 innate lymphoid cells, and cytokine production.

A. Periodic acid-Schiff (PAS) staining of formalin-fixed lung section from 8 dpi. Bar, 200 μm. ↑ indicates the detection of mucin, and ˄ designates mononuclear cells aggregates.

B. Gob5 expression in the lung at 8 dpi.

C. RSV-F viral protein expression in lung at 8 dpi. ND: non-detectable.

D. Numbers of CD4 T cells in lung at 8dpi.

E. Numbers of CD44^hiCD62L^lo effector CD4 T cells in lung at 8 dpi.

F. Numbers of Group 2 Innate lymphoid cells (ILC2) in lung at 6 dpi.

G. Numbers of ILC2 in mLN at 6 dpi.

H. mLN cells were isolated from mice at 8 dpi and subject to RSV re-stimulation for 48 hours. Indicated cytokines in supernatant were measured.

I. mLN cells were isolated from mice at 8 dpi and re-stimulated with PMA and Ionomycin for 5 hours. Viable CD4 T cells were gated, and intracellular IL-13 and IL-17A were stained.

Data represent mean ± s.e.m. N=7~10 each group. Each symbol represents individual mouse processed and analyzed separately. Data are representative of at least two experiments * P<0.05; ** P<0.005; *** P<0.0005; NS: no significance (unpaired two-tailed t-test)

Excessive cytokine production is one of the key features leading to RSV pathogenesis. The IFNγ promotes viral clearance while IL-4, IL-5, IL-13 and IL-17A are pathogenic (35–37). Here we determined the effect of Dll4 blockade on cytokine profiles in draining lymph nodes. Equal numbers of mLN cells were cultured and re-stimulated with 10⁵ pfu RSV. In vivo Dll4 blockade during RSV infection led to significantly increased Th2 cytokines IL-5 and IL-13 as well as IL-17A while IFNγ, IL-4, IL-9, and IL-10 were not significantly affected in RSV re-challenged lymph node cells (Fig 2H). To specify if Dll4 regulated T helper cell cytokine production in CD4 T cells, mLN cells were re-stimulated with PMA + Ionomycin and CD4 T cells were examined by flow cytometry. Dll4 blockade significantly increased IL-13+ and IL-17A+ CD4 helper T cells in mLN in both 6 dpi and 8 dpi (Fig 2I and data not shown). These data indicated that Dll4 neutralization exacerbated RSV-induced immunopathology, as shown by elevated mucus production, activated CD4 T cells, and Th2-Th17 cytokine over-production.

Dll4 neutralization reduces central T_reg and increases Th17-like T_reg cells during RSV infection

Several studies demonstrated that T_reg cells quickly accumulate in lymphoid tissues and in the airway to dampen activated T cell infiltration and Th2 responses (24, 25, 38), and RSV infection drove T_reg cells to Th2-like effector cells with impaired function (39). Our group showed that Dll4 inhibition exacerbated type 2 cytokine production and activated T cells infiltration, but little is known about the role of Dll4 in T_reg cell development, stability and function. After targeting Dll4 as previously described, total T_reg cells did not change in mLN at 6 dpi (Fig 3A). However, when gating on CD62L^hiCD44^lo central CD4 T cells (cT) (Fig 3B), Dll4 inhibition decreased Foxp3⁺ cell frequency and expression (Fig 3C, D), while decreasing expression of the CCR7 chemokine receptor on cT cells (Fig 3E, F). CCR7-mediated signals recruit CD62L^hiCD44^lo T cells to the T cell zone, and CCR7 is a marker of cT_R cells (40). Dll4 neutralization decreased the cT_R cell populations (Fig 3G), while eT_R cells were not altered in mLN (Fig 3H). These data suggested that Dll4 specifically sustained cT_R cells in draining lymph node during RSV infection.

Figure 3. Dll4 inhibition impairs the maintenance of central T_reg cells in lymph node in vivo.

