Figure 1.
hMSCs reduces proliferation of stimulated lymphocytes in a contact-independent manner.
(A–B) A significant proliferation reduction of PHA stimulated lymphocytes (CD3) (38.58±12.39%) assessed by expression of KI-67 was observed in presence of hMSCs (CD3: hMSC) (24.39±12.39%), the same was observed when absolute number KI-67 expressing cells was evaluated decreasing from (3.0×105±1.3×105) up to (1.7×105±0.9×105) (n = 8). (C) A significant proliferation reduction of DC stimulated lymphocytes (CD3: DC) (12.95±2.23%) assessed by expression of KI-67 was also observed in presence of hMSCs (CD3:DC:hMSC) (6.5±2.23%). (n = 8). (D) There was significant reduction of lymphocytes proliferation assessed by expression of KI-67, when in presence of hMSCs (CD3: hMSC) (16.18±4.99%) and with transwell (CD3:T:hMSC) (12.67±0.83%) if compared to the control (CD3) (37.67±6.63) (n = 6). (E–G) Figures showing the cells after 72 hs of culture (E) Lymphocytes without stimulation (F) Lymphocytes PHA stimulated (G) Lymphocytes PHA stimulated in co-cultures with hMSCs (H–J) Figures showing the (%) of BrdU (H) Lymphocytes without stimulation (I) Lymphocytes PHA stimulated (J) Lymphocytes PHA stimulated in co-culture with hMSCs (K) A significant proliferation reduction of PHA stimulated lymphocytes (CD3) (7.89±1.05%) assessed by BrdU incorporation was observed in presence of hMSCs (CD3:hMSC) (3.37±1.12%), (L) the same was observed when absolute number of BrdU expressing cells were evaluated in absence (0.6×105±0.2×105) or presence of hMSCs (0.2×105±0.1×105) (n = 6). Significant p-values showed in the graphic.
Figure 2.
Indomethacin reduces PHA stimulated lymphocytes proliferation in presence of hMSCs and tryptophan addition to these cultures recovery the lymphocytes proliferation.
(A) Proliferation control (CD3) (21.35±3.62%) is enhanced if compared to stimulated lymphocytes co-cutivated with hMSCs (CD3:M) (16.75±2.81). In addition, we observed that CD3:hMSC have their proliferation even more reduced when added of 50 ng (11.99±2.92%), or 100 ng (8.92±2.32%) of indomethacin. (B) Proliferation control (CD3) (21.43±3.38%) is enhanced if compared with stimulated lymphocytes co-cutivated with hMSCs (CD3:M) (7.55±1.66). However, tryptophan addition to hMSCs condition (CD3:M:TRY) recovered the proliferation (20.33±5.03) (n = 4). Significant p-values showed in the graphic.
Figure 3.
hMSCs presence reduces apoptosis and/or necrosis of stimulated lymphocytes and it is partially-contact dependent.
(A) Early apoptosis in PHA stimulated lymphocytes co-cultivated (CD3/hMSC) or not (CD3) with hMSCs. There is a significant reduction after 24 and 48 hs of cultivation in hMSCs presence (15.61±3.55; 9.17±2.85%) as compared to hMSCs absence (20.28±8.03; 12.71±2.0%) respectively. (B) Late apoptosis/necrosis in PHA lymphocytes stimulated in presence (CD3/hMSC) or not (CD3) of hMSCs. There is a significant reduction after 24 and 48 hs of cultivation in hMSCs presence (19.05±10.83; 28.38±13.47%) as compared to hMSCs absence (24.00±9.91; 41.95±11.73%) respectively. (C) Early apoptosis in DC stimulated lymphocytes co-cultivated (CD3:DC/hMSC) or not (CD3:DC) with hMSCs. There is a significant reduction after 24 and 48 hs of cultivation in hMSCs presence (9.80±2.42; 12.09±1.87%) as compared to hMSCs absence (13.30±1.82; 15.49±21.82%) respectively. (D) Late apoptosis/necrosis in DC stimulated lymphocytes co-cultivated (CD3:DC/hMSC) or not (CD3:DC) with hMSCs. There is a significant reduction after 24 and 48 hs of cultivation in hMSCs presence (7.79±0.93; 11.46±2.64%) as compared to hMSCs absence (17.38±1.83; 19.09±4.64%) respectively. (E) and (F) show early apoptosis and the late apoptosis, respectively of stimulated lymphocytes (CD3/PHA) and co-cultivated with hMSCs (CD3/hMSC) using a 0.2 um transwell (CD3T/hMSC) for 72 hours. (E) There is a significant reduction of early apoptosis in presence of hMSCs (17.08±4.05%) if compared to control (31.84±8.65%) and in presence of transwell (25.85±7.39%). (F) A significant reduction of late apoptosis/necrosis of stimulated lymphocytes (36,41+6.42) is observed if compared to values obtained in presence of hMSCs (19.19±7.51%) and transwell (25.73±9.34%). (n = 8) Significant p-values showed in the graphic.
