C57BL/6 mice, 6–8 weeks were procured from the Institute of Microbial Technology (IMTECH), Chandigarh, India.

All experiments were approved by the Institutional Animal Ethics Committee of IMTECH and performed according to the National Regulatory Guideline issued by Committee for the Purpose of Supervision of Experiments on Animals (No. 55/1999/CPCSEA), Ministry of Environment and Forest, Government of India and Institutional Biosafety Committee.

All the recombinant cytokines, antibodies (Abs) and ELISA reagents were purchased from BD Biosciences (San Diego, CA, USA) and unless mentioned. TLR-4L (LPS EK ultrapure) was procured from Invivogen (San Diego, CA, USA). Abs for Western blotting, such as anti-mouse LC3 and anti-mouse inducible nitric oxide synthase (iNOS) Abs were purchased from Sigma (St. Louis, MO, USA) and AbCam (Cambridge, England), respectively.

Bone marrow-derived DCs were cultured according to the protocols adapted from Lutz et al. (24). Briefly, bone marrow cells (BMC) were flushed aseptically from femurs and tibia of mice. For DC culture, cells were grown in RPMI-1640 (Invitrogen Life Technologies, Eugene, OR, USA) containing FBS (10%) (GIBCO, Grand Island, NY, USA) with penicillin (100 U/ml), streptomycin (100 mg/ml), and l-glutamine (100 mM) and supplemented with recombinant murine granulocyte-macrophage-colony-stimulating factor (rGMCSF) (2 ng/ml) and recombinant murine interleukin (rIL)-4 (4 ng/ml). Cultures were maintained in a humidified atmosphere at 5% CO₂/37°C. The medium was replenished after 3 days.

On 6 days, BMDCs were incubated with biotin conjugated anti-CD40 Ab (CD40A) or isotype control Abs (IgG2a,κ) (4 μg/10⁶ cells) for 30 min on ice followed by cross-linking with equivalent concentration of streptavidin, under similar conditions. The cells were washed and then plated in culture plates (5 × 10⁵ cells/ml) containing either medium alone or medium containing TLR-4 agonist (5 ng/ml). Suitable controls such as cells alone, cells stimulated with anti-CD40 Abs or isotype-matched Abs, with streptavidin or with TLR-4 agonist alone were also included in all experiments. For macrophages, BMC were grown in RPMI-1640 + FBS-10% and replenished with L929 supernatant (SN) (20%), as a source of macrophage-colony-stimulating factor (M-CSF). These cells were stimulated as described above for BMDCs.

Dendritic cells harvested at 24 h were resuspended in FACS buffer (2% FBS, 2 mM sodium azide in PBS). To inhibit non-specific staining, cells were incubated with anti-CD16/32 Ab for 25 min at 4°C. Later, cells were stained with fluorochrome-conjugated Abs specific for mouse CD80, CD86, MHCII, or isotype-matched control Abs at a recommended concentration (0.5 μg/10⁶ cells). The cells were fixed with 1× paraformaldehyde. Regular steps of washing were followed at each step. Data were collected using FACS ARIA and analyzed with the BD DIVA software. The integrated MFI (IMFI) was calculated by multiplying the MFI with percentage of cells.

Cytokines viz IL-6, IL-12, TNF-α, and IFN-γ were detected in culture SNs at indicated time points (24 h) by standard ELISA, according to manufacturer’s instruction.

Mtb strains (H37Rv, H37Ra) were provided by Dr. VM Katoch, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra and GFP-H37Ra by Dr. P Gupta, IMTECH, Chandigarh, India. Mtb was cultured in Middlebrook 7H9 broth containing glycerol (0.2%) and Tween-80 (0.05%), supplemented with albumin, dextrose, and catalase. The viability of bacteria was determined by plating on Middlebrook 7H11 medium supplemented with oleic acid, albumin, dextrose, and catalase and counting the number of colony-forming units (CFUs).