A. Percentage of Foxp3⁺ T_reg in mLN at 6 dpi

B. Representative flow cytometric analysis showing Foxp3 expression in CD62L^hiCD44^lo central CD4 T cells in mLN at 6 dpi

C. Percentage of Foxp3⁺ cells in CD62L^hiCD44^lo central CD4 T cells harvested from the mLN at 6 dpi

D. Mean fluorescence intensity of Foxp3 in CD62L^hiCD44^lo central CD4 T cells in mLN at 6 dpi

E. Representative flow cytometric analysis showing CCR7 expression in CD62L^hiCD44^lo central CD4 T cells in mLN at 6 dpi

F. Mean fluorescence intensity of CCR7 in CD62L^hiCD44^lo central CD4 T cells in mLN at 6 dpi

G. Percentage of CD62L^hiCD44^loFoxp3⁺ central T_reg in mLN at 6 dpi

H. Percentage of CD44^hiCD62L^loFoxp3⁺ effector T_reg in mLN at 6 dpi

Data represent mean ± s.e.m. Each symbol represents individual mouse processed and analyzed separately. Data are representative of at least two independent experiments * P<0.05; ** P<0.005; *** P<0.0005; NS: no significance (unpaired two-tailed t-test)

In contrast its effects in lymph nodes, Dll4 neutralization enhanced the abundance of Foxp3⁺ T_reg cells in the lungs of infected mice (Fig 4A) and CD44^hi CD62L^lo Foxp3⁺ eT_R cells in lung at 8 dpi (Fig 4B) without affecting cT_R cells (data not shown). Based on our previous finding that Dll4 blockade enhanced IL-13 and IL-17A production in CD4 T cells (Fig 2I), we examined if Dll4 can also perturb cytokine production in Foxp3⁺ CD4 T cells. Lung cells harvested at 8 dpi were re-stimulated and stained for intracellular Foxp3, IL-13 and IL-17A before flow cytometric analysis. In vivo Dll4 blockade significantly increased the abundance of IL-17A⁺Foxp3⁺ (Fig 4C) and IL-13⁺Foxp3⁺ (Fig 4D) but not IL-13⁻, IL-17A⁻, Foxp3⁺ T_reg cells (data not shown). To further characterize if Dll4 regulates either Th17 or Th2 transcription factor co-expression in T_reg cells, RORγt or GATA3 were co-stained with Foxp3 after re-stimulation. Dll4 neutralization increased the percentage of RORγt⁺Foxp3^lo cells and RORγt⁺Foxp3⁻ but not RORγt⁻Foxp3⁺ after 8 dpi (Fig 4E, F). We did not detect significant changes in either GATA3⁺ T_reg cells or total GATA3+ CD4 T cells in both lung and LN after Dll4 inhibition (data not shown). These data suggested that Dll4 inhibition drove a Th17-like effector phenotype in T_reg cells during RSV infection in vivo.

Figure 4. Dll4 blockade drives effector-like, inflammatory, and less functional T_reg cells in the lungs during RSV infection.

A. Percentage of Foxp3⁺ T_reg in lung were analyzed at 8 dpi

B. Percentage of CD44^hiCD62L^loFoxp3⁺ effector T_reg in lung at 8 dpi

C. Percentage of IL-17A-producing T_reg cells in lung from 8 dpi after PMA and Ionomycin re-stimulation were measured.

D. Percentage of IL-13-producing T_reg cells in lung from 8 dpi after PMA and Ionomycin re-stimulation were measured.

E. Percentage of RORγt⁺Foxp3^lo CD4 T cells in lung from 8dpi after PMA and Ionomycin re-stimulation were measured.

F. Percentage of RORγt⁻Foxp3⁺ CD4 T cells in lung from 8dpi after PMA and Ionomycin re-stimulation were measured.

G. Mean fluorescence intensity of GzmB in Foxp3⁺ T_reg in the lung at 8 dpi

H. Representative flow cytometry showed the frequency of GzmB+ in Foxp3⁺ T_reg in the lung at 8 dpi

I. Percentage of GzmB⁺ effector T_reg (eT_R), central T_reg (cT_R), CD62L^hiCD44^hi T_reg, and CD62L^loCD44^lo T_reg in the lung after 8 dpi

