Figure 1.
PHA induced Apoptosis of CD4+ T cells.
A.CD4+ T cells were purified from PBMC from healthy donors. B. Cells were cultured 24 h in presence of 2.5 µg/ml of PHA and observed by microscopy (20×). PHA-treated cells are aggregated in multicellular blast. C. After 24 h, cells were analyzed by FACS. CD4+ T cells were cultured in presence or absence of increasing concentration of PHA and apoptosis was quantified by cytometry using Annexin-V staining. D. Apoptosis of CD4+ T cells cultured in presence or absence of 2.5 µg/ml was quantified using late (7-AAD, Topro-III) apoptotic markers by cytometry. E. Dot plots analysis of unstimulated or PHA-activated CD4+ T cells showing Annexin-V/Topro-III or 7AAD/Topro-III double staining. F. Percentage of Annexin-V, 7-AAD, Topro-III expressed by CD4+ T cells cultured in presence or absence of PHA for 24 h analyzed by cytometry. G. CD69 activation marker and Topro-III double staining of untreated or PHA-activated CD4+ T cells. P values (p) were determined using a two-tailed Student's t test comparing unstimulated versus PHA stimulation (p<0.05 one star, p<0.01 two stars, p<0.001 three stars). Data of panels C, D, E and G are representative of 4 independent experiments.
Figure 2.
TNF family member expression by PHA-activated CD4+ T cells.
A. CD4+ T cells from healthy donors were cultured 24 h in presence (red histograms) or absence (solid grey histograms) of PHA (2.5 µg/ml) and were phenotyped for TRAIL, DR4, DR5, Tweak-R and Fas expressions by FACS. B. Quantification of soluble TRAIL production by ELISA of PHA-activated CD4+ T cells after 1, 2 and 5 days of culture. C. TRAIL mRNA quantification of unstimulated or PHA-activated CD4+ T cells. mRNA levels were analyzed by quantitative RT-PCR. Results are expressed as the ratio of TRAIL to GAPDH gene reporter. D. CD4+ T cells were cultured 24 h in presence of increasing concentration of PHA. Cells were stained with 7-AAD and membrane TRAIL (mTRAIL) antibody and were analyzed by flow cytometry. DR5 mRNA quantification of unstimulated or PHA-activated CD4+ T cells. E. Dot-Plot analysis of 7-AAD and mTRAIL stainings. Cells were cultured in absence or presence of PHA (2.5 µg/ml) and stained with 7-AAD and membrane TRAIL antibody. F. mRNA levels were quantified by quantitative RT-PCR. Results are expressed as the ratio of DR5 to GAPDH gene reporter. G. Quantification of DR5 content in PHA activated CD4+ T cells after 24 h of culture. Cells were lysed and DR5 levels in lysates were quantified by ELISA. H. FACS analysis of DR5 membrane expression by primary CD4+ T cells stimulated with 1 µg/ml and 2.5 µg/ml of PHA (red histograms) compared to untreated CD4+ T cells (grey). I. Dot plot cytometry analysis of resting or PHA-activated CD4+ T cells showing 7-AAD and DR5 expression. J. CD4+ T cells were cultured in absence or presence of PHA for 24 h. Graphics show the percentage of 7-AAD positive cells among DR5 expressing cells under, and proportion of DR5+ among 7-AAD expressing cells. Data of panels A, D, E, H and I are representative of 4 independent experiments. P values (p) were determined using a two-tailed Student's t test (p<0.05 one star, p<0.01 two stars, p<0.001 three stars).
Figure 3.
3D microscopy study of DR5 expression by CD4+ T cells.