Dendritic cells and macrophages were infected with Mycobacterium smegmatis or Mtb at MOI 1:5 and harvested after 3 or 4 h, respectively. The extracellular bacteria were removed by four to five times washing with 1× PBS. M. smegmatis and Mtb-infected DCs were stimulated with C40.T4 [CD40A (4 μg/10⁶ cells) and TLR-4L (5 ng/ml)] and cultured in the presence of amikacin (2 μg/ml) for 16 or 24 h, respectively, in 48 W plate to kill extracellular bacteria. Where indicated, isoniazid (INH) (25 μg/ml) and rifampicin (RIF) (0.5 μg/ml) were added along with C40.T4.

Bacterial growth in DCs and macrophages was quantified after 24 h of infection. Cell SNs were removed and 0.1% saponin was added to lyse the cells and plating was done with 10-fold serial dilution on 7H11 agar plate. The colonies were enumerated 3 weeks after incubation at 37°C/5% CO₂.

Supernatants of Mtb-infected DCs were harvested after 48 h of infection and nitric oxide (NO) was measured by Griess method (25, 26). Briefly, SNs (50 μl) were incubated with an equal volume of Griess reagent for 5 min at RT. Later, absorbance was measured at 550 nm.

Dendritic cells were stimulated with C40.T4 for 24 h. The cells were harvested, washed, and then pulsed with HRP (50 μg/ml). Antigen was chased for 30–60 min and uptake was arrested by adding chilled PBS (1×) followed by transferring cells on ice. Cells were washed extensively with ice cold PBS-FBS-1%. Subsequently, cells were lysed using Tris-HCl (10 mM) and Triton X-100 (0.05%) for 30 min on ice, with intermittent vortexing. Intracellular HRP was estimated colorimetrically in the cell lysates using OPD-H₂O₂ chromogen-substrate. Concentration of HRP uptake was measured with respect to standard. Cells maintained at 4°C were used as control. HRP activity in test samples was suitably normalized with controls.

For confocal analysis, DCs were stimulated as mentioned above for 24 h. The cells were infected with GFP-Mtb (H37Ra) at (MOI 1:5) for 4 h, washed extensively (4×) with ice cold PBS (1×) and fixed with paraformaldehyde (4%). The cells were washed and placed on poly-l-lysine coated cover slips and imaged using Zeiss Confocal Laser microscope. Z-stacks were taken to exclude the interference of extracellular bacteria. Results were examined by image analysis software.

For bacterial uptake estimation through CFU, DCs were stimulated with C40.T4 for 24 h. Later, DCs were harvested and infection was given at MOI (1:5) for 4 h. It was followed by lysis of cells with 0.1% saponin and plating was done with 10-fold serial dilution on 7H11 agar plate. The colonies were enumerated 3 weeks after incubation at 37°C/5% CO₂.

Dendritic cells were incubated with ON TARGET plus CD40 and beclin siRNA (Dharmacon, Chicago, IL, USA), according to manufacturer’s protocol. Briefly, DCs were incubated with CD40 (20 nM) and beclin (20 nM) siRNA in serum-free media in the presence of transfection reagent 2 for 72 h. Later, level of inhibition in CD40 expression was estimated through flowcytometry and beclin at mRNA level by RT-qPCR. CD40 or beclin knock down DCs were infected with Mtb following the same protocol as mentioned above.

Mtb-infected DCs were stimulated through C40.T4 in the presence or absence of iNOS inhibitor (N-monomethyl-l-arginine) (1 μM) for 24 h. Later, cells were lysed and CFU plating was done to quantify the viable bacteria.

Dendritic cells were stimulated through C40.T4 for 2 h to detect LC3 and 18 h after Mtb infection for iNOS expression by Western blotting. Cells were washed and lysed with radioimmunoprecipitation assay (RIPA) buffer. Cellular extracts (40 μg protein) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Blots were incubated with anti-LC3 and iNOS Abs, followed by HRP-conjugated secondary Abs. Regular steps of incubation and washing were followed at each stage. Membranes were developed using a chemiluminescent reagent and subsequently exposed to film (Pharmacia-Amersham, Freiburg, Germany). Starving DCs were used as positive controls.

