This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experimental animal protocols received prior approval from the Institutional Animal Ethical Committee (Bose Institute, Registration Number: 95/99/CPCSEA).

BALB/c mice were purchased from the National Center for Laboratory Animal Sciences, India. For each experiment, 8–10 mice (4–6 weeks old) were used, regardless of sex. L. donovani strain AG-83 (MHOM/IN/1983/AG-83) was maintained in vitro in Medium 199 (Sigma) containing 10% fetal calf serum (FCS; Gibco BRL). Experiments were performed with stationary phase promastigotes. The CpG-ODN 1826 (5′-TCCATGACGTTCCTGACGTT-3′) and the control-ODN (non-CpG-ODN, 5’-TCCATGAGCTTCCTGAGCTT-3’) was obtained from InvivoGen. CXCL10-depleting antibody was obtained from R&D Systems.

Bone marrow-derived DCs from BALB/c mice were generated as described previously (28). Non-adherent cells were collected, and 1 × 10⁶ cells were placed in plates containing 1 ml of complete medium with GM-CSF (150 U/ml; R&D Systems) and IL-4 (75 U/ml; R&D Systems) as originally described earlier (5). Half of the medium was replaced on day 3, 5, and 7 and fresh medium containing GM-CSF and IL-4 was added. On day 8 of culture, most cells had acquired typical dendritic morphology. These cells were used as the source of DCs in subsequent experiments.

For DC-based vaccination, DCs were pulsed with both SLA and CpG-ODNs (29) as originally described earlier (5). In case of dual stimulation, CpG-ODN (10 μg/ml) or control-ODN (10 μg/ml) was added to the media for last 6 h after 12 h of SLA stimulation. DCs were then washed with PBS thrice and injected i.v. (10⁶ cells in 100 μl of PBS/mouse) into mice through the tail vein. One week later, mice were infected intravenously with 1 × 10⁷ stationary phase L. donovani promastigotes. Mice were sacrificed on day 56 post-infections. Spleen and liver parasitic loads were determined from Giemsa-stained impression smears, calculated as the number of parasites per 1000 nucleated cells × organ weight (in milligrams) and expressed in Leishman Donovan Units (LDU) (30). After 28 days of infection, spleens from infected BALB/c mice were removed aseptically, and a single-cell suspension was prepared. Briefly, spleen homogenate was subjected to centrifugation on a Histopaque-1077 (Sigma) gradient and splenocytes were collected, washed, and resuspended in RPMI-1640 complete medium supplemented with 10% FCS.

For in vivo depletion of CXCL10, anti-mouse CXCL10 mAb (R&D Systems) were injected intraperitoneal (i.p.) on day 0 (250 mg), day 2 (100 mg), and day 4 (100 mg) after SLA–CpG–DCs vaccination as originally described earlier (5). These mice were subsequently infected with 1 × 10⁷ stationary phase L. donovani promastigotes after 7 days of initial vaccination. Two hundred fifty milligrams of anti-CXCL10 mAb was again injected i.p on days 10, 15, and 24 of initial vaccination. Depletion efficiencies were assessed at regular intervals.

CD4⁺ T cells were purified from splenocytes from differently treated mice by positive selection using magnetic beads as originally described earlier (9). CD4⁺ T cells were purified by anti-mouse CD4 (L3T4)-magnetic particles (BD Biosciences). To further separate CD4⁺ T cells into CD25⁺ and CD25⁻ populations, total CD4⁺ T cells were isolated by negative selection using magnetic beads followed by positive selection using anti-CD25 magnetic beads on a magnetic separator column into CD4⁺CD25⁺ and CD4⁺CD25⁻ populations as per manufacturer’s suggested protocol (MagCellect Treg isolation kit, R&D Systems). The cells were stained with anti-CD25 mAb, and the purity of cell preparations was determined by using FACS analysis (FACSCalibur; BD Labware). The purities of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were routinely >90 and 99%, respectively.

Additional analyses of T cell phenotypes were also performed using FACS where splenocytes were stained using 1 μg Ab/1 × 10⁶ cells and either run immediately or fixed (3% paraformaldehyde in PBS). In some cases, the splenocytes were first stained with CD4 FITC followed by permeabilization using FACS permeabilizing solution (BD Pharmingen) and FOXP3-PE staining. The panel of Abs used for T cell phenotyping included the following: CD4, CD25, GITR, Foxp3, and CTLA4 (BD Biosciences). The dot plots were derived from the gated events based on the region encircling lymphocytes, which was set using a forward versus side scatter display, and all fluorescent parameters were gated on this population and analyzed on a Flow cytometer (FACSCalibur), using the Cell Quest program on at least 10,000 events.

