Mycobacteria-responsive sonic hedgehog signaling mediates programmed death-ligand 1- and prostaglandin E2-induced regulatory T cell expansion

CD4+CD25+FoxP3+ regulatory T cells (Tregs) are exploited by mycobacteria to subvert the protective host immune responses. The Treg expansion in the periphery requires signaling by professional antigen presenting cells and in particularly dendritic cells (DC). However, precise molecular mechanisms by which mycobacteria instruct Treg expansion via DCs are not established. Here we demonstrate that mycobacteria-responsive sonic hedgehog (SHH) signaling in human DCs leads to programmed death ligand-1 (PD-L1) expression and cyclooxygenase (COX)-2-catalyzed prostaglandin E2 (PGE2) that orchestrate mycobacterial infection-induced expansion of Tregs. While SHH-responsive transcription factor GLI1 directly arbitrated COX-2 transcription, specific microRNAs, miR-324-5p and miR-338-5p, which target PD-L1 were downregulated by SHH signaling. Further, counter-regulatory roles of SHH and NOTCH1 signaling during mycobacterial-infection of human DCs was also evident. Together, our results establish that Mycobacterium directs a fine-balance of host signaling pathways and molecular regulators in human DCs to expand Tregs that favour immune evasion of the pathogen.


SHH and NOTCH signaling regulate M. bovis BCG-induced Treg expansion.
To investigate the molecular circuitry regulating mycobacteria-mediated Treg expansion, role for signaling pathways like NOTCH, WNT and SHH were assessed. Twenty-four hours post-infection with M. bovis BCG, DCs were washed and co-cultured with autologous CD4 + T cells for 5 days to analyze the expansion of Treg population. As shown in Fig. 1a,b, M. bovis BCG-infected DCs induced significant expansion of CD4 + CD25 + FoxP3 + Treg cells. However, DCs pretreated with cyclopamine (SHH pathway inhibitor) failed to induce Treg expansion. Further, treatment of DCs with GSI (NOTCH signaling activation inhibitor) enhanced their ability to expand Tregs. Interestingly, no significant difference was observed on perturbation of WNT pathway using IWP-2 (a WNT secretion inhibitor). These results suggest that SHH signaling positively regulates M. bovis BCG-mediated CD4 + CD25 + FoxP3 + Treg expansion and on the contrary, NOTCH signaling was found to exhibit negative regulation. Importantly, no definite roles for SHH and NOTCH signaling was found in mediating Th1 cell cytokine IFN-γ on M. bovis BCG infection (Fig. 1c,d). However, NOTCH signaling was required for infection-induced IL-2 (Fig. 1d). These data thus suggest selective role of SHH and NOTCH signaling pathways in modulating human Treg responses without disturbing IFN-γ responses. In line with these observations, perturbation of SHH and NOTCH signaling did not directly modulate either M. bovis BCG-induced DC maturation (Fig. 2a), or infection-stimulated secretion DC cytokines like TNF-α and IL-6 ( Fig. 2b). Interestingly, infection-induced Th1-polarizing IL-12p70 was found to be SHH and NOTCH signaling dependent. Together, results suggest that while SHH signaling was found to be essential for M. bovis BCG-induced CD4 + CD25 + FoxP3 + Treg expansion, NOTCH signaling suppress Treg expansion.