Dendritic cells stimulated through C40.T4 were fixed on poly-l-lysine coated cover slips for 15 min. Later, cells were fixed with paraformaldehyde (4×) for 10 min followed by treatment with Tween-20 (0.1%) for 30 s. To block non-specific sites, cells were incubated with BSA (5%) for 3 h, followed by rabbit anti-mouse LC3 Ab for 4 h. Subsequently, cells were incubated with anti-rabbit FITC for 1 h. Regular washings were done followed at each step. The cells were imaged through confocal microscopy. Starving DCs were also kept as positive control. Punctas were counted manually.

Total RNA was isolated by Trizol reagent from DCs incubated with beclin siRNA according to the manufacturer’s instructions (Dharmacon, Chicago, IL, USA). RNA was quantified with the help of NanoDrop. A260/A280 ratio of all samples was in the range of 1.90–2.00. Intactness of RNA samples was determined with the help of formaldehyde denaturing agarose gel-electrophoresis. DNA contamination from RNA samples was removed by amplification grade DNase (Sigma, St Louis, MO, USA). Briefly, RNA samples (1 μg) were incubated with DNase (1U) for 15 min in the reaction buffer. After the incubation, DNase activity was terminated by stop solution (Sigma, St Louis, MO, USA). Furthermore, the samples were heated to 70°C for 10 min to inactivate DNase activity. Results are represented in the form of re-expression (fold) relative to untreated controls. Analysis was done by comparative Ct method, whereas Ct values were normalized against house-keeping control actin. RT-qPCR and data analysis were done by Realplex Mastercycler (Eppendorf, Hamburg, Germany).

Mice were aerosol challenged with Mtb for the deposition of 100 CFUs in lungs. At 24 h after aerosol challenge, three mice were sacrificed for quantification of pathogen delivery to lungs by measuring CFU in lung homogenates. Mice were found to be infected with the 100–120 CFU of H37Rv in their lungs. After 21 days, animals were administered twice with the CD40A (50 μg/300 μl/mouse) in conjunction with TLR-4L (1 μg/300 μl/mouse) (C40.T4) i.p. with an interval of 15 days (18). The control groups were injected with either CD40A (50 μg/300 μl/mouse) or TLR-4L (1 μg/300 μl/mouse) or placebo (PBS). After 50 days of aerosol challenged, animals were sacrificed and the bacterial burden was enumerated in the lungs by CFU counting.

Mice were treated with C40.T4 or controls with CD40A or TLR4L or PBS after 21 days of aerosol challenged with Mtb. Fifty days later, mice were sacrificed. The animals were perfused with cold PBS-heparin and spleen and mediastinal lymph nodes were harvested and single cell suspension was prepared. Briefly, lymphocytes from spleen were prepared by lysing RBCs with ACK lysis buffer (NH₄Cl 0.15 M, KHCO₃ 10 mM, EDTA 88 mM), washed 3× with PBS and resuspended in RPMI-1640-FBS-10%. Viability of the cells was assessed by trypan blue dye-exclusion method. The experiments were performed to detect intracellular cytokines and effector/central memory markers on T cells upon in vitro restimulation with purified protein derivative (PPD).

Splenocytes of Mtb-challenged mice treated with C40.T4 were in vitro stimulated with PPDs to expand Mtb reactive T cells. Later, CD4 T cells and CD8 T cells were purified by negative selection using MACS sorter, as per manufacturer’s instructions (BD Biosciences, San Diego, CA, USA). CD4 T cells or CD8 T cells (5 × 10⁶) were administered into recipient mice after 24 h of sub-lethal irradiation (500R). Within 24 h of cell transfer, recipient mice were aerosol challenged with Mtb (27).

For in vitro assay, purified CD4 T cells and CD8 T cells isolated as mentioned above were co-cultured with Mtb-infected DCs for 48 h. Later, cells were lysed with 0.1% saponin and plating was done with 10-fold serial dilution on 7H11 agar plate. The colonies were enumerated 3 weeks after incubation at 37°C/5% CO₂.

The GraphPad Prism software program was used to perform all statistical analysis. Statistical testing was performed by one way ANOVA for group analysis and Student’s t-test for comparing two groups. Differences were considered significant at a level of p < 0.05.