Splenic responder CD4⁺CD25⁻ T cells (5 × 10⁵) and T-depleted, mitomycin C-treated syngeneic APCs (5 × 10⁵) were cultured in the absence or presence of increasing numbers of splenic CD4⁺CD25⁺ Treg cells for 4 days in 96-well, round-bottom plates as originally described earlier (9). Soluble leishmanial antigen (SLA) (10 μg/ml) was added to the culture for stimulation. One microcurie of [3H] thymidine was added 18 h before harvesting, and incorporated radioactivity was determined on a beta emission reader. Supernatants were collected from the co-culture of responder CD4⁺CD25⁻ and CD4⁺CD25⁺ Treg cells (1:1) at 24 h (for IL-2) or 72 h (for other cytokines). In some cases, splenocytes (2 × 10⁶ cells/ml per well), CD4⁺CD25⁻ T cells (1 × 10⁶ cells/ml per well), or CD4⁺CD25⁺ Treg cells (1 × 10⁶ cells/ml per well) from different sets of treatment were stimulated with SLA (10 μg/ml) for 24 h (IL-2) or 72 h (for other cytokines). The levels of cytokines in supernatants were determined by specific ELISAs using paired mAbs for IL-2, IL-10, IFN-γ, and TGF-β along with the appropriate mouse cytokine controls (BD Biosciences and R&D Systems).

For intracellular cytokine analysis, flow cytometry was performed for the determination of IFN-γ, IL-12, TGF-β, and IL-10 produced by CD4+ T cells in differently treated mice at the single-cell level as originally described earlier (9). In another experiment, CD4⁺CD25⁺ Treg cells were analyzed for Foxp3-positive Treg cells producing TGF-β. Splenocytes or MACS-purified CD4⁺CD25⁺ Treg cells from different groups of experimental mice were stimulated for 20–24 h with SLA (10 μg/ml). Brefeldin A (10 μg/ml) was added to the cultures 2 h before harvesting. The cells were washed in PBS containing 0.1% NaN₃/1% FCS at 4°C, and some sets were stained with FITC-conjugated anti-CD4. Cells were then permeabilized by treatment with FACS permeabilizing solution (BD Pharmingen) and stained with PE-conjugated IFN-γ, FITC-conjugated IL-12, FITC or PE-conjugated anti-mouse TGF-β mAb, PE-conjugated anti-mouse IL-10 mAb, PE-conjugated Foxp3 anti-hamster mAb, or isotype-matched control mAb and analyzed on a Flow cytometer (FACSCalibur) using the Cell Quest program on at least 10,000 events.

Total RNA was extracted from 2 × 10⁶ CD4⁺CD25⁺ T cells or CD4⁺CD25⁻ T cells with use of TRI Reagent (Sigma), according to the manufacturer’s protocol as originally described previously (9). Isolated RNA (1 μg) was then reverse transcribed using Revert Aid M-MuLV Reverse Transcriptase (Fermentas). The resulting complementary DNA was used for real-time (RT) PCR to detect different Treg cell-specific markers with use of the ABI 7500 RT-PCR system with the DNA-binding SYBR green dye (Applied Biosystems). Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) was used as a reference. The forward- and reverse-specific primer sequences used were as follows: CTLA4 Forward, 5′-GGACGCAGATTTATGTCATTGATC-3′, CTLA4 Reverse, 5′-CCAAGCTAACTGCGAC AAGGA-3′; Foxp3 Forward, 5′-CGTACACCCAGGAAAGACAG-3′, Foxp3 Reverse, 5′-ATCCAGGAGATGATCTGCTTG-3′; GITR Forward, 5′-GACGGTCACTGCAGACTTTG-3′; GITR Reverse, 5′-GCCATGACCAGGAAGATGAC-3′. The reaction conditions involved an initial activation step (5 min at 95°C) and a cycling step (denaturation for 30 s at 94°C, annealing for 30 s at 58.5°C, and extension for 1 min at 72°C for 40 cycles), followed by melting curve analysis. Detection of dequenched probe, calculation of threshold cycles, and further analysis of these data were performed using the Sequence Detector software (Applied Biosystems). Relative changes in CTLA4, Foxp3, and GITR messenger RNA (mRNA) expression were compared with unstimulated control, normalized to GAPDH, and quantified by the 2^−ddCt method.