M. bovis BCG-responsive SHH signaling is PI3K-mTOR-NF-κB pathway dependent.
To further understand the molecular mechanism involved in M. bovis BCG-mediated SHH signaling, the activation status of SHH signaling in the DCs was assessed. Stimulation of DCs with M. bovis BCG or M. tuberculosis induced significant activation of SHH signaling pathway (Fig. 3a,b). Activation of canonical SHH pathways is marked by elevated transcripts of SHH, GLI1 and PTCH1 and increased levels of SHH, GLI1, pGSK-3β (Ser9) and decreased NUMB expression. Mycobacteria-stimulated activation of SHH signaling was found to be dependent on PI3K-mTOR-NF-κ B pathway as pretreatment of DCs with specific inhibitors of PI3K (LY294002), mTOR (Rapamycin) and NF-κ B (BAY 11-7085) led to significant reduction of M. bovis BCG-induced SHH signaling (Fig. 3c,d). Corroborating these results, DCs pretreated with mTOR or NF-κ B specific inhibitors failed to expand CD4 + CD25 + FoxP3 + Treg cells (Fig. 3e). However, PI3K-specific inhibitor had no effect on M. bovis BCG-induced CD4 + CD25 + FoxP3 + Treg cells expansion (Fig. 3e).

SHH signaling-mediated expression of PD-L1 and COX-2-PGE 2 is required for Treg expansion.
Having established that mycobacteria activate the PI3K-mTOR-NF-κ B-SHH signaling cascade in human DCs to induce Treg expansion, we attempted to identify the molecular regulators that mediate the process. Utilizing cues from previous investigations [15][16][17] , we analyzed the role for co-stimulatory molecules PD-L1, PD-L2 and a soluble factor such as COX-2-PGE 2  resulted in increased expression of PD-L1 and PTGS2/COX-2 ( Fig. 4a, left panel). In line with our previous results, PD-L2 was not induced in stimulated DCs. Further, enhanced COX-2 expression was also associated with concomitant increase in PGE 2 secretion by DCs (Fig. 4a, right panel). Following the previous results, expression of PD-L1 and PTGS2/COX-2 transcripts was found to be PI3K-mTOR-NF-κ B-SHH dependent (Fig. 4b). Further, surface expression of PD-L1 (Fig. 4c), expression of COX-2 in the whole cell lysate (Fig. 4d) and secretion of PGE 2 by DCs (Fig. 4e) followed the similar trend. SHH-mediated expression of these mediators was confirmed by reduced expression of PD-L1, COX-2 and PGE 2 on M. bovis BCG infection in the presence of SHH-specific siRNA ( Fig. 4f-i). Finally, inhibition of either PD-L1 or COX-2 by specific blocking antibody (anti-PD-L1) or pharmacological inhibitor (NS-398, COX-2 inhibitor) could partially inhibit the M. bovis BCG-induced Treg expansion (Fig. 5a). Of note, inhibition of both PD-L1 and COX-2 in DCs significantly suppressed the ability of mycobacteria to expand Tregs (Fig. 5b,c). GLI1, a zinc-finger protein, is a dedicated transcription factor for SHH signaling-mediated gene expression 31 . ChIP analysis revealed M. bovis BCG-induced recruitment of GLI1 to the promoter of PTGS2/COX-2 in DCs suggesting a SHH-dependent transcriptional regulation of COX-2 expression (Fig. 6a). However, no apparent change was observed on PD-L1 promoter (Fig. 6a). In this context, we speculated the role for modulation of negative regulators of PD-L1 by infection-responsive SHH signaling. MiRNAs belong to one such class of post-transcriptional regulators. Extensive bioinformatic analysis (TargetScan, miRanda, miRWalk and RNAhybrid) together with available cues on downregulated miRNAs in tuberculosis patient 20,32 or M. tuberculosis or M. bovis BCG samples 33 identified miR-15b, miR-324-5p, miR-338-5p and miR-425-5p as candidate miRNAs that could target PD-L1. In line with this, DCs infected with M. tuberculosis or M. bovis BCG exhibited reduced levels of the identified miRNAs. MiR-155 was utilized as a known positive control (Fig. 6b). However, among the M. bovis BCG-downregulated miRNAs, expression of miR-324-5p and miR-338-5p was found to be SHH-regulated as