Although, signaling to DCs was delivered through CD40, CD80, and CD86 molecules but CD40-stimulated DCs exhibited optimal activation (Figure S1A in Supplementary Material). Furthermore, CD40-mediated signaling was significantly amplified in conjunction with TLR-4 (Figure S1B in Supplementary Material). Hence, in the subsequent experiments, CD40 and TLR-4 (C40.T4) were used to activate DCs. We used Abs to CD40 (CD40A) and highly pure LPS (EK ultrapure) as an agonist for TLR-4 (TLR-4L) signaling. The optimum concentration of CD40A (4 μg/10⁶ cells) and TLR-4L (5 ng/ml) was selected on the basis of dose-dependent response in releasing IL-6 and IL-12 (Figure S1B in Supplementary Material). IL-6 and IL-12 are the major cytokines produced by DCs in response to infection by intracellular pathogens. These cytokines have potent effect on T cells activation and other cells of immune system. Therefore, they are considered as an important bridge between innate and adaptive immunity. Upregulation of costimulatory molecules is a characteristic feature of mature DCs (28). Interestingly, DCs triggered through C40.T4 showed significant enhancement in IMFI of CD80 (p < 0.05), CD86 (p < 0.05) and MHCII (p < 0.05) molecules (Figures S1C–E in Supplementary Material). No change was observed with isotype-matched control Abs of CD40. It was of concern for us whether the influence of signaling through C40.T4 is restricted to DCs alone or can stimulate the macrophages as well. Similarly, we observed that triggering C40.T4 substantially (p < 0.01) enhanced the production of IL-6 by macrophages (Figure S2A in Supplementary Material).

The aim of the current study was to activate Mtb-infected DCs through C40.T4 and, consequently, to control the growth of the pathogen. We observed that C40.T4 stimulation improved the potency of infected DCs to produce cytokines, such as IL-6 (p < 0.001), IL-12 (p < 0.0001), and TNF-α (p < 0.001) (Figures 1A–C). No change was noted in IL-10 (Figure 1D). Similarly, infected macrophages stimulated with C40.T4 augmented the secretion of IL-6 (p < 0.05) (Figure S2B in Supplementary Material). Furthermore, Mtb-infected DCs stimulated through C40.T4 expressed considerably higher levels of costimulatory molecules CD80 (p < 0.01) and CD86 (p < 0.01), as compared to control (Figures 1E–G). However, not much change was observed in the expression of MHCII. It may be due to saturation in the expression of MHCII on DCs upon infection with Mtb.

Dendritic cells are highly efficient in antigen uptake (28). Therefore, we next studied whether the C40.T4 stimulation enhanced the capacity of DCs for antigen uptake. Interestingly, C40.T4 signaling showed significant (p < 0.05) augmentation in the antigen (HRP) uptake as observed by colorimetric method (Figure 2A). It is known that DCs endocytose HRP chiefly through fluid phase pinocytosis driven by constitutive membrane ruffling activity and traces of it is taken up by receptor-mediated endocytosis (29, 30). It suggests that DCs activated through C40.T4 stimulation exhibited substantial enhancement in HRP uptake through pinocytosis. Furthermore, we studied the phagocytic ability of DCs by confocal microscopy and CFU plating assay. Our results demonstrate higher uptake of GFP⁺Mtb by DCs upon stimulation through C40.T4 and noteworthy (p < 0.05) increase in the Mtb uptake by confocal microscopy and CFU plating, respectively (Figures 2B,C). These results suggest that the signaling of DCs through C40.T4 improves their endocytic capacity.

Next, we were interested to study the effect of C40.T4 on the bactericidal activity of DCs. Mtb-infected DCs were triggered through C40.T4 for 24 h. We observed enhanced bactericidal activity of C40.T4 stimulated DCs, as evidenced by significant killing of virulent strain (H37Rv) of Mtb (p < 0.05). These results were further substantiated using non-virulent strains of mycobacteria, such as H37Ra (p < 0.05) and M. smegmatis (p < 0.05) (Figures 3A–C). Not much change was observed in CD40 or TLR-4 or isotype-matched controls. Similar results were observed in the case of macrophages (Figure S2C in Supplementary Material). It is important to mention that the difference in uptake of Mtb as shown in (Figure 2C) had no impact on their bactericidal activity. In the case of phagocytic assay, DCs were first stimulated with C40.T4 and then infected with Mtb. By contrast, bactericidal activity of DCs was demonstrated by first infecting them with Mtb and then stimulating through C40.T4.