Cell lysates were prepared as described earlier (31). Equal amounts of protein (30 μg) in each lane were subjected to SDS-10% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked overnight with 3% BSA in Tris–saline buffer (pH7.5), and immunoblotting was carried out for detecting phosphorylated or dephosphorylated forms of SMAD4 as described previously (32).

A minimum of five mice were used per group for in vivo experiments. The data, represented as mean ± standard deviation (SD), is from one experiment, which was performed at least three times. Student’s t-test was employed to assess the significance of the differences between the mean values of control and experimental groups. A P value of 0.05 was considered significant and <0.001 was considered highly significant.

Our results demonstrated that SLA-pulsed DCs in the presence of TLR9 agonist, CpG-ODN (SLA–CpG–DCs), acquired the ability to induce complete protection against L. donovani. SLA–CpG–DCs-vaccinated BALB/c mice on day 56 of infection, showed marked decrease in parasitic burden; 96 ± 2.6 and 97 ± 1.9% reduction in hepatic and splenic parasite burden, respectively, compared with PBS-treated infected controls (Figure 1A). Mice vaccinated with SLA and control-ODN-stimulated dendritic cells (SLA-Cont. ODN–DCs) were unable to give any protection against the leishmanial challenge (Figure 1A). Moreover, the protection conferred by SLA–CpG–DC vaccination against L. donovani infection is significantly dependent on a Th1 polarized anti-parasitic immune response (Figures 1B,C). Splenocytes from differently vaccinated mice at 28 days post-treatment were re-stimulated with SLA to evaluate the percentages of T cells secreting various pro-inflammatory or anti-inflammatory cytokines. There was about sixfold increase in IFN-γ secreting CD4⁺ T cells along with nearly fivefold increase in IL-12 secreting CD4⁺ T cells in splenocytes from SLA–CpG–DC-vaccinated parasitized mice compared with only infected mice (Figures 1B,C). Moreover, there was nearly fourfold decrease in TGF-β secreting CD4⁺ T cells along with nearly threefold decrease in IL-10-secreting CD4⁺ T cells in splenocytes from SLA–CpG–DC-vaccinated parasitized mice compared with only infected mice (Figures 1D,E).

To check whether SLA–CpG–DCs vaccination of L. donovani-infected mice can modulate the Treg cells, which have been previously reported to play a crucial role in disease progression (33), FACS analysis was performed to evaluate the frequency and phenotype of splenic Treg cell population. We observed a significant decrease from 23% to 11% in CD4⁺CD25⁺Foxp3⁺ Treg cells in splenocytes isolated from SLA–CpG–DCs-vaccinated infected mice compared to only infected mice (Figures 2A,B). Other Treg cell-specific markers like CTLA4 and GITR showed a significant decrease in splenocytes isolated from SLA–CpG–DC-vaccinated infected mice compared to infected sets (Figures 2C,D). We observed 2.5-fold decrease in GITR and threefold decrease in CTLA4 mRNA levels in SLA–CpG–DC-vaccinated infected sets in comparison with only infected sets.

To study whether the Treg cells isolated from the Leishmania-infected BALB/c mice can suppress the function of effector T cells, we performed co-culture experiments using CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells purified from spleen of differently treated mice. We observed that CD4⁺CD25⁺ Treg cells isolated from untreated infected mice can efficiently suppress the proliferation of responder CD4⁺CD25⁻ T cells in a dose-dependent manner. Whereas, CD4⁺CD25⁺ Treg cells, isolated from SLA–CpG–DC-vaccinated infected mice, could not suppress the proliferation of responder CD4⁺CD25⁻ T cells (Figure 3A).

Moreover, CD4⁺CD25⁺ Treg cells from SLA–CpG–DC-vaccinated mice failed to suppress the release of IL-2 when co-cultured with CD4⁺CD25⁻ responder T cells (1:1) compared to CD4⁺CD25⁺ Treg cells isolated from infected mice (Figure 3B). Additionally, CD4⁺CD25⁺ Treg cells from infected mice significantly abrogated the IFN-γ secretion by responder CD4⁺CD25⁻ T cells, while CD4⁺CD25⁺ Treg cells from SLA–CpG–DC-vaccinated parasitized mice, could not suppress the IFN-γ production from responder CD4⁺CD25⁻ T cells demonstrating parasite-specific T cell responses (Figure 3C).

