Journal: Frontiers in Immunology

Article Title: Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

doi: 10.3389/fimmu.2021.650731

Figure Lengend Snippet: CADA strongly suppresses the upregulation of the co-stimulatory molecule 4-1BB in activated PBMCs. (A, B) PBMCs were pre-incubated with CADA (10 μM) or DMSO for 3 days, after which they were activated by CD3/CD28 beads or PHA. Cell surface CD28 expression was measured on gated CD4 + T cells by flow cytometry on d3 post activation. Cell surface expression of OX40 and 4‑1BB was measured on total PBMCs by flow cytometry on d2 post activation. Bar diagrams are showing the MFI for the indicated receptor, with DMSO in white and CADA in red. Paired t-tests were performed to compare CADA to DMSO with *p<0.05. Values represent mean ± SD of four different donors (n=4). (C) PBMCs were pre-incubated with CADA (10 μM) or DMSO for 3 days, after which they were activated by CD3/CD28 beads or PHA. Cell surface 4‑1BB expression was measured on gated CD4 + and CD8 + T cells by flow cytometry on the indicated time points post activation. Multiple t-tests were performed to compare CADA to DMSO for each condition with *p<0.05 and with Holm-Sidak method as correction for multiple comparison. Curves are showing mean values ± SD of six different donors (n=6). (D) PBMCs were pre-incubated with CADA (10 μM) or DMSO for 2 hours, and subsequently activated by anti-CD3 antibodies only. Cell surface CD4 and 4‑1BB expression was measured by flow cytometry at 24h post activation. Representative dot plots for CD4 and 4‑1 BB double staining are shown for unstimulated and stimulated cells. Do note that this short CADA-treatment has neglectible effect on the CD4 expression in unstimulated cells (see lower right quadrants), but blocks the expression of 4‑1BB in activated cells, both in CD4 negative (upper left quadrant) and CD4 positive cells (upper right quadrant). See also Supplementary Figure 6 .

Article Snippet: Flow cytometry antibodies were purchased from (i) eBioscience (Thermo Fisher Scientific): APC-labeled anti-mouse CD4 (clone GK1.5) and APC-labeled anti-human phospho-STAT5 (Tyr694) (clone SRBCZX); (ii) BioLegend: PE-labeled anti-human CD4 (clone SK3), PE-labeled anti-human CD4 (clone OKT4), APC-labeled anti-human CD4 (clone SK3) and PE-labeled anti-human CD69 (clone FN50); (iii) BD Biosciences: FITC-labeled anti-CD3 (clone UCHT-1), BV510-labeled anti-human CD8 (clone SK1), PE-labeled anti-human CD25 (clone 2A3), FITC-labeled anti-human CD25 (clone 2A3), PE-labeled anti-human CD28 (clone CD28.2), PE-labeled anti-human TCRα/β (clone IP26), PE-labeled anti-human OX40 (clone ACT35), PE-labeled anti-human 4-1BB (clone 4B4-1) and BD Horizon Fixable Viability Stain 780.

Techniques: Incubation, Expressing, Flow Cytometry, Activation Assay, Double Staining

Journal: Frontiers in Immunology

Article Title: Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

doi: 10.3389/fimmu.2021.650731

Figure Lengend Snippet: CADA suppresses lymphocyte proliferation and inhibits upregulation of CD4 and CD8 after activation by CD3/CD28 beads or PHA. (A) PBMCs were pre-incubated with CADA (10 μM) or DMSO during 3 days, after which they were activated by CD3/CD28 beads (left panels) or PHA (right panels). At 4h, 1d, 2d, 3d or 4d post activation, [ 3 H]-thymidine was added and proliferation response was measured by detecting counts per minute (cpm) 22h later. Individual cpm values are shown for stimulated PBMCs with DMSO-treated cells as open symbols and CADA-treated cells as solid red dots. A paired t-test was performed to compare CADA to DMSO with *p<0.05. Horizontal lines indicate the mean values of 4 to 6 donors (n=6, except for time point 4d where n=4). (B, C) Cell surface CD4 (B) and CD8 (C) receptor expression was measured by flow cytometry just before activation (0h) and 4h, 1d, 2d, 3d or 4d post activation with CD3/CD28 (left) or PHA (right). Mean fluorescence intensity (MFI) of human CD4 or CD8 receptor expression is shown for different donors of PBMCs (indicated separately) with DMSO-treated samples as a black dotted line and CADA-treated samples as a red line. Curves are showing single MFI values of the indicated receptor (acquired from at least 5,000 cells per donor) for four different donors (n=4). (D) PBMCs were incubated with CADA (10 μM) or DMSO for 3 days, and subsequently analyzed for cell surface receptors CD3, CD4, CD8 and TCRα/β. Bar diagrams are showing the MFI of the indicated receptor on gated CD4 + or CD8 + subpopulations, with DMSO in white and CADA in red. Welch’s corrected t-tests were performed to compare CADA to DMSO with *p<0.05. Values represent mean ± SD of two different donors (n=2). (E) PBMCs were stimulated with PHA for 3 days and were subsequently exposed to CADA (10 µM) or DMSO for another 2 days. Dot plot represents cell surface expression of CD3 and CD4, with DMSO as black dots and with CADA as red overlay. Do note that the CADA-treated cells have similar CD3 expression levels as DMSO, but reduced CD4 levels (see difference in black and red population in upper right quadrant). See also Supplementary Figure 3 .

