Patient profiles.

<p>Patient profiles.</p

Combination of high IL-12 and low IL-10 can skew induction of Th1 response.

<p>A) RT-PCR. B) Densitometric analysis. C, D, E & F) Flow cytometry. CD4⁺ population from TALs (obtained from untreated EAC/Dox bearing mice) was purified and challenged either with single or combine dose of recombinant IL-12 and IL-10 and cultured for 96 h. Purified CD4⁺ population from TALs derived from untreated EAC/Dox bearing mice, cultured without any treatment was taken as untreated control. Equivalent amount of mRNA (2 µg) from each experimental group was used for semi-quantitative RT-PCR analysis and in all cases GAPDH was used as housekeeping gene control (A). Densitometry analysis of mRNA expression of each gene transcript was expressed as a ratio of cytokine mRNA to GAPDH mRNA (B). Intracellular cytokines specific for Th1 (IFN-γ) or Th2 (IL-4) or suppressive (TGF-β) production profile in the above mentioned experimental groups were also analyzed by flow cytometry and a representative data is shown (C). CD4+ TALs were co-cultured with untreated TAMs or CuNG treated TAMs either unfixed or fixed with paraformaldehyde or CuNG treated fixed TAMs along with high rIL-12 and low rIL-10. In some cases CD4+ TALs and CuNG treated unfixed TAMs were separated by transwell insert (0.45 µ Meter pore) in culture. After 96 h of culture intracellular IFN-γ and TGF-β production pattern were studied by flow cytometry (D). Mean fluorescence intensity for IFN-γ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007048#pone-0007048-g005" target="_blank">Fig. 5E</a>) and TGF-β (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007048#pone-0007048-g005" target="_blank">Fig. 5F</a>) production by these experimental groups were also analyzed from the flow cytometric statistical data and represented graphically. Representative data from three independent experiments is presented.</p

In vitro CuNG treatment caused reprogramming of Treg.

<p>A, B & C) Flow cytometry. CD4⁺CD25⁺ Treg populations were purified from TALs of untreated EAC/Dox bearing mice. (A) Treg cells were labeled with CFSE and then cultured for 96 h with cell free supernatant of TAMs (isolated from untreated EAC/Dox bearing mice) either kept untreated or treated in vitro with CuNG for 48 h. Intracellular IFN-γ and TGF-β production was analyzed with respect to specific isotype control by flow cytometry. Representative data of three independent experiments is shown. (B) Treg cells were cultured for 96 h with supernatant of 48 h culture of untreated or in vitro CuNG treated TAMs and fresh medium (1∶1). Intracellular IFN-γ and TGF-β production versus FoxP3 expression was analyzed with respect to specific isotype control by flow cytometry. Representative data of four independent experiments is shown. (C) Tregs (CD4⁺CD25⁺ cells) isolated from ascitic fluid of untreated EAC/Dox bearing mice were cultured for 96 h in presence of cell-free supernatants from 48 h cultures of untreated or CuNG treated TAMs (culture supernatant: fresh medium being 1∶1). Now, these cells were washed and cultured with CFSE loaded CD4⁺ T cells isolated from inguinal and axillary lymph nodes of normal mice (Treg and CD4⁺ T cells were taken in a proportion of 1∶5) for 96 h. Fluorescence levels of CFSE were measured by flow cytometry. Proliferation of normal CD4⁺ T cells either in the presence or absence of Treg cells were also analyzed by CFSE fluorescence level. Representative data of 3 independent experiments is presented here.</p

Culture supernatant of TAMs, treated with CuNG, caused altered cytokines production by TALs.

<p>A) RT-PCR. B) Densitometric analysis. C) Flow cytometry. CD4+ T cells were purified from TALs obtained from untreated EAC/Dox bearing mice and cultured for 96 h with cell free supernatant of TAMs obtained from untreated EAC/Dox bearing mice that were either kept untreated for 48 h or treated with CuNG (48 h treatement) in vitro or with cell free supernatant of TAMs (cultured for 48 h in absence of CuNG) obtained from in vivo CuNG (15 days after treatment, i.e., when tumors start regressing prominently) treated EAC/Dox bearing mice. Cytokine profile was analyzed by semi-quantitative RT-PCR. Purified CD4⁺ population from TALs (derived from untreated EAC/Dox bearing mice) cultured without any treatment were used as untreated control. After completion of 96 h of incubation equivalent amount of mRNA (2 µg) from TALs of each experimental group was used for RT-PCR analysis and representative data from three independent experiment is presented (A). In all cases GAPDH was used as housekeeping gene control. Densitometry analysis of mRNA expression of each gene transcript was expressed as a ratio of cytokine mRNA to GAPDH mRNA (B). Intracellular cytokines specific for Th1 (IFN-γ) or Th2 (IL-4) or suppressive (TGF-β) production profile in the above mentioned experimental groups were also analyzed by flow cytometry and representative data of three independent experiments is presented here (C).</p

Both in vitro and in vivo CuNG treatment caused alteration of cytokines profile of TAMs.