Figure 4.
hMSCs produces Interleukin-7 which effect in PHA stimulated lymphocytes is likely hMSCs presence in early and late apoptosis.
(A) Early apoptosis in PHA stimulated lymphocytes (CD3) is 34.7±7.05% and in presence of hMSC is reduced (CD3:hMSC) (24.6±5.11%) or stimulated lymphocytes plus interleukin-7 (CD:3IL-7) (29.12±6.4), but no difference is observed if compared with hMSCs co-cultivated cells plus IL-7 block agent (CD3:hMSC:IL-7Bl) (36.27±3.8%). (B) Late apoptosis/necrosis in PHA stimulated lymphocytes (CD3) is 38.55±1.76% and in presence of hMSCs is reduced (CD3:hMSC) (19.85±4.72%), in presence of IL-7 block agent (CD3:hMSC:IL-7Bl) (26.97±2.12%) or when stimulated lymphocytes plus interleukin-7 (CD3:IL-7) (21.52±3.65%). (C) The first plot show the gate on hMSCs by SSC vs FSC (D) the hMSCs gated cells are analyzed for isotype controls (E) the hMSCs gated cells are analyzed for CD105 and IL-7, we observed that 80% of the cells expressed CD105 and co-expressed IL-7. (n = 4) Significant p-values are showed in the graphic.
Figure 5.
Naïve T cells differentiated into Th1 and Th17 in presence of hMSCs secrete approximately 50% less INF-γ and IL-17.
(A) Naïve T cells differentiated for Th1 in presence of hMSCs secrete less IL-17 (3.17±0.86%) than the ones differentiated in their presence (6.25±0.63%) (B) Naïve T cells differentiated for Th1 in presence of hMSCs secrete less INF-y (23.53±2.21%) than the ones differentiated in their presence (40.97±7.41%) (C) Naïve T cells differentiated for Th17 in presence of hMSCs secrete less IL-17 (15.97±0.95%) than the ones differentiated in their presence (26.53±3.97) (n = 3). Significant p-values showed in the graphic.
Figure 6.
Presence of hMSCs do not switch the differentiation of naive T cells from IL-17A/IFN-γ secreting cells into regulatory T cells phenotype.
(A) During Th1 differentiation, there is no significant differences between regulatory T cells numbers in presence of hMSCs (Treg:M) (2.38×103±1.43×103) and in absence (Treg) (2.02×103±1.25×103). (B) During Th17 differentiation, there is no significant differences between regulatory T cells numbers in presence of hMSCs (Treg:M) (0.66×103±0.22×103) and in absence (Treg) (1.38×103±0.78×103).
Figure 7.
Schematic representation of hMSC interaction with lymphocytes.
(A) Soluble molecule, but not prostaglandin E2, that will maybe activate and enhance IDO pathway on hMSCs (B) As IDO activation consequence there is tryptophan depletion from the environment leading to down regulation in lymphocytes proliferation (C) hMSCs IL-7 secretion will lead lymphocytes to survive without interfering with proliferation (D) For unknown reasons hMSCs will lead to a down regulation in INF-γ and IL-17 secretion by T lymphocytes altering the environmental ratio between Th1:IFN-γ, Th17:IL-17 secreting cells and T regulatory cells.