the downregulation of miR-324-5p and miR-338-5p was significantly rescued in presence of a SHH inhibitor, cyclopamine (Fig. 6c). The target sites located at the residues spanning from 99-106 (for miR-324-5p) and 526-533 (for miR-338-5p) of the 3′UTR of PD-L1 were identified as critical for miRNA-3′UTR interactions (Fig. 6d). To establish that PD-L1 is the bonafide targets of miR-324-5p and miR-338-5p, we utilized the classical 3′UTR luciferase assays. Transfection of a monocytic cell line THP-1 with miR-324-5p or miR-338-5p (a,b) Immature DCs were cultured in GM-CSF and IL-4 alone (Med) or along with pharmacological inhibitors of SHH signaling pathway like Cyclopamine (SMO inhibitor), WNT signaling pathway like IWP-2 (a WNT secretion inhibitor) or NOTCH signaling like γ -secretase inhibitor (GSI) for 1 h followed by infection with M. bovis BCG (MOI 1:10) for 24 h. After extensive wash, DCs were co-cultured with autologous CD4 + T cells. CD4 + CD25 + FoxP3 + Tregs were analyzed by flow cytometry. (a) Representative dot blot of 6 independent experiments is shown. (b) Percentage of CD4 + CD25 + FoxP3 + cells in the DC-CD4 + T cell co-cultures (mean ± SEM, n = 8). (c) In a similar set up as panels (a,b) percentage of IFN-γ + CD4 + cells in the DC-CD4 + T cell co-cultures (mean ± SEM, n = 7). (d) T cell cytokines IL-2, TNF-α and IFN-γ were analyzed in the cell-free culture supernatants of DC:T cell co-culture (mean ± SEM, n = 6-7) by cytokine bead array. Med, Medium. *P < 0.05; **P < 0.005; ***P < 0.001 (one-way ANOVA followed by Turkey's multiple-comparisons test). mimics markedly reduced WT PD-L1 3′UTR luciferase activity. However, no significant reduction was observed when mutant construct for miR-324-5p and miR-338-5p binding on PD-L1 3′UTR was utilized (Fig. 6e). These results thus validate that PD-L1 is a direct target of miR-324-5p and miR-338-5p. In accordance with this observation, we found that DCs expressing miR-324-5p or miR-338-5p miRNAs displayed reduced ability to induce the surface expression as well as total protein levels of PD-L1 on M. bovis BCG infection (Fig. 6f,g). Together, these results highlight a dichotomous role for M. bovis BCG-induced SHH signaling in DCs during expression of COX-2 and PD-L1. NOTCH1 signaling-dependent PI3K-mTOR-NF-κB axis regulates the Th1 responses. After establishing the role for SHH signaling, the other identified signaling pathway (Fig. 1a,b) in DCs that affected the Treg expansion, NOTCH signaling, was analyzed. NOTCH1 signaling activation in DCs-infected with mycobacteria was assessed using transcript analysis of HES1, NOTCH1, JAG1 and JAG2 and generation of NICD. Elevated levels of HES1 and JAG2 transcripts and NICD marked the activation of NOTCH1 signaling on M. tuberculosis or M. bovis BCG infection (Fig. 7a,b). Exploring the role for other NOTCH receptor, it was found that while no increase in the transcripts of NOTCH2-4 was observed on M. bovis BCG stimulation, intracellular domain of NOTCH2 and NOTCH4 were induced on mycobacterial infection (Fig. 7c,d). However, we chose to analyze the function of NOTCH1 signaling in the current study as its activation was found comparatively more robust. While inhibition of PI3K-mTOR-NF-κ B axis did not alter the M. bovis BCG-induced NICD generation (Fig. 7e), pharmacological intervention of NOTCH1 signaling using GSI significantly abrogated the M. bovis BCG-mediated activation of PI3K-mTOR-NF-κ B pathway suggesting that NOTCH1 pathway regulates the PI3K-mTOR-NF-κ B signaling cascade (Fig. 7f). In line with this observation, NOTCH1-responsive PI3K-mTOR-NF-κ B axis was found essential for generation of M. bovis BCG-induced inflammatory cytokines like IL-6, TNF-α and IL-12 despite for the fact that no change in the infection-induced DC maturation was observed when PI3K-mTOR-NF-κ B pathway was inhibited (Fig. 7g).