Article Snippet: Flow cytometry antibodies were purchased from (i) eBioscience (Thermo Fisher Scientific): APC-labeled anti-mouse CD4 (clone GK1.5) and APC-labeled anti-human phospho-STAT5 (Tyr694) (clone SRBCZX); (ii) BioLegend: PE-labeled anti-human CD4 (clone SK3), PE-labeled anti-human CD4 (clone OKT4), APC-labeled anti-human CD4 (clone SK3) and PE-labeled anti-human CD69 (clone FN50); (iii) BD Biosciences: FITC-labeled anti-CD3 (clone UCHT-1), BV510-labeled anti-human CD8 (clone SK1), PE-labeled anti-human CD25 (clone 2A3), FITC-labeled anti-human CD25 (clone 2A3), PE-labeled anti-human CD28 (clone CD28.2), PE-labeled anti-human TCRα/β (clone IP26), PE-labeled anti-human OX40 (clone ACT35), PE-labeled anti-human 4-1BB (clone 4B4-1) and BD Horizon Fixable Viability Stain 780.

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

doi: 10.3389/fimmu.2021.650731

Figure Lengend Snippet: CADA suppresses lymphocyte proliferation and inhibits upregulation of CD4 and CD8 after activation by CD3/CD28 beads or PHA. (A) PBMCs were pre-incubated with CADA (10 μM) or DMSO during 3 days, after which they were activated by CD3/CD28 beads (left panels) or PHA (right panels). At 4h, 1d, 2d, 3d or 4d post activation, [ 3 H]-thymidine was added and proliferation response was measured by detecting counts per minute (cpm) 22h later. Individual cpm values are shown for stimulated PBMCs with DMSO-treated cells as open symbols and CADA-treated cells as solid red dots. A paired t-test was performed to compare CADA to DMSO with *p<0.05. Horizontal lines indicate the mean values of 4 to 6 donors (n=6, except for time point 4d where n=4). (B, C) Cell surface CD4 (B) and CD8 (C) receptor expression was measured by flow cytometry just before activation (0h) and 4h, 1d, 2d, 3d or 4d post activation with CD3/CD28 (left) or PHA (right). Mean fluorescence intensity (MFI) of human CD4 or CD8 receptor expression is shown for different donors of PBMCs (indicated separately) with DMSO-treated samples as a black dotted line and CADA-treated samples as a red line. Curves are showing single MFI values of the indicated receptor (acquired from at least 5,000 cells per donor) for four different donors (n=4). (D) PBMCs were incubated with CADA (10 μM) or DMSO for 3 days, and subsequently analyzed for cell surface receptors CD3, CD4, CD8 and TCRα/β. Bar diagrams are showing the MFI of the indicated receptor on gated CD4 + or CD8 + subpopulations, with DMSO in white and CADA in red. Welch’s corrected t-tests were performed to compare CADA to DMSO with *p<0.05. Values represent mean ± SD of two different donors (n=2). (E) PBMCs were stimulated with PHA for 3 days and were subsequently exposed to CADA (10 µM) or DMSO for another 2 days. Dot plot represents cell surface expression of CD3 and CD4, with DMSO as black dots and with CADA as red overlay. Do note that the CADA-treated cells have similar CD3 expression levels as DMSO, but reduced CD4 levels (see difference in black and red population in upper right quadrant). See also Supplementary Figure 3 .

