CD8+ T-cells counting and purity assessment
CD8+ T-cells were counted after isolation using flow cytometry analysis as shown in (Supplementary Figure 1A). The mean percentage of CD8+ T-cells in PBMCs was 40% and the mean percentage of eluted CD8+ T-cells in unwanted cells during the isolation was 10%. PBMCs and eluted unwanted cells were counted using Trypan blue. We used the following formula to calculate the mean number of CD8+ T-cells in 50 ml of whole blood:
CD8+ T-cells count = (PBMCs count*percentage of CD8+ T-cells) – (eluted unwanted cells count *percentage of CD8+ T-cells).
For example: CD8+ T-cells count= (83.5*106*0.4) – (43*106*0.1) = 28.9*106 cells.
CD8+ T-cells were counted after activation using CFSE analysis and measuring the mean number of divisions. For the co-culture experiment, CFSE was read after 3 days of activation. The mean number of divisions was 0.25 and the fold increase in cell number was 1.20.
T-cells purity was checked by flow cytometry after cell isolation from the donors as shown in (Supplementary Figure 1B). The mean percentage of the isolated CD8+ T-cells was 99.0% and the CD3+ T-cells was also 99.0%, while the mean percentage of CD4+ T-cells was less than 0.5%. Cell purity was also assessed after three days of CD8+ T-cells activation. The purity of CD8+ T-cells was 99.0% and the CD4+ T-cells were also less than 0.5% in the culture (Supplementary Figure 1C). On day 5 of CD8+ T-cells activation, the purity was also 99.0% as presented in (Supplementary Figure 1D).
Effect of PANC1-derived CMs on the proliferation of activated CD8+ T-cells
To determine if the hypoxic conditions induced the immunosuppressive effect of PANC-1 cells on cytotoxic CD8+ T-cell proliferation, CD8+ T-cells were activated for three days and then cultured in 50% and 100% ratios of HCM or NCM. HCM was derived from PANC-1 cells exposed to 10 and 20 cycles of hypoxia for 72 hours, while NCM was derived from PANC-1 cells cultured under normoxic conditions. A control CD8+ T-cell culture media was used as a negative control. CFSE dye was used to measure the mean number of CD8+ T-cells divisions, which was significantly higher in cells treated with 100% hypoxic and normoxic CM in comparison with untreated cells. No significant difference between cells treated with HCM or NCM was observed in all ratios (Figure 1. A&B).
Effect of HCM vs. NCM on Activated CD8+ T-cells Viability
After treating CD8+ T-cells with HCM and NCM, cell viability was assessed using the 7-AAD assay. The percent of cells stained with 7-AAD represents the non-viable cells. All CD8+ T-cells viability measurements were more than 95% when cultured either in RPMI-1640 media, NCM or HCM. The mean percentages of non-viable cells are shown in (Supplementary Figure 2).
Effect of CD8+ T-Cells Coculture and CM on Hypoxic and Normoxic PANC-1 Cell Viability
A co-culture experiment was performed to test the sensitivity of hypoxic PANC-1 cells to cytotoxic CD8+ T-cells and an MTT assay was carried out to measure cell viability. CD8+ T-cells were collected from the donors and activated for three days, then incubated for 72 hours with either hypoxic PANC-1 cells, previously exposed to 10 or 20 hypoxic shots, or with normoxic cells in an effector: target ratio (E:T; 20:1, 10:1 or 5:1). The untreated hypoxic and normoxic PANC-1 cells were used as controls.
The cytotoxic effect was positively correlated with E:T ratios. Significantly higher resistance was seen in hypoxic PANC-1 cells relative to normoxic cells at ratios E:T 20/1 and 10/1 after 10 cycles of hypoxia. While hypoxic cells after 20 cycles showed a statistically significant difference at all ratios as indicated in Figure 2A.
Isolated CD8+ T-cells were activated with CD3/CD28 antibodies and rhIL-2 for 5 days and then the CM was collected. MTT assay was performed to measure cell viability after culturing the hypoxic PANC-1 cells with different ratios of CD8+ T-cells CM (100%, 75%, 50%, and 25%). Untreated normoxic and hypoxic cells were used as controls.
The resulting lytic effect on PANC1 cells was directly correlated with an increasing concentration of CM. Additionally, hypoxic cells displayed a statistically significant higher resistance to activated CD8+ T-cells secretory proteins in cells that underwent 10 cycles of hypoxia and treated with 100%, 75%, and 50% of CM; as well as cells that underwent 20 cycles of hypoxia at all ratios as presented in Figure 2.B.