It was quite important for us to check the specificity of signals delivered through CD40 and TLR-4. CD40 gene in DCs was knock down by siRNA (CD40^KD). Efficacy of the assay, as observed by flowcytometry, was 60% (Figures 4A,B). In addition, the specificity of TLR-4 signaling was established using TLR-4 inhibitor (TLR-4ⁱ) CLI-095. Next, CD40^KD DCs infected with Mtb were stimulated with CD40A and TLR-4L in the presence or absence of TLR-4ⁱ. Interestingly, such CD40^KD DCs cultured in the presence of TLR-4ⁱ followed by their stimulation through C40.T4 showed no decline in the growth of Mtb, when compared with Mtb-infected DCs triggered through C40.T4. We also observed no change in the control DCs with or without mock transfection (Figure 4C). Hence, establishing the specificity of these experiments.

The lengthy drug-regime for treating TB patients not only inflicts side-effects but also other serious problems like emergence of drug-resistant strains of Mtb. Therefore, we hypothesized to explore adjunct therapy with drugs in combination with immunomodulators to minimize the dose and duration of anti-TB drugs and increase their bactericidal activity. Mtb-infected DCs activated through C40.T4 considerably bolstered the bactericidal potency of anti-TB drugs RIF (p < 0.001) and isoniazid (p < 0.05), in comparison to drugs alone (Figures 5A,B). It may be concluded from these results that adjunct therapy of boosting immunity via CD40 and TLR-4 may be an important strategy to enhance the killing efficacy of drugs.

Importantly, our time kinetics study established that adjunct therapy employing immunomodulators and drugs could substantially reduce the duration of drug regime for killing Mtb (Figure 5C). Mtb-infected DCs activated through C40.T4, upon treatment with RIF exhibited reduction in the intracellular burden of Mtb (1.8 × 10⁵ to 1 × 10⁵) at a significantly lesser time (16 h) than treatment with RIF alone (72 h) (Figure 5C). Thus, our observation establishes the importance of adjunct therapy utilizing CD40A and TLR-4L along with drugs. This adjunct therapy could be further explored as immunotherapeutic regimen for reducing the duration of TB treatment.

Involvement of NO is a classical mechanism related with the killing of intracellular pathogens. Hence, we thought to monitor the change in the NO secretion. Importantly, magnitude of NO secretion was significantly (p < 0.01) enhanced in Mtb-infected DCs triggered through C40.T4, as compared to untreated or CD40 or TLR-4 triggered DCs (Figure 6A). Furthermore, our Western blotting experiments also showed considerable enhancement in the expression of iNOS (Figure S3A in Supplementary Material). We confirmed the specificity of NO on the survival of Mtb by culturing C40.T4 activated DCs with iNOS inhibitor (N-monomethyl l-arginine). We observed the recovery of viable bacteria in the presence of iNOS inhibitor (Figure S3B in Supplementary Material). However, only marginal restoration in the survival of Mtb was achieved, indicating the involvement of an additional mechanism. Hence, we thought to monitor autophagy, which is considered to be quite crucial in inhibiting the intracellular survival of Mtb (31, 32). Intriguingly, C40.T4 triggered DCs exhibited significantly higher conversion of LC3I to LC3II, a hallmark phenomenon of autophagy (Figure 6B). This information was corroborated with intracellular LC3 staining for puncta formation (Figures 6C–E). Furthermore, acidic vacuoles were stained with acridine orange dye to show the induction of autophagy (Figure 6F). In addition, we have knocked down the expression of beclin in DCs through siRNA prior to Mtb infection (Figure S3C in Supplementary Material). We observed that beclin knock down DCs if triggered through C40.T4 fails to restrict the Mtb growth (Figure 6G). These results suggest that C40.T4-induced autophagy significantly contributes in constraining the Mtb growth.