Article Snippet: Flow cytometry antibodies were purchased from (i) eBioscience (Thermo Fisher Scientific): APC-labeled anti-mouse CD4 (clone GK1.5) and APC-labeled anti-human phospho-STAT5 (Tyr694) (clone SRBCZX); (ii) BioLegend: PE-labeled anti-human CD4 (clone SK3), PE-labeled anti-human CD4 (clone OKT4), APC-labeled anti-human CD4 (clone SK3) and PE-labeled anti-human CD69 (clone FN50); (iii) BD Biosciences: FITC-labeled anti-CD3 (clone UCHT-1), BV510-labeled anti-human CD8 (clone SK1), PE-labeled anti-human CD25 (clone 2A3), FITC-labeled anti-human CD25 (clone 2A3), PE-labeled anti-human CD28 (clone CD28.2), PE-labeled anti-human TCRα/β (clone IP26), PE-labeled anti-human OX40 (clone ACT35), PE-labeled anti-human 4-1BB (clone 4B4-1) and BD Horizon Fixable Viability Stain 780.

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

doi: 10.3389/fimmu.2021.650731

Figure Lengend Snippet: CADA dose-dependently inhibits CD8 + T cell proliferation and cytotoxic T cell function. (A) PBMCs (red), purified CD4 + T cells (blue) or purified CD8 + T cells (green) were co-cultured with mitomycin C inactivated RPMI1788 cells in the presence of different doses of CADA. At day 5, [ 3 H]-thymidine was added and proliferation response was measured by detecting counts per minute (cpm) 18h later. Lymphocyte proliferation is given as percentage of the corresponding DMSO control (mean ± SD of six different donors; n=6). (B) Purified CD8 + T cells were pre-incubated with CADA (10 μM) or DMSO during 3 days, after which they were activated by PHA or CD3/CD28 beads. At 24h post activation, [ 3 H]-thymidine was added and proliferation response was measured by detecting cpm 20h later. Graphs show intra-donor treatment effect on the proliferation response (each donor is indicated separately). A paired t-test was performed to compare CADA to DMSO with *p<0.05; n=4. (C) PBMCs were cultured in medium alone (black) or were co-cultured with inactivated RPMI1788 cells in the absence (white) or presence (red) of increasing doses of CADA during 6 days. Next, PBMCs were exposed to 51 Cr-loaded RPMI1788 cells for 4h, after which supernatant was collected and 51 Cr release was quantified. To measure spontaneous and maximum release of 51 Cr, medium or saponin was added to the 51 Cr-loaded RPMI1788 cells, respectively. The mean percentage of specific lysis was calculated by using the following formula: % specific lysis = (experimental release – spontaneous release)/(maximum release – spontaneous release) x 100. A t-test was performed to compare CADA to DMSO with *p<0.05. Bars represent mean ± SD of 4 technical replicates of one donor.

Article Snippet: Flow cytometry antibodies were purchased from (i) eBioscience (Thermo Fisher Scientific): APC-labeled anti-mouse CD4 (clone GK1.5) and APC-labeled anti-human phospho-STAT5 (Tyr694) (clone SRBCZX); (ii) BioLegend: PE-labeled anti-human CD4 (clone SK3), PE-labeled anti-human CD4 (clone OKT4), APC-labeled anti-human CD4 (clone SK3) and PE-labeled anti-human CD69 (clone FN50); (iii) BD Biosciences: FITC-labeled anti-CD3 (clone UCHT-1), BV510-labeled anti-human CD8 (clone SK1), PE-labeled anti-human CD25 (clone 2A3), FITC-labeled anti-human CD25 (clone 2A3), PE-labeled anti-human CD28 (clone CD28.2), PE-labeled anti-human TCRα/β (clone IP26), PE-labeled anti-human OX40 (clone ACT35), PE-labeled anti-human 4-1BB (clone 4B4-1) and BD Horizon Fixable Viability Stain 780.

Techniques: Cell Function Assay, Purification, Cell Culture, Incubation, Activation Assay, Lysis

Journal: Frontiers in Immunology

Article Title: Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

doi: 10.3389/fimmu.2021.650731

Figure Lengend Snippet: CADA affects cytokine release by activated PBMCs. PBMCs were stimulated with mitomycin C inactivated RPMI1788 cells (MLR), CD3/CD28 beads or PHA and exposed to CADA (10 µM) as described in the legend to Figures 1 and 2 . Supernatants were collected on day 5 (MLR) or day 3 (beads and PHA) post stimulation and cytokine levels were determined by Bio-Plex assay. Bar diagrams are showing the cytokine concentration (mean ± SD), with DMSO in white and CADA in red, and with individual values shown as open (DMSO) or solid (CADA) symbols. Note that cytokine levels in the MLR samples are plotted on a logarithmic scale. Welch’s corrected t-tests were performed to compare CADA to DMSO with *p<0.05. Samples were taken from four (beads and PHA; n=4) or five (MLR; n=5) different donors. See also Supplementary Figure 4 .