Data represent mean ± s.e.m. Each symbol represents individual mouse processed and analyzed separately. Data are representative of at least two independent experiments. * P<0.05; ** P<0.005; *** P<0.0005; NS: no significance (unpaired two-tailed t-test)

To further investigate if Dll4 regulated T_reg cell functional markers, several were examined—Inducible T cells co-stimulator (ICOS), Programmed cell death protein 1 (PD-1), Granzyme B (GzmB), and Neuropilin-1 (Nrp1). Dll4 inhibition decreased the expression of GzmB, while ICOS, PD-1 and Nrp1 expression was unchanged in Foxp3+ T_reg cells at 8 dpi (Fig 4G and data not shown). It was previously shown that during RSV infection, GzmB was the critical functional molecule for T_reg cell function to limit the associated immunopathology (26). We found that Dll4 neutralization decreased the frequency of GzmB⁺ in Foxp3+ T_reg cells in the lung after 8 dpi (Fig 4H), especially prominently for GzmB⁺ eT_R cells (Fig 4I). Our results indicate that Dll4 prevented Foxp3 T_reg cell acquisition of Th17-like effector phenotype, and it supported GzmB expression in T_reg cells in the lung during RSV infection.

Dll4 activates and maintains inducible T_reg cells in vitro

Our data reveal differential effects of Dll4 in Foxp3+ T_reg cells in lymphoid vs. non-lymphoid tissue during pulmonary infection. To further investigate Dll4 effects on T_reg cell differentiation in a controlled context, splenic CD4⁺CD25⁻CD62L^hiCD44^lo naïve CD4 T cells were activated with anti-CD3/anti-CD28 with or without plate-bound recombinant Dll4, and skewed toward T_reg cells with TGF-β and IL-2. We first confirmed that Dll4 was able to activate expression of the Notch target gene Hes1 in naïve CD4 T cells, Th0 and T_reg cells (Sup Fig 2A,B,C) in a dose-dependent manner (Sup Fig 2C) and activated intracellular domain of Notch1 (N1ICD) cleavage (Sup Fig 2D). Subsequently, naïve T cells were skewed toward Th0 or iT_reg cells. Foxp3 expression was significantly increased with Dll4 activation under iT_reg cell skewing conditions (Fig 5A). Importantly, Dll4 increased CD25⁺Foxp3⁺ iT_reg cells in a dose-dependent manner only in the presence of TGF-β (Fig 5B). Furthermore, Foxp3 expression was significantly decreased with homozygous ROSA26-driven expression of the pan-Notch-inhibitor dominant negative Mastermind-like 1 (DNMAML1) (Fig 5C), and with inactivation of the Rbpj gene, encoding RBP-Jκ (Fig 5D). Since MAML1 and RBP-Jκ mediate transcriptional activation from Notch signaling, these data revealed that Dll4-activated Foxp3 expression was dependent upon canonical RBP-Jκ/MAML-dependent Notch signaling. To further characterize Foxp3+ cells in this differentiation system, primary Dll4-activated iT_reg cell cultures were rested in IL-2 for 3 days without Dll4 (Fig 5E). Dll4-treated iT_reg cells consistently maintained a higher percentage of Foxp3+ (Fig 5F) and higher Foxp3 expression (Fig 5G). To investigate whether Dll4 affected homeostatic features on T_reg cells during in vitro skewing, CD62L and CD44 expression was evaluated before or after the IL-2 rest phase. Naïve CD4 T cells that were exposed to Dll4 during primary iT_reg cell skewing maintained more CD62L^hiCD44^loFoxp3+ cells consistently independent of the dose of IL-2 during the rest period (Fig 5H) and at multiple time points after primary skewing (Fig 5I). These data suggest that the presence of Dll4 during iT_reg cell activation and differentiation stabilized Foxp3 expression and sustained more T_reg cells with CD62L^hiCD44^lo phenotype in vitro.

Figure 5. Dll4 promotes Foxp3 expression and sustains central T_reg cells during iT_reg differentiation.