Discussion
Induction and expansion of an inhibitory CD25 + FoxP3 + Treg population reckons as one of the immune evasion strategies that mycobacteria employ to combat the protective immune responses 5,9,15,16 . In the current investigation, we found novels roles for SHH and NOTCH1 pathways in modulating M. bovis BCG-induced Treg expansion. We attribute the current observation to Treg expansion and not Treg induction as co-culture experiments with naïve CD4 + T did not show significant change in Treg population on mycobacterial infection (data not shown).While no previous reports were available implicating SHH signaling regulating Treg functions, multiple studies in mice and humans have identified NOTCH signaling to modulate Treg population. Overexpression of JAG1 signaling in DCs induced a regulatory phenotype in CD4 + T cells in both humans and mice 34,35 . However, direct role of JAG1 assisting the differentiation of naive CD4 + T to Tregs is not established, underscoring the differential functions of JAG1 in regulating Treg generation in humans and mice 36 . In another investigation, NOTCH1 signaling was found to promote TGF-β -mediated Treg functions in both humans and mice 26 . On the contrary, DLL4-mediated NOTCH signaling inhibits TGF-β -mediated Treg development and JNK-responsive STAT5 activation, a requisite for FoxP3 expression and maintenance 25,37 . In line with latter observation, mycobacteria-responsive activation of NOTCH1-JAG2 axis in DCs was found to suppress CD25 + FoxP3 + Treg population. Supporting our observation, while JAG2 was found to expand Tregs during graft rejection in mice 38 , elevated JAG2 levels in hematopoietic progenitor cells was required for Treg expansion in mice to suppress T cell-mediated diseases 39 . Significant contribution of the WNT pathway in Treg expansion was not observed. However, previous reports suggest both positive and negative regulation of Treg functions by the canonical WNT signaling pathway. While inhibition of β -CATENIN in DCs comprised their ability to induce Tregs 27 , WNT signaling activation inhibited the transcriptional activity of FoxP3 in T cells 28 . bovis BCG alone or after pretreatment of LY294002 (PI3K inhibitor), Rapamycin (mTOR inhibitor) or BAY 11-7085 (NF-κ B inhibitor) were co-cultured with autologous CD4 + T cells. Percentage of CD4 + CD25 + FoxP3 + T cells (mean ± SEM, n = 4) were analyzed by flow cytometry. All RT-PCR data represents the mean ± SEM from at least 3 independent experiments and all blots are representative of 3 independent experiments. Images have been cropped for presentation; full-size blot is shown in Supplementary Fig. S1. Med, Medium. *P < 0.05; **P < 0.005; ***P < 0.001 (one-way ANOVA followed by Turkey's multiple-comparisons test).
Scientific RepoRts | 6:24193 | DOI: 10.1038/srep24193 M. bovis BCG-induced NOTCH1-PI3K-mTOR-NF-κ B signaling in DCs was also found to promote inflammatory cytokines like IL-1β , IL-6, TNF-α and IL-12. Multiple investigations in both humans and mice have subscribed a NOTCH signaling ligand-specific T cell differentiation 40 : Th1 41-43 , Th2 44-46 and Th17 47,48 . However, in the current investigation, mycobacterial infection of human DCs induced NOTCH1 signaling to program the cells towards pro-inflammatory in nature. This observation is in accordance with our previous report that suggested a function of a mycobacterial immunodominant protein, Rv0754, in regulating a NOTCH1-PI3K pathway-dependent pro-inflammatory environment to subvert CTLA-4-and TGF-β -induced suppression of DC maturation 22 . Interestingly, though infection-induced signaling pathways regulated the DC functions in terms of modulating the specific cytokines and T cell phenotype, no apparent role for these identified pathways was found for DC maturation.