SLA–CpG–DC vaccination of L. donovani-infected mice results in a significant decrease in Th2 cytokine secreting CD4⁺ T cells, i.e., IL-10 and TGF-β (Figures 4B,C). As expected, FoxP3 levels were higher in CD4⁺CD25⁺ Treg cells isolated from infected group of mice. On the contrary, CD4⁺CD25⁻ T cells showed lower expression of Foxp3 mRNA during infection (Figure 4A). Now to delineate which of these two cells are the major producer of IL-10 and TGF-β, we evaluated the anti-inflammatory cytokines secreted by CD4⁺CD25⁺ Treg cells and CD4⁺CD25⁻ T cells from different groups of mice. CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells from control mice showed lower levels of IL-10 and TGF-β (Figures 4B,C).

In infected mice, CD4⁺CD25⁻ T cells are the major producers of IL-10. On the other hand, CD4⁺CD25⁺ Treg cells are the major source of TGF-β release. Interestingly, significant decrease in TGF-β release from CD4⁺CD25⁺ Treg cells was observed in CD4⁺CD25⁺ Treg cells isolated from SLA–CpG–DC-vaccinated infected sets compared with infected mice, while CD4⁺CD25⁻ T cells showed a significant reduction in IL-10 release in vaccinated group of mice compared with infected sets (Figures 4B,C).

In another experiment, CD4⁺CD25⁺ Treg cells, isolated from different groups of animals, were analyzed for TGF-β secreting Foxp3⁺ Treg cells. We observed higher levels (69.24%) of TGF-β-secreting Foxp3⁺ Treg cells in infected mice, which were significantly abridged (24.56%) in SLA–CpG–DC-vaccinated infected mice (Figure 4D), indicating decrease of TGF-β secretion from CD4⁺CD25⁺Foxp3⁺ Treg cells following SLA–CpG–DC vaccination during VL.

We have previously demonstrated that the induction of anti-leishmanial protective immunity by SLA–CpG–DCs is entirely dependent on IP-10 (5). Moreover, our lab has also shown that exogenously administered IP-10 can decrease the TGF-β secretion in Leishmania-infected mice (9). So to examine possible involvement of IP-10 in the secretion of TGF-β from CD4⁺CD25⁺ Treg cells following SLA–CpG–DCs vaccination, we vaccinated the mice, infected it with Leishmania and monitored the course of infection in the presence or absence of IP-10 (Figure 5A).

CD4⁺CD25⁺ Treg cells from IP-10 depleted SLA–CpG–DC-vaccinated mice produced significantly higher amount of TGF-β together with fourfold increase in their suppressive property compared with IP-10 non-depleted vaccinated parasitized mice, which showed significant reduction in TGF-β secretion along with clear decrease in the suppressive property of the CD4⁺CD25⁺ Treg cells (Figures 5B,C). These findings clearly demonstrate the involvement of IP-10 in the regulation of CD4⁺CD25⁺ Treg cell functioning in SLA–CpG–DC-vaccinated parasitized mice.

Efficient TGF-β signaling requires proper activation of SMAD4 (27). To establish whether SMAD4 is involved in the SLA–CpG–DC-mediated regulation of TGF-β signaling in CD4⁺CD25⁺ Treg cells, we studied the phosphorylation of SMAD4 in CD4⁺CD25⁺ Treg cells during SLA–CpG–DC vaccination. Increased levels of phospho-SMAD4 was observed in CD4⁺CD25⁺ Treg cells isolated from infected mice, however CD4⁺CD25⁺ Treg cells isolated from SLA–CpG–DC-vaccinated mice showed lower levels of phospho-SMAD4 indicating SLA–CpG–DC-mediated regulation of TGF-β signaling through SMAD4 modulation (Figure 5D). Though, increased levels of phospho-SMAD4 in CD4⁺CD25⁺ Treg cells in IP-10-depleted vaccinated parasitized mice suggest the involvement of IP-10 in the SLA–CpG–DCs-mediated suppression of TGF-β.