Article Snippet: Flow cytometry antibodies were purchased from (i) eBioscience (Thermo Fisher Scientific): APC-labeled anti-mouse CD4 (clone GK1.5) and APC-labeled anti-human phospho-STAT5 (Tyr694) (clone SRBCZX); (ii) BioLegend: PE-labeled anti-human CD4 (clone SK3), PE-labeled anti-human CD4 (clone OKT4), APC-labeled anti-human CD4 (clone SK3) and PE-labeled anti-human CD69 (clone FN50); (iii) BD Biosciences: FITC-labeled anti-CD3 (clone UCHT-1), BV510-labeled anti-human CD8 (clone SK1), PE-labeled anti-human CD25 (clone 2A3), FITC-labeled anti-human CD25 (clone 2A3), PE-labeled anti-human CD28 (clone CD28.2), PE-labeled anti-human TCRα/β (clone IP26), PE-labeled anti-human OX40 (clone ACT35), PE-labeled anti-human 4-1BB (clone 4B4-1) and BD Horizon Fixable Viability Stain 780.

Techniques: Plex Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

doi: 10.3389/fimmu.2021.650731

Figure Lengend Snippet: CADA treatment results in decreased CD25 upregulation and reduced intracellular pSTAT5 and CTPS1 levels in activated PBMCs. (A) PBMCs were pre-incubated with CADA (10 μM) or DMSO for 3 days, after which they were activated with CD3/CD28 beads. Cellular surface CD25 expression was measured on gated CD4 + T cells by flow cytometry at 4h, 1d, 2d, 3d or 4d post activation. Representative dot plots show CD25/CD4 double staining at 1d post activation (for comparison, a dot plot at 4h is presented in Supplementary Figure 5C ). Graph shows individual MFI values of CD25 expression (acquired from at least 5,000 cells per donor) from different donors with DMSO-treated cells as open symbols and CADA-treated cells as solid red dots. A paired t-test was performed to compare CADA to DMSO with *p<0.05. Horizontal lines indicate the mean values of 4 different donors (n=4). (B–D) PBMCs were pre-incubated with CADA or DMSO for 3 days, after which they were left unstimulated or were activated with CD3/CD28 beads or PHA. (B, C) At day 2, half of the samples were boosted with IL-2. Cell surface CD25 receptor (B) and intracellular pSTAT5 (C) expression were simultaneously measured by flow cytometry. Bar diagrams are showing the MFI values for CD25 and pSTAT5 (mean ± SD). Multiple t-tests were performed to compare CADA (red bars) to DMSO (white bars) for each condition with *p<0.05 and with Holm-Sidak method as correction for multiple comparison. Data were collected from four different donors (n=4). (D) At day 2 post activation, cells were lysed and CTPS1 expression was detected by western blotting. Clathrin was used as protein loading control. Graph shows quantification of protein bands, normalized to the clathrin internal control. Normalized CTPS1 level of unstimulated DMSO control was set as 1. (E) PBMCs were pre-incubated with CADA or DMSO for 2 hours (2h) or for 3 days (3d), and subsequently left unstimulated (no IL-2) or boosted with exogenous IL-2 (+ IL-2). After 15 minutes, intracellular pSTAT5 expression was measured by flow cytometry. Bar diagrams are showing the MFI for pSTAT5 (acquired from at least 5,000 cells per donor), with DMSO in white and CADA in orange or red. Welch’s corrected t-tests were performed to compare CADA to DMSO with *p<0.05. Values represent mean ± SD of two different donors (n=2). (F) PBMCs were pre-incubated with CADA or DMSO for 2 hours (2h) or for 3 days (3d), and subsequently activated with CD3/CD28 beads, PHA or anti-CD3 antibodies. At 24h post activation, [ 3 H]-thymidine was added and proliferation response was measured by detecting cpm 18h later. Lymphocyte proliferation was calculated as percentage of DMSO control. Bar diagrams are showing the relative cell proliferation, with DMSO in white and CADA in orange or red. Welch’s corrected t-tests were performed to compare CADA to DMSO with *p<0.05. Values represent mean ± SD of three different donors (n=3). See also Supplementary Figure 5 .