To identify the molecular mechanism that mediates SHH and NOTCH signaling-responsive T cell phenotype during mycobacterial infection, we chose to analyze the contributions of PD-L1, PD-L2 and PGE 2 . The co-stimulatory molecules on the APCs, PD-L1 and PD-L2 signal to the PD-1 on the T cells to orchestrate a Treg differentiation and expansion 49 . Formation of the PD-L1 and PD-1 ligand-receptor complex triggers the SHP1/2 activation that suppresses the STAT1 activity, thereby abrogating IFN-γ -mediated responses. STAT1 is (d,e) DCs were treated as explained above. Immunoblotting for COX-2 from total cell lysate (d) and ELISA for measuring PGE 2 in the cell-free supernatant (e). (f-i) Immature human DCs were transiently transfected with NT or SHH siRNA. 48 h post transfection, cells were infected with M. bovis BCG for 24 h to assess the surface expression of PD-L1 by flow cytometry (mean ± SEM, n = 4) (f) or 12 h to estimate COX-2 protein by immunoblotting (g) and PGE 2 in the cell-free supernatant by ELISA (h). Validation of SHH siRNA was performed by immunoblotting for SHH in the siRNA-transfected DCs (i). All RT-PCR and ELISA data represents the mean ± SEM from at least 3 independent experiments and all blots are representative of 3 independent experiments. Images have been cropped for presentation; full-size blot is shown in Supplementary  Fig. S1. Med, Medium; NT, Non-targeting. *P < 0.05; **P < 0.005; ***P < 0.001 (one-way ANOVA followed by Turkey's multiple-comparisons test). otherwise known to inhibit FoxP3 expression 50 . Importantly, reports suggest that induced expression of PD-L1 in human DCs is necessary for mycobacteria-induced Treg expansion 15,16 . However, PD-L1 KO and PD-1 KO mice strangely displayed exacerbated tuberculosis disease with excessive inflammatory responses and increased susceptibility to infection underscoring the crucial role for PD-L1 signaling during mycobacterial infection 14,51 . Interestingly, inhibition of PD-L1 in DCs also promoted mycobacteria-induced IFN-γ production in T cells 52 . Of note, COX-2 catalyzed PGE 2 serves as a cue for Treg expansion and functions 53,54 . PGE 2 -EP signaling is known to aid in FoxP3 expression 55 . Importantly, in humans, mycobacterial infection triggers PGE 2 -dependent expansion of CD25 + FoxP3 + Tregs 17 . In agreement with all these observations, our results revealed that SHH-dependent expression of PD-L1 and COX-2-PGE 2 during mycobacterial infection induces the Treg expansion. It was also noted that there was synergistic effect of PD-L1 and COX-2 in mediating mycobacteria-induced Treg expansion. In fact, upon inhibition of both PD-L1 and COX-2, the Treg frequency reached close to medium control conditions. Interestingly, a recent report showed similar synergistic effects of COX and PD-1 for eradication of tumors and its usefulness as adjuvants for immune-based therapies 56 . Further, while expression of COX-2 was a transcriptional regulation by SHH signaling, post-transcriptional regulation of PD-L1 by SHH signaling-regulated miRNAs was observed.
Based on available information on genome-wide miRNA profiling in tuberculosis patients vs healthy individuals 20,32,57,58 and ex-vivo infection studies 33 , a panel of miRNAs that were downregulated on mycobacterial infection and served as putative miRNAs that target PD-L1 were chosen for the study. MiR-155 was utilized as a positive control as it is not only known to be induced to several manifolds during mycobacterial infection, but also associated with maturation of DCs 59,60 . Among the tested miRNAs, miR-324-5p and miR-338-5p were identified as the bonafide miRNAs that target PD-L1 in a SHH-dependent manner and hence, downregulated during mycobacterial infection. However, the mechanism of SHH-mediated transcriptional regulation of miR-324-5p and miR-338-5p needs to be studied further.