A. Inducible T_reg (iTreg) and activated T cells (Th0) were skewed from naïve CD4 T cells for 48 hours with or without plate-bound Dll4 in vitro. Foxp3 expression were measured.

B. CD25⁺Foxp3⁺ iT_reg were skewed from naïve CD4 T cells for 72 hours with or without 2 ng/mL of TGF-β and indicated concentration of Dll4 (μg/mL)

C. Foxp3 expression after 48 hours skewing from either wild type naïve CD4 T cells or naive T cells expressing dominant negative MAML1 (DNMAML)

D. Foxp3 expression after 48 hours skewing from either wild type or CD4-specific Rbpj-deficient naïve CD4 T cells

E. Naïve CD4 T cells from wild type B6 mice were skewed toward iT_reg with or without Dll4 stimulation for 72 hours (d3), then rested in 10 ng/mL of IL-2 for another 72 hours. Representative flow cytometry showed the expression of Foxp3 in viable CD4 T cells at day 6 (d6).

F. Percentage of Foxp3⁺ in viable CD4 T cells at day 6.

G. Mean fluorescence intensity of Foxp3 in viable Foxp3+ CD4 T cells at day 6

H. Percentage of CD62L^hiCD44^loFoxp3⁺ central T_reg (cT_R) after 0 ng/mL, 2 ng/mL and 10 ng/mL of IL-2 resting at day 6 iT_reg culture

I. Percentage of cT_R at day 3 and day 6 with 2 ng/mL of IL-2 in iT_reg culture

Data represent mean ± s.e.m. Each symbol represents individual mouse processed and analyzed separately. Data are representative of at least two experiments * P<0.05; ** P<0.005; *** P<0.0005; NS: no significance (unpaired two-tailed t-test)

Dll4-exposed iT_reg are more suppressive in vitro

Our data showed that Dll4 inhibition decreased T_reg cell functional markers during RSV infection (Fig 4), and Dll4/Notch directly sustained Foxp3 expression and cT_R cells (Fig 5). These findings imply that Dll4 may change T_reg cell function. To further evaluate the function of iT_reg cells after Dll4 exposure, we sorted viable, Foxp3-eGFP⁺ iT_reg cells from iT_reg cultures on day 6 and co-cultured these cells with CD45.1⁺ naïve CD4 T cells as responders. CD45.1⁺ naïve T cells were labeled with CellTrace Violet (CTV) and stimulated with α-CD3/α-CD28 mouse T activator for 72 hours. Viable CD45.1⁺ cells were gated to examine CTV dilution as readout of cell proliferation (Fig 6A). Responder cells that were co-cultured with Dll4-treated iT_reg cells were less proliferative, especially at the 1:1 and 1:2 cell ratio of T_reg: T_naive (Fig 6B), suggesting that Dll4-treated iT_reg cells were more suppressive than iT_reg cells without Dll4. Intriguingly, CD45.1⁺ responder cells were less viable when co-culture with Dll4-exposed iT_reg cells (Fig 6C). Because one of the functions of T_reg cells is to consume survival signals from other T cells, we evaluated IL-2Rα (CD25) expression after 6 days of iT_reg cell skewing. Co-stimulation of iT_reg cells with Dll4 enhanced CD25 expression on Foxp3+ T_reg in vitro (Fig 6D). There data indicated that Dll4-exposed iT_reg were more functional in vitro.

Figure 6. Dll4-exposed iT_reg are more suppressive in vitro.

A. Schematic representation of the in vitro suppression assay. Naïve CD4 T cells from Foxp3-eGFP knock-in mice underwent iT_reg differentiation with or without Dll4 for 72 hours, and resting in 2 ng/mL IL-2 for 72 hours. Viable iT_reg or Dll4-exposed iT_reg were sorted out as Foxp3-eGFP⁺ DAPI⁻, and co-cultured with CellTrace violet (CTV) labeled CD45.1⁺ naïve T cells with anti-CD3/anti-CD28 beads.