Article Snippet: Flow cytometry antibodies were purchased from (i) eBioscience (Thermo Fisher Scientific): APC-labeled anti-mouse CD4 (clone GK1.5) and APC-labeled anti-human phospho-STAT5 (Tyr694) (clone SRBCZX); (ii) BioLegend: PE-labeled anti-human CD4 (clone SK3), PE-labeled anti-human CD4 (clone OKT4), APC-labeled anti-human CD4 (clone SK3) and PE-labeled anti-human CD69 (clone FN50); (iii) BD Biosciences: FITC-labeled anti-CD3 (clone UCHT-1), BV510-labeled anti-human CD8 (clone SK1), PE-labeled anti-human CD25 (clone 2A3), FITC-labeled anti-human CD25 (clone 2A3), PE-labeled anti-human CD28 (clone CD28.2), PE-labeled anti-human TCRα/β (clone IP26), PE-labeled anti-human OX40 (clone ACT35), PE-labeled anti-human 4-1BB (clone 4B4-1) and BD Horizon Fixable Viability Stain 780.

Techniques: Incubation, Expressing, Flow Cytometry, Activation Assay, Double Staining, Western Blot

Journal: Frontiers in Immunology

Article Title: Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

doi: 10.3389/fimmu.2021.650731

Figure Lengend Snippet: CADA strongly suppresses the upregulation of the co-stimulatory molecule 4-1BB in activated PBMCs. (A, B) PBMCs were pre-incubated with CADA (10 μM) or DMSO for 3 days, after which they were activated by CD3/CD28 beads or PHA. Cell surface CD28 expression was measured on gated CD4 + T cells by flow cytometry on d3 post activation. Cell surface expression of OX40 and 4‑1BB was measured on total PBMCs by flow cytometry on d2 post activation. Bar diagrams are showing the MFI for the indicated receptor, with DMSO in white and CADA in red. Paired t-tests were performed to compare CADA to DMSO with *p<0.05. Values represent mean ± SD of four different donors (n=4). (C) PBMCs were pre-incubated with CADA (10 μM) or DMSO for 3 days, after which they were activated by CD3/CD28 beads or PHA. Cell surface 4‑1BB expression was measured on gated CD4 + and CD8 + T cells by flow cytometry on the indicated time points post activation. Multiple t-tests were performed to compare CADA to DMSO for each condition with *p<0.05 and with Holm-Sidak method as correction for multiple comparison. Curves are showing mean values ± SD of six different donors (n=6). (D) PBMCs were pre-incubated with CADA (10 μM) or DMSO for 2 hours, and subsequently activated by anti-CD3 antibodies only. Cell surface CD4 and 4‑1BB expression was measured by flow cytometry at 24h post activation. Representative dot plots for CD4 and 4‑1 BB double staining are shown for unstimulated and stimulated cells. Do note that this short CADA-treatment has neglectible effect on the CD4 expression in unstimulated cells (see lower right quadrants), but blocks the expression of 4‑1BB in activated cells, both in CD4 negative (upper left quadrant) and CD4 positive cells (upper right quadrant). See also Supplementary Figure 6 .

Article Snippet: Flow cytometry antibodies were purchased from (i) eBioscience (Thermo Fisher Scientific): APC-labeled anti-mouse CD4 (clone GK1.5) and APC-labeled anti-human phospho-STAT5 (Tyr694) (clone SRBCZX); (ii) BioLegend: PE-labeled anti-human CD4 (clone SK3), PE-labeled anti-human CD4 (clone OKT4), APC-labeled anti-human CD4 (clone SK3) and PE-labeled anti-human CD69 (clone FN50); (iii) BD Biosciences: FITC-labeled anti-CD3 (clone UCHT-1), BV510-labeled anti-human CD8 (clone SK1), PE-labeled anti-human CD25 (clone 2A3), FITC-labeled anti-human CD25 (clone 2A3), PE-labeled anti-human CD28 (clone CD28.2), PE-labeled anti-human TCRα/β (clone IP26), PE-labeled anti-human OX40 (clone ACT35), PE-labeled anti-human 4-1BB (clone 4B4-1) and BD Horizon Fixable Viability Stain 780.

Techniques: Incubation, Expressing, Flow Cytometry, Activation Assay, Double Staining