Mycobacteria harbors numerous antigens that are recognized by several pattern recognition receptors; TLR2 being the dominant one. Various studies, including ours, have suggested that mycobacteria and its antigens After extensive washing, these DCs were incubated with anti-PD-L1 blocking antibody or isotype antibody and co-cultured with autologous CD4 + T cells for five days. CD4 + CD25 + FoxP3 + Tregs were analyzed by flow cytometry. Representative dot-plots showing frequency of CD4 + CD25 + FoxP3 + Tregs were presented (b). (c) Percentage of CD4 + CD25 + FoxP3 + cells in the DC-CD4 + T cell co-cultures (mean ± SEM, n = 5). *P < 0.05 (one-way ANOVA followed by Holm-Sidak's multiple comparisons test).
Scientific RepoRts | 6:24193 | DOI: 10.1038/srep24193 induce TLR2 signaling in both macrophages and DCs 16,18,19,[21][22][23]57,61 . Importantly, many of these previous studies have implicated that mycobacteria-induced activation of NOTCH in both macrophages 18,62 and DCs 22 and mycobacteria-induced activation of SHH pathways in macrophages 20,21 were TLR2-dependent. Further, while current and previous results 18,22 suggest that mycobacteria-induced NOTCH1 signals the activation of PI3K-mTOR-NF-κ B axis, current and a previous study 20 also suggests that mycobacteria-induced SHH signaling is PI3K-mTOR-NF-κ B dependent. Keeping these results in mind, TLR2-dependent activation of NOTCH1 signaling during mycobacterial infection could play a dominant role in DCs. However, we have not assessed the role of TLR2 in the current study.
In summary, establishing novel roles for SHH and NOTCH1 signaling pathways, we found mycobacteria-activated SHH signaling induces PD-L1 and COX-2-PGE 2 to mediate the expansion of CD4 + CD25 + FoxP3 + Tregs whereas infection-induced NOTCH1 signaling was found to suppress the Treg expansion (Fig. 8f). Thus, mycobacteria modulate the host signaling pathways and molecular regulators in DCs to determine the functional outcome of the immune responses including Tregs expansion.

Generation of Human DCs. Human Peripheral blood mononuclear cells (PBMC) were isolated from buffy
coats of the healthy blood donors purchased from Centre Necker-Cabanel, Etablissement Français du Sang, Paris, France. Ethical committee permission was obtained for the use of buffy bags of healthy donors (N°12/EFS/079). All samples were analyzed anonymously. Circulating monocytes were isolated from these PBMCs using CD14 magnetic beads. The purity was more than 98%. Monocytes were cultured in RMPI-1640 medium containing and CD80 were examined by flow cytometry (mean ± SEM, n = 7-10). Data is represented as % positive cells or MFI ((g), top panels). Cell-free supernatants from the above-said experiment were assessed for secretion of IL-6, TNF-α , IL-12p70 by cytokine bead array ((g), lower panels) (mean ± SEM, n = 5-7). All RT-PCR data represents the mean ± SEM from at least 3 independent experiments and all blots are representative of 3 independent experiments. Images have been cropped for presentation; full-size blot is shown in Supplementary  Fig. S2. Med, Medium. *P < 0.05; **P < 0.005; ***P < 0.001 (one-way ANOVA followed by Turkey's multiplecomparisons test). Transient transfection studies. Transient transfection of immature DCs with 100 nM siRNA or 150 nM miRNA mimics were carried out utilizing Oligofectamine reagent (Life Technologies). SHH, non-targeting siRNA and siGLO Lamin A/C were obtained from Dharmacon as siGENOME ™ SMARTpool reagents, which contain a pool of four different double-stranded RNA oligonucleotides. MiR-324-5p, miR-338-5p mimics and negative control mimics were purchased from Ambion (Life Technologies). Transfection efficiency was found to be more than 50% in all the experiments as determined by counting the number of siGLO Lamin A/C positive cells in a  Supplementary Fig. S2. Med, Medium. *P < 0.05; **P < 0.005; ***P < 0.001 (one-way ANOVA followed by Turkey's multiple-comparisons test).