B. After 3 days in co-culture, proliferation was assessed by CTV dilution in CD45.1⁺ responder cells.

C. After 3 days co-culture, viability was characterized by Live/Dead staining (LD) in CD45.1 responder cells

D. CD25 expression by viable iT_reg at day 6

Dll4 and Notch activation strengthened iT_reg to be less plastic toward Th17

Data above showed that Dll4 inhibition increased Th17-like T_reg cells during RSV infection. To examine whether Dll4 and Notch signaling could inhibit acquisition of a Th17 effector phenotype in iT_reg cells, naïve CD4 T cells were purified and cultured in iT_reg skewing conditions. Il17a expression was examined during iT_reg cell skewing. Inactivation of Notch increased Il17a expression during iT_reg cell skewing (Fig 7A), while Foxp3 transcripts were decreased (Fig 7B). Both DNMAML1 expression and Rbpj inactivation in CD4 T cells led to increased IL-17A in iT_reg+ Dll4 skewed cultures (Fig 7C). These data suggest that intrinsic Notch activation inhibited Il17a expression while enhancing Foxp3 expression in iT_reg cells during differentiation. To further investigate whether Dll4 affects IL-17A production after T_reg cell differentiation, an equal number of cells were challenged under IL-6 + TGFβ Th17 conditions for 3 more days after the standard 6 day iT_reg cell skewing culture. In Th17 re-stimulation, Dll4-treated iT_reg cells secreted significantly less IL-17A (Fig 7D) with associated decreased RORγt+ cells (Fig 7E). Furthermore, Dll4-exposed iT_reg cells retained a higher percentage and expression level of Foxp3 (Fig 7F, 7G). These data suggest that Dll4-mediated signals during iT_reg cell differentiation led to less inflammatory Th17 skewing while maintaining T_reg cell commitment. To clarify if Dll4-educated Foxp3⁺ iT_reg cells had less plasticity to become RORγt⁺, 10⁵ viable Foxp3-eGFP⁺ iT_reg cells were sorted after 6 days of skewing and cultured in Th17 conditions. Approximately 40% of eGFP⁺ iT_reg cells without Dll4 acquired RORγt⁺ expression, whereas Dll4-skewed eGFP⁺ iT_reg cells expressed significantly less and RORγt⁺Foxp3⁺ (Fig 7H). While a similar amount of eGFP⁺ cells lost their Foxp3 expression to be Foxp3⁻ in either non-Dll4 or Dll4-educated iT_reg cells (data not shown), Dll4-treated iT_reg cells still retained more Foxp3⁺RORγt⁻ during Th17 skewing (Fig 7I). These data suggest that Dll4-educated iT_reg cells had less plasticity in an inflammatory Th17 environment.

Figure 7. Dll4/Notch activated iT_reg cells retain Foxp3 expression and are resistant to Th17 skewing in vitro.

A. Splenic naïve CD4 T cells from DNMAML or wild type (wt) B6 mice were skewed toward iT_reg for 48 hours. Il17a expression in iT_reg culture were measured.

B. Foxp3 expression in iT_reg culture were measured by realtime PCR after 48 hours of skewing.

C. IL-17A secretion from iT_reg were measured by luminax system after 48 hours of skewing

D. Naïve CD4 T cells from wild type B6 mice were skewed toward iT_reg with or without Dll4 stimulation for 72 hours, then rested in 10 ng/mL of IL-2 for another 72 hours. At day 6, 5 × 10⁵ of viable cells were re-stimulated in Th17 conditions. After 3 days, IL-17A in the supernatant was accessed

E. Percentage of RORγt⁺ in viable CD4 T cells after Th17 skewing

F. Percentage of Foxp3⁺ in viable iT_reg or Dll4-exposed iT_reg after Th17 skewing

G. Expression level of Foxp3 in viable Foxp3⁺CD4 T cells in iT_reg or Dll4-exposed iT_reg culture after Th17 skewing

H. Naïve CD4 T cells from wild type B6 mice were skewed toward iT_reg with or without Dll4 stimulation for 72 hours, then rested in 10 ng/mL of IL-2 for another 72 hours. 5 × 10⁵ of viable Foxp3-eGFP⁺ cells were sorted out and re-stimulated in Th17 conditions at day 6. After 3 days, the percentage of RORγt⁺Foxp3⁺ was determined