Finally, we used this strategy in the experimental model of TB. The animals were aerosol challenged with Mtb and 21 days later inoculated with CD40A and TLR-4L. After 50 days, mice were sacrificed. The cells were isolated from the lungs and spleen and cultured with PPD for 48 h. Later, we monitored the expression of activation markers viz. CD44 and CD69 on CD4 T cells. Interestingly, we observed that animals injected with C40.T4, displayed higher levels of CD44 and CD69 on CD4 T cells (Figures 7A–D). Similar results were noted in the case of CD8 T cells (Figure 7B). Control groups constituting of individually administered CD40A or TLR-4L showed marginal change in the expression of activation markers. It implies the importance of synergism between C40.T4 molecules in reinvigorating immunity during Mtb infection.

CD4 T cells and CD8 T cells are crucial in providing protection against Mtb (33). IFN-γ released by CD4 and CD8 T cells play a pivotal role in restricting the growth of Mtb (34). IFN-γ^–/– mice are fatally susceptible and humans deficient for IFN-γ or IFN-γR are prone to Mtb infection (35). Although, IFN-γ is necessary for immunity against TB, but its presence alone is not sufficient for protection. Recently, IL-17 has been proven to be efficient in reducing the bacterial burden (36, 37). Therefore, we examined the expression of IFN-γ and IL-17. Mice inoculated with anti-CD40 Abs and TLR-4L showed predominance of Th1 and Th17 response on in vitro stimulation with PPD, as evident by significantly higher production of IFN-γ (p < 0.05) and IL-17 (p < 0.05), respectively (Figures 7E–G). Similar results were noted for IFN-γ (p < 0.01) release by CD8 T cells (Figure 7F). Furthermore, we observed significant upregulation in the expression of CD86 (p < 0.05) and CD40 on DCs isolated from the lungs of Mtb-infected animals injected with C40.T4 (Figures 7H,I).

Next, we monitored the CFU burden in the lungs of C40.T4 treated mice. We observed significant (p < 0.01) decline in the mycobacterial burden compared to control mice treated with PBS, TLR-4L and CD40A (Figure 8A). Furthermore, the data were validated by histopathology samples. We observed that C40.T4 treated lungs showed less granulomatous lesions. The lungs were less consolidated with more normal alveolar structures. The number of granulomas were decreased in the lungs of C40.T4 immunized mice, as compared to placebo control (Figure 8B). These experiments showed the potential of C40.T4 immunotherapy in boosting the immunity of the Mtb-infected animals along with reduction in the bacterial load.

Next, we have demonstrated the vis-à-vis role of CD4 T cells and CD8 T cells in limiting Mtb growth. Hence, we purified CD4 T cells and CD8 T cells from Mtb aerosol challenged mice treated with C40.T4 and adoptively transferred in sub-lethally γ-irradiated animals. Mice were aerosol challenged with Mtb. After 15 days, animals were sacrificed and significant reduction (p < 0.05) in the bacterial load in the lungs of mice was noticed. Similar results were observed with CD8 T cells (p < 0.001) (Figure 8C). Likewise, to prove the influence more precisely, CD4 T cells and CD8 T cells were co-cultured with Mtb-infected DCs. We observed significant decline in the CFUs in cultures incubated with CD4 T cells (p < 0.05) and CD8 T cells (p < 0.05) (Figures S4A,B in Supplementary Material). These results suggest that C40.T4 triggering augments the T cell response, which contributes in inhibiting the growth of bacterium.

Mtb-infected mice treated with INH in conjunction with C40.T4 showed significant (p < 0.001) reduction in the mycobacterial load in the lungs (Figure 8D). Most importantly, the dose of 5 mg/kg bwt of INH administered to mice as an adjunct therapy with C40.T4 imparted considerably (p < 0.05) better decline in the CFUs, as compared to 25 mg/kg bwt of INH alone. It is worth to mention here that the C40.T4 + INH was administered only twice over a period of 30 days after Mtb challenge. These results support our hypothesis that C40.T4 significantly reduces the dose of anti-TB drug INH.