A. CD4+ T cells were cultured overnight in presence or absence of PHA (2.5 µg/ml) and stained for DR5 (green), nuclear staining DAPI (blue) and CD4 (red) to study DR5 protein expression and localization. Unstimulated CD4+ T cells exhibited DR5 protein expression in the cytoplasm at close membrane proximity, defined by CD4 staining (upper panels). In contrast, PHA-activated CD4+ T cells expressed intracellular and membrane DR5. DR5/CD4 colocalization appears in yellow. To better visualize DR5 localization, a zoom of the dotted line defined zone from CD4/DR5/DAPI overlay picture was added (extreme right panels). B. DR5 analysis using 3D interactive surface plot. Previous 3D microscopy pictures were analyzed using 3D interactive surface plot. Representation allowed a clear visualization of intracellular DR5 (green) and membrane CD4 (red). Unstimulated CD4+ T cells harbored only intracellular DR5 inside the CD4 (membrane) delimitation. PHA stimulation of CD4+ T cells induced the export of DR5 protein at the surface delineated by CD4 staining (red) but some DR5 also remained intracellular. Yellow spots represent DR5/CD4 colocalization. C. FACS analysis of intracellular staining of DR5 and compilation of all plans from previous microscopic images were used to perform 3D reconstruction. This representation illustrates the difference of DR5 quantity between unstimulated and activated CD4+ T cells. PHA-stimulated cells expressed more DR5 (green) and show more colocalization (yellow) with CD4 than unstimulated cells. Each picture is representative of the vast majority of the observed cells on the slides.
Figure 4.
3D microscopy quantification of DR5 expression by CD4+ T cells.
A. CD4+ T cells were cultured overnight in presence or absence of PHA (2.5 µg/ml) and stained for DR5 (green), nuclear staining DAPI (blue) and CD4 (red) to study DR5 protein expression and localization. 10 cells per experiment were quantified for their DR5 spot number in 3 independent experiments for a total of 30 cells per condition (untreated or PHA-stimulated). White arrows show some examples of the DR5 spots that were counted B. Total number of DR5 spots in 30 untreated and 30 PHA-stimulated cells. Results show the number of DR5 spots per cell C. Percentage of membrane DR5 expression in 30 untreated and 30 PHA-stimulated cells. P values (p) were determined using a two-tailed Student's t test (p<0.05 one star, p<0.01 two stars, p<0.001 three stars).
Figure 5.
Apoptosis inhibition of CD4+ T cells by soluble DR5 and DR5 siRNA.
A. CD4+ T cells were cultured 24 h in presence of increasing concentration of PHA and with or without 5 µg/ml of DR5. Apoptosis was quantified using Annexin-V staining by flow cytometry. B. CD4+ T cells were cultured 24 h in presence of 5 µg/ml of PHA and with or without soluble DR5 (5 µg/ml). Apoptosis was quantified using Annexin-V staining by flow cytometry. C. Efficiency of siRNA transfection in CD4+ T cells. Cells were transfected with unlabelled or Alexa-488 scrambled siRNA overnight. Dot plots show transfection efficiency of CD4+ T cells by Scr-Alexa-488 siRNA (right panel). D. Transfection of CD4+ T cells with DR5 siRNA significantly inhibited DR5 expression by PHA-stimulated CD4+ T cells in contrast to scrambled (Scr) siRNA (left panel). Inhibition of DR5 expression at the surface of PHA-stimulated CD4+ T cells using DR5 siRNA also dramatically inhibited Annexin-V positive cells induced by PHA compared to Scr siRNA (right panel). E. Dot plot analysis of untransfected and transfected CD4+ T cells with scrambled siRNA (Scr siRNA), DR5 siRNA (DR5 siRNA). After overnight transfection, CD4+ T cells were cultured in absence (upper panels) or presence (lower panels) of PHA. Images show dot plots of DR5/Annexin-V staining. F. TRAIL, DR5 and CD69 FACS analysis by CD4+ T cells stimulated overnight in presence of anti-CD3 and anti CD28 antibodies. G. FACS dot plot analysis of CD69-Annexin-V and CD69-Topro-III staining of CD4+ T cells cultured in presence or absence of anti-CD3/CD28 activation overnight. Data of panels C, D, E, F and G are representative of 4 independent experiments. P values (p) were determined using a two-tailed Student's t test (p<0.05 one star, p<0.01 two stars, p<0.001 three stars).