I. Percentage of RORγt⁻Foxp3⁺ after Th17 re-stimulation on Foxp3-eGFP⁺ cells

DISCUSSION

The objective of this study was to determine if the Notch ligand Delta-like 4 (Dll4) influenced Foxp3⁺ T_reg cell development, homeostasis, and function during pulmonary infection. Notch signaling has been suggested to be instrumental in Foxp3+ T_reg cell formation and maintenance (18, 41). Notch ligands—Dll1, Jagged1 and Jagged2—have been shown to regulate T_reg cell expansion and elicit suppressive activity (14, 17, 42). The present study suggests that Dll4 mechanistically regulates the stability of iT_reg cells. Previous studies showed that primary RSV infection enhanced Foxp3⁺ T_reg cells in the airway (25, 38). Our data indicate that Dll4 sustained Foxp3 expression especially in central T_reg cells, and altered homeostatic features and functional markers of T_reg cells. In vivo Dll4 inhibition allowed the acquisition of effector cytokine production in T_reg cells, increased their effector phenotype (CD44^hiCD62L^lo), and decreased functional T_reg cell markers (GzmB) during infection. In vitro skewing of iT_reg cells costimulated with Dll4 from naïve T cells had enhanced regulatory function, retained their central T_reg cell phenotype, and were more stable with less susceptibility to become RORγt⁺ Th17 cells. Importantly, stable and functional T_reg cells prevent autoimmunity and enhance immunity against chronic infection (43). Thus, identifying Dll4 as one stabilizing factor in autologous T_reg cells could potentially further optimize clinical efficacy of treatment protocols (44, 45).

Dll4/Notch signaling appears to have differential functions in various disease models. Using systemic blockade, Dll4 inhibition ameliorated graft-versus-host disease, Type 1 diabetes, and experimental autoimmune encephalomyelitis (20, 22, 46–48). In contrast, Dll4 blockade leads to exacerbated allergic airway disease and pulmonary RSV infection responses, as well as to increased BCG-induced granuloma formation (9, 49). These different results could be reconciled with a revised model that Notch signaling fine-tunes T cell responses to different environmental cues (5), and thereby plays a distinct role in different diseases, such as antigen driven/infectious vs. autoimmune or allogeneic responses. Recently, a novel study demonstrated that intrinsic Notch signaling limited peripheral T_reg cell maintenance during steady state and in Graft-versus-host disease (GvHD) (18). In contrast to these latter finding, our results suggests that Dll4 and canonical Notch signaling sustained iT_reg cell differentiation and stability to prevent immunopathogenesis of pulmonary infection. Recent evidence supports that iT_reg cells are important for mucosal homeostasis, suppression of immune responses to environmental and food antigens, and to impede mucosal inflammation. In contrast, natural occurring T_reg (nT_reg) cells prevent autoimmunity and alter the threshold of immune activation (50, 51). Notch signaling and Dll4 restrain nT_reg development in vivo (20), and GvHD was mainly prevented by nT_reg (52, 53) but not iT_reg due to lineage instability (54). To answer the question if Dll4 differentially affects nT_reg versus iT_reg cells, we used Neuropilin-1 (Nrp1) to distinguish nT_reg versus iT_reg cells in vivo as described (55–58). Our preliminary findings showed that Dll4 inhibition decreased Nrp1⁻ T_reg cells during RSV infection but not Nrp1⁺ T_reg cells in the spleen (data not shown). Therefore, we speculate that Dll4/Notch may have a differential role on nT_reg versus iT_reg, thereby limiting nT_reg cells in GvHD but enhance iTreg cells in RSV infection. Additional research needs to be done to elucidate this intriguing hypothesis.

