Table 1.
Sequences of the PCR primers.
Fig 1.
Characterization of CD8+ T cells at 28 days postinfection upon Ara-LAM treatment in Leishmania donovani infected BALB/c mice.
(A) CD8+ T from differently treated BALB/c mice 28 days postinfection were subjected to FACS analyis to check the expression of CD25+IL12R+, CD25+CD28+, CD25+IFN-γR+ cells. Data are from one of three representative experiments. (B) In separate set of experiment, CD8+ T cells from differently treated mice group were isolated and cultured in presence of plate-bound anti-CD3ε mAbs (5μg/mL) and CD28 (1μg/mL) and expresion of perforin and granzyme-B was done by conventional RT PCR. Data are from one of three representative experiments.
Fig 2.
Ara-LAM facilitates TLR2 dependent activation and expansion of CD8+ T-cells in Leishmania donovani infected BALB/c mice.
(A) Purified CD8+ T-cells were subjected to FACS analysis for TLR2 expression. Separately, purified CD8+ T-cells from differently treated mice were co-cultured with autologous infected macrophages (10:1) for 48hrs and IFN-γ, perforin, granzyme-B expression were determined by intracellular FACS. (B) CD8+ T-cells from differently treated mice groups were stimulated as described previously and conventional RT PCR was done after RNA extraction. (C) Purified CD8+ T-cells from differently treated mice and autologous L. donovani–infected macrophages were co-cultured for 72 hours. Proliferation was determined by an 18 h [3H] thymidine incorporation assay. Data were presented as count/million (×103). Results were mean value ±SD. from triplicate wells. The asterisk indicated a statistically significant induction (**P<0.001) of T-cell proliferation, compared with that in infected mice.
Fig 3.
Histone H3 modifications at the IFN-γ, Perforin, Granzyme-B promoter of CD8+ T-cells in different groups of BALB/c mice.
(A-B) CD8+ T cells from differently treated mice groups were co-cultured with autologous infected macrophages for 45 min and chromatin immunoprecipitation (ChIP) assays were conducted. Immunoprecipitations were performed using Abs specific to phosphorylated H3 histone (IP: phospho-H3), acetylated H3 histone (IP: acetyl-H3) and conventional RT PCR was performed using primers specific to the IFN-γ, perforin and granzyme-B promoter region. (C) CD8+ T-cells were co-cultured with infected macrophages, lysed and the nuclear protein extracts were analyzed for the activation of T-bet by Western blot. (D) CD8+ T-cells from differently treated mice group were co-cultured with autologous L. donovani–infected macrophages, Immunoprecipitations were conducted using T-bet (IP: T-bet) specific Abs. Conventional RT-PCR was performed for amplifying the putative T-bet binding sites of the IFN-γ, perforin, granzyme-B promoter. Data represented were one of the three indepenedent experiments with similar results performed in the same way.
Fig 4.
Ara-LAM activates TLR2 signalling via activation of NF-κB and p38 MAPK in naive CD8+ T-cells.
(A) Purified CD8+ T-cells (1×106/mL) were stimulated with plate-bound anti-CD3ε mAbs (5μg/mL) and CD28 (1μg/mL) for 24 hrs and transfected with control siRNA or TLR2 siRNA, followed by Ara-LAM (3μg/mL) treatment for 24 hr. The cells were then lysed and subjected to immunoprecipitation with anti-TLR2 antibody, and the blots were probed with anti-MyD88 antibody. (B) Cells were lysed and immunoprecipitated with anti-MyD88 antibody; the blots were probed with anti-IRAK1 antibody. Cytosolic and nuclear protein extracts were analyzed for nuclear translocation of NF-κB. (C-D) In yet separate experiments, CD8+ T cells were treated by Ara-LAM for 5, 15, 30, and 60 min, and lysed. The lysate was subjected to Western blot analysis for the expression of p38MAPK, phospho-p38MAPK and ERK1/2, phospho-ERK1/2. Data represented were one of the three indepenedent experiments with similar results performed in the same way.
Fig 5.
Ara-LAM promptly regulates effector functions in CD8+ T-cells through NF-κB and p38MAPK mediated T-bet signalling.
(A) CD8+ T-cells were isolated by MACS from the spleen BALB/c mice. Purified CD8+ T cells were stimulated as described previously and allowed to transfect with control siRNA or T-bet siRNA, or treated with SB203580 (SB) (5μg/ml), or SN50 (SN) (20μg/ml), subsequently followed by Ara-LAM (3μg/mL) treatment for 24 hr. The cells were then lysed and nuclear protein extracts were prepared, followed by subjected to Western blot with anti-T-bet. (B) The blot shown is representative of triplicate experiments that yield similar type of results. In a separate set of experiments, after the treatment schedule, the cells were collected in Trizol for RNA extraction, and conventional RT PCR analysis was performed to determine the expression of T-bet, IFN-γ, perforin, granzyme-B. Data represented were one of the three indepenedent experiments with similar results performed in the same way.
Fig 6.
Ara-LAM treatment in L. donovani infected BALB/c mice showed reduced parasite burden.
Mice were treated as described in Fig 1 legend. Mice from different groups were sacrificed on day 14 and 28 post-infection. The parasite burden in liver and spleen were expressed in Leishman-Donovan units (LDUs). Results were for 3 independent experiments and represented the mean values ± SD for four animals per group. **P<0.001 for the comparison with infected mice.
Fig 7.
Ara-LAM up-regulates IFN-γ secreting CD8+ T-cells in an organ-dependent manner during L. donovani infection.
28days post-infection, different groups of mice were sacrificed. Splenocytes and hepatocytes were stimulated with soluble leishmanial antigen (SLA, 10 μg/mL) for 48hrs. Before harvesting, cells were incubated with brefeldin A (10 μg/mL) for 4 hrs, CD8+ T cells were MACS sorted (see Materials and Methods), permeabilized (0.1% saponin) and stained with anti-mouse IFN-γ-FITC and anti-mouse IL-10-PE antibodies and were analyzed by flow cytometry. Data are from 1 of 3 experiments conducted in the same way with similar results.