<p>A) ELISA. B) Flow cytometry. C) Fluorometric analysis. D, E & F) ELISA. In vitro CuNG treatment (2.5 µg/ml) did not change IFN-γ production from CD4⁺ T cells of TALs of untreated EAC/Dox bearing mice (A). TAMs were purified from peritoneal ascitic fluid of both untreated and 15 days of CuNG treated EAC/Dox bearing mice and labeled with anti F4/80 antibodies and with either intracellular IL-10 or IL-12 or TGF-β or with specific isotype control Abs. Immunofluorescence analysis were performed by flow cytometry. Representative data of 3 independent experiments is presented (B). Purified TAMs were either kept untreated or treated with CuNG in vitro and ROS was measured [in terms of peroxide using dichlorofluorescein diacetate (DCF-DA)] at different time points. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007048#s2" target="_blank">Results</a> are presented as mean±SD of 3 independent experiments (C). Purified TAMs from untreated EAC/Dox bearing mice were plated (2×10⁶ cells/500 µl). Cells were either kept untreated or pretreated with tocopherol (50 µM) for 1 h. Then the cells were further cultured for 12 h, 24 h and 48 h in the presence or absence of CuNG (2.5 µg/ml). The culture supernatants were collected and analyzed for cytokines IL-10 (D), IL-12 (E) and TGF-β (F) by ELISA and results are presented as mean±SE of 3 independent experiments, each experiment having every measurement in triplicate.</p

Treatment of CuNG upregulates IL-12 production by adherent population of PBMC from different cancer patients sample.

<p>Flow cytometry. PBMC from different cancer patients were isolated and only adherent population was either treated with CuNG (2.5 µg/ml) or kept untreated for 48 hr. Cells was labeled with Abs specific for surface CD14 and intracellular IL-10 or IL-12 or TGF-β and analyzed by flow cytometry.</p

Presence of small amount of IL-10 prolonged Th1 response by delaying T cells apoptosis.

<p>A) Flow cytometry. B) Fluorometric analysis. C & D) Flow cytometry. Purified CD4⁺ population from TALs of untreated EAC/Dox bearing mice cultured in the presence of either only rIL-12 or the combination of high rIL-12 (2.7 ng/ml) and low rIL-10 (0.35 ng/ml) or the culture supernatant of in vitro CuNG treated (48 h of CuNG treatment) TAMs or IL-10 neutralized culture supernatant of in vitro CuNG treated (48 h of CuNG treatment) TAMs or 48 h of culture supernatant from in vivo CuNG treated TAMs or IL-10 neutralized culture supernatant of in vivo CuNG treated TAMs, for 72 h, 96 h and 120 h. Purified CD4⁺ population without any treatment was taken as untreated control. Levels of apoptosis were estimated by PI/Annexin V-FITC staining and flow cytometry. Representative data of 3 independent experiments is presented here (A). Purified CD4⁺ population pre-treated with H2O2 for 30 mins was taken as positive control for active caspase 3 level (Mean fluorescence intensity value of H2O2 control was 39.25±0.67 that was taken as 100% for active caspase 3 level). In each experimental group active caspase 3 levels was represented by % of H₂O₂ positive control. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007048#s2" target="_blank">Results</a> presented are of 4 independent experiments (B). Expression of Fas by CD4⁺ population of above mentioned experimental groups were also analyzed by flow cytometry. Cells were labeled with Abs specific for CD4 and for surface Fas or with specific isotype Abs and immunofluorescence analysis was performed. Representative result of 4 independent experiments is presented (C). CD4⁺ TALs were cultured with or without different combinations of rIL-12 and rIL-10 in absence or presence of neutralizing antibody against IFN-γ for 72 h. Fas expression was studied by flow cytometry and representative data of 3 independent experiments is presented here (D).</p