Gene expression alterations in hypoxic and normoxic PANC-1 cells co-cultured with CD8+ T-cells
qRT-PCR was used to assess gene expression of P53, TNF-α, and IL-6 in hypoxic and normoxic PANC-1 cells after cycle 10 and 20 of hypoxia (Norm cyc10, Hyp cyc10, Norm cyc20, and Hyp cyc20). PANC-1 cells co-cultured with CD8+ T-cells in an E:T ratio of 20:1 and 10:1. CT values were normalized to the GAPDH gene. Untreated normoxic and hypoxic cells were also used as controls.
A statistically significant increase in TNF-α expression was observed in all hypoxic and normoxic PANC-1 cells after 10 cycles of hypoxia, and it was significantly increased after 20 cycles of hypoxia in normoxic cells only at E:T ratio of 10:1. A significant difference was detected between treated hypoxic and normoxic cells at cycle 10 of E:T ratio 20:1 and at cycle 20 of E:T ratio 10:1 (Figure 3. A&D).
For IL-6 expression, it was significantly increased in all hypoxic and normoxic cells in comparison with untreated cells except normoxic cells after 20 cycles of hypoxia at E:T ratio of 20:1. A statistically significant difference was noticed in PANC-1 cells exposed to 10 and 20 cycles of hypoxia relative to normoxic cells in an E:T ratio of 10:1 (Figure 3. B&E).
TP53 expression was significantly increased in all treated normoxic PANC-1 cells in comparison with untreated cells except at cycle 20 of E:T ratio 10:1. TP53 expression was significantly decreased in all treated hypoxic cells relative to hypoxic untreated cells in all treatment conditions. TP53 expression levels were significantly increased in all normoxic treated cells relative to hypoxic treated cells (Figure 3. C-F).
Alterations in gene expression in hypoxic and normoxic PANC-1 cells treated with CD8+ T-cells CM
Quantitative real-time RT-PCR was used to assess the change in gene expression of P53, TNF-α, and IL-6 in hypoxic and normoxic PANC-1 cells treated with either 100% or 75% CD8+ T-cells CM. CT values were normalized to GAPDH gene and normoxic and hypoxic untreated cells were used as controls.
On the other hand, PANC-1 cells treated with CD8+ T-cells secretory proteins showed a significant increase in TNF-α expression which was observed in all treated hypoxic and normoxic PANC-1 cells relative to untreated cells. A statistically significant difference was seen in TNF-α expression in hypoxic PANC-1 cells exposed to 10 cycles of hypoxia relative to normoxic cells in the 100% CM treatment, while no significant difference was observed in all other ratios used (Figure 4. A&D).
IL-6 expression was significantly increased in all hypoxic and normoxic treated cells in comparison with untreated cells in a ratio-dependent manner except at cycle 20 in a 75% CM ratio. Also, a statistically significant difference was noticed in hypoxic PANC-1 cells exposed to 10 cycles of hypoxia relative to normoxic cells in 100%, and 75% CM ratios. However, when PANC-1 cells were exposed to 20 cycles of hypoxia a significant difference in 100% CM ratio was observed relative to the normoxic cells (Figure 4. B&E).
On the other hand, TP53 expression was significantly increased in all treated normoxic cells of cycle 10, with no significant difference in any of the treated normoxic cells at cycle 20. In hypoxic cells, TP53 expression was significantly decreased in untreated hypoxic cells in all conditions. In comparison between treated hypoxic and normoxic cells, a significant decrease in TP53 expression was observed in hypoxic cells treated with 100% CM ratios (Figure 4. C&F).
Cytokine production in CD8+ T-cells cultured in HCM or NCM of PANC-1 cells
For further assessment of the effect of hypoxic conditions on PANC-1 cells immunosuppression, cytokines production from CD8+ T-cells was measured after culturing the activated cells in HCM or NCM for 72 hours in 100% and 50% ratios.
TNF-α, IFN-γ and IL-6 Cytokines were detected using the LEGENDplex Multi-Analyte Flow Assay Kit. For all cytokine measurements, a standard curve was obtained by using seven diluted concentrations ranging between 2.44 pg/ml to 10,000 pg/ml with a sample of zero concentration was used. Three standard curves were obtained for the three detected cytokines TNF-α, IFN-γ and IL-6 with LOD value 0.48, 0.33, and 6.49 respectively. R2 values and standard curves are plotted in Figure 5. Cytokines concentration was calculated by LEGENDplex software supplied by the manufacturer. Then the percentage of concentration change was calculated relative to the control using the following equation.
