Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A, B) TRPM7 current densities and (B) TRPM7 I/V relationship of Jurkat T cells