RSV infection can induce IL-17A production (37, 59) and IL-17A facilitates mucus production to promote Th2-driven immunopathology (37). Furthermore, T_reg cells could be reprogrammed to Th17-like in the presence of appropriate inflammatory cytokines (60). iT_reg cell plasticity is critical for conversion to IL-17A-producing Foxp3⁺ and RORγt⁺Foxp3⁺ populations under multiple microenvironments and diseases (62–64). In this current study, Dll4 prevented IL-17A⁺ T_reg cell development in infected mice. Moreover, Dll4-"educated" Foxp3-GFP+ iT_reg cells retained higher Foxp3 expression and resisted skewing toward Th17 in vitro. These results indicate that Dll4 modulates T_reg cell stability and plasticity toward Th17 in inflammatory environments.

Pathogenic RSV infection also exhibits a type 2 biased immune response (65). Our group has shown that Dll4 prevented Th2 cytokine secretion in CD4 T cells during RSV infection. Since both Notch signaling and T_reg cells have been reported to be involve in Th2 programs, it raised a question if Dll4 regulates Th2 cytokine production directly or indirectly through T_reg cell effects. Surprisingly, our data did not demonstrate a significant perturbation in GATA3⁺ Th2 lineage after Dll4 inhibition (data not shown). Instead, our data presented a dysfunctional T_reg cell and Th17-like effector phenotype after Dll4 inhibition. Since IL-17A can enhance Th2 responses in RSV infection and other pulmonary inflammation, these results may suggest that the Th2 response is a result of an indirect effect of reduced T_reg cell function and increased IL-17.

Beside Th2 cells, recent studies have demonstrated that RSV infection enhances type 2 innate lymphoid cells (ILC2) with a concurrent IL-13 increase (34). The present study indicated that Dll4 inhibition increased the number of ILC2 corresponding with elevated IL-5 and IL-13 levels. Notch activation has been shown to support ILC2 progenitor development in vitro (41, 66), but the in vivo role of Dll4 in ILC2 biology during infection has not been described. Our novel findings further highlight the importance of Dll4 in ILC2 homeostasis in vivo, but it is not clear whether the regulation is direct or indirect. Since iT_reg cells inhibit ILC2 and attenuate pulmonary inflammation (67), future studies will be necessary to specify the indirect contribution of iT_reg cells in restraining ILC2 during pulmonary infection.

The homeostatic features of Foxp3⁺ T_reg cells were characterized with CD62L, CD44, and CCR7. CD62L^hiCD44^loCCR7⁺ cT_R cells are quiescent, long-lived, and dependent on IL-2, whereas CD44^hiCD62L^loFoxp3⁺ eT_R appear to be shorter lived (40, 68). These two subpopulations favored distinct compartments, as cT_R cells homing to secondary lymphoid organs (SLO) to be suppressive for autoimmunity (69). Lymph node sensitization is critical to mount a proper immune response since insufficient lymph node formation led to impaired Foxp3 T_reg cell function, promoted IL-17A-mediated pulmonary pathology, and initiated lymphoid cell clusters in the lung (70, 71). In the present study, data showed that Dll4 sustained the cT_R cell population in SLO while preventing Th17 effector phenotype during RSV infection, representing a novel role of Dll4 in cT_R cell maintenance.

In addition to the alteration of the T_reg cell cytokine phenotype, the present study further suggests that Dll4 sustained T_reg cell function by influencing GzmB in vivo and CD25 in vitro. Intriguingly, GzmB in Foxp3+ T_reg cells controlled RSV-induced T cells infiltration in airways (26), and GzmB has been postulated to be a better functional marker of iT_reg cells during viral infection (72). Further investigation on the function of T_reg subsets during RSV infection needs to be characterized in the future. Here, our finding in GzmB may provide a mechanistic explanation for Dll4 inhibition in exacerbating RSV-induced CD4 and CD8 T cells infiltration (9).

Taken together, our study demonstrates that Dll4/Notch promotes iT_reg cell development, homeostasis, stability/plasticity, and function in vitro and in vivo during RSV infection. Dll4 prevented RSV pathogenesis and supported iT_reg cell development in vitro. The effects of Dll4 on T_reg cells are an example of how Notch signaling can promote T cell homeostasis and disease regulation. By extending our understanding of Dll4 in T_reg cell stability and function, our results may provide a road for future translational research leading to a better therapeutic strategy.