Percentage of concentration change (%) = The concentration of treated sample/concentration of untreated (control) * 100%.
TNF-α was significantly increased when cells were cultured in 100% CM in comparison with control untreated cells. Except for cells cultured in 100% HCM after PANC-1 cells were exposed to 20 cycles of hypoxia. On the other hand, TNF-α showed a significant production when CD8+ T-cells cultured in HCM of PANC-1 cells exposed to 20 cycles of hypoxia at a 100% ratio in comparison with NCM with no significant difference between cells cultured on HCM or NCM in all other conditions (Figure 5. A&D).
For IL-6 production, significantly lower production was detected when cells were cultured in 50% NCM of cycle 10, 50% HCM of cycle 10, 50% NCM of cycle 20, and 100% HCM of cycle 20. However, no significant difference was observed in production between cells cultured in HCM or NCM in all culture conditions (Figure 5. B&E).
IFN-γ was significantly increased when cytotoxic cells were cultured in all ratios of HCM and NCM. Except for cells cultured in 100% HCM after PANC-1 cells were exposed to 20 cycles of hypoxia. A significant reduction in IFN-γ production was measured when immune cells were cultured with 50% NCM of cycle 10 (Figure 5. C&F).
Also, significantly higher production was detected when CD8+ T-cells cultured in HCM were exposed to 10 hypoxia cycles at a 50% ratio in comparison with NCM. But a significantly lower production was detected when CD8+ T-cells cultured in HCM were exposed to 10 of hypoxia at a 50% ratio in comparison with NCM (Figure 5. C&F).
Cytokines Production in CD8+ T-Cells and PANC-1 Cells Coculture
TNF-α, IL-6, and IFN-γ production were measured using LEGENDplex Multi-Analyte Flow Assay Kit. The supernatant of the co-culture experiment was harvested, and the cytokine concentrations were measured. Then the percentage change relative to the control was measured. The cytokine concentrations in untreated hypoxic and normoxic PANC-1 cells were lower than the detection limit, the control used in this assay was the activated CD8+ T-cells in basic culture media RPMI-1640 (Figure 6).
The concentration of TNF-α was lower than the control in a ratio-dependent manner. A statistically significant reduction in TNF-α concentration was observed in all co-culture conditions in comparison with the control. No significant difference was observed in TNF-α between hypoxic and normoxic PANC-1 cells after 10 and 20 cycles of hypoxia when co-cultured with CD8+ T-cells (Figure 6. A&D).
For IL-6 concentration, a statistically significant increment was detected in all co-culture conditions compared to the control in a ratio-dependent manner. Except for hypoxic cells at (E:T ratio 10:1) and hypoxic cells underwent 20 cycles of hypoxia at all ratios. No significant difference was observed in IL-6 percentage between normoxic and hypoxic PANC-1 cells in all ratios except for hypoxic PANC-1 cells that underwent 20 cycles of hypoxia at an E:T ratio 10:1 (Figure 6. B&E).
There was a statistically significant reduction in IFN-γ in all treated conditions in comparison with the control. No significant difference was observed between hypoxic and normoxic PANC-1 cells after 10 and 20 cycles of hypoxia (Figure 6. C&F).
Cytokines Production in PANC-1 Cells Cultured in CD8+ T-Cells Conditioned Media
CD8+ T-cells were activated for 5 days, and the CM was collected and cultured with hypoxic and normoxic PANC-1 cells for 72 hours. The supernatants were harvested, and the concentrations of the cytokines were measured. Then the concentration changes in percentage relative to the control were evaluated in hypoxic and normoxic PANC-1 cells and showed a lower level than the detection limit. The control in this assay was the CM cultured with PANC-1 cells.
A statistically significant reduction in TNF-α concentration was observed in all co-culture conditions in comparison with the control. No significant difference between hypoxic and normoxic PANC-1 cells after 10 and 20 cycles of hypoxia was observed (Figure 7. A&D).
For IL-6 concentration, a statistically significant increment was detected in all culture conditions relative to the control after 10 cycles of hypoxia. On the other hand, only the normoxic cells at 100% CM showed a significantly higher production of IL-6 after 20 cycles. There was no significant difference in IL-6 percentage between normoxic and hypoxic PANC-1 cells exposed to 10 and 20 cycles of hypoxia at all ratios (Figure 7. B&E).
A statistically significant reduction in IFN-γ was observed in the culture in comparison with the control. However, there was no significant difference between hypoxic and normoxic PANC-1 cells after 10 and 20 cycles of hypoxia at all ratios (Figure 7. C&F).