Scientific RepoRts | 6:24193 | DOI: 10.1038/srep24193 microscopic field using fluorescent microscope. 48 h post siRNAs transfection or 24 h post miRNAs transfection, the cells were treated or infected as indicated and processed for analysis.
Quantification of miRNA expression. For detection of miRNAs, total RNA was isolated from infected or treated DCs using the TRI reagent. Quantitative real time RT-PCR for miR-155, miR-15b, miR-324-5p, miR-338-5p and miR-425-5p was performed using TaqMan miRNA assays (Ambion) as per manufacturer's instructions. U6 snRNA was used for normalization.
Immunoblotting. Infected or treated DCs were lysed in RIPA buffer constituting 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/ml of each aprotinin, leupeptin, pepstatin, 1 mM Na 3 VO 4 and 1 mM NaF. Equal amount of protein from each cell lysate was resolved on a 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (PVDF) (Millipore) by the semi-dry transfer (Bio-Rad) method. Nonspecific binding was blocked with 5% nonfat dry milk powder in TBST [20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20] for 60 min. The blots were incubated overnight at 4 °C with primary antibody followed by incubation with anti-rabbit-HRP or anti-mouse-HRP secondary antibody in 5% BSA for 2 h. After washing in TBST, the immunoblots were developed with enhanced chemiluminescence detection system (Perkin Elmer) as per manufacturer's instructions. β -ACTIN was used as loading control. For probing another protein in the same region of the PVDF membrane, the blots were stripped in the stripping buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS and 0.7% β -mercaptoethanol] at 60 °C on a shaker, blocked with 5% nonfat dry milk powder and probed with antibodies as mentioned above. DC:CD4 + T cell co-cultures. Autologous CD4 + T cells were obtained using T cell isolation kit II. The treated DCs were co-cultured with CD4 + T cells at 1:10 for 5 days. Cell-free culture supernatants were collected for the analysis of T cell cytokines. Cells were treated with PMA-Ionomycin and Golgistop for 5 h. Following this, the T cells were processed for staining with fluorchrome-conjugated mAbs for flow cytometry. γ-irradiated BCG-stimulated DC:CD4 + T cell co-culture. Immature DCs treated with DMSO or 25 μM NS-398 for 1 h were stimulated with 20 μg/ml γ -irradiated BCG (Strain AF 2122/97 (ATCC ® BAA-935 ™ ) obtained through BEI Resources, NIAID, NIH) for 24 h. Post stimulation, DCs were thoroughly washed with RPMI, counted and blocked with isotype control or anti-PD-L1 (10 μg/ml) for 1 h. DCs were then co-cultured with autologous CD4 + T cells at 1:10 ratio for 5 days. On day 0 and day 3 of the co-culture, 25 μM NS-398 was added for efficient COX-2 inhibition. T cells were processed for staining with fluorchrome-conjugated mAbs for flow cytometry.
Enzyme immunoassay for PGE 2 . Enzyme immunoassays for quantitation of PGE 2 were carried out in 96-well microtiter plates (Nunc) using culture supernatant. ELISA plates were incubated with culture supernatant overnight at 4 °C followed by three washes with 1X PBST. After blocking with 1% BSA for 1 h at 37 °C, wells were incubated with anti-PGE 2 antibod y for 6 h at 37 °C followed by washing with 1X PBST. The plates were further incubated with HRP conjugated anti-rabbit secondary antibody for 2 h at 37 °C. The assay was developed with 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich). The absorbance values were measured at 450 nm by using ELISA reader (Molecular Devices).
Statistical analysis. Levels of significance for comparison between samples were determined by the Student's t test distribution and one-way ANOVA. The data in the graphs are expressed as the mean ± S.E for values from 3 independent experiments and P values < 0.05 were defined as significant. Graphpad Prism 5.0 software (Graphpad software) was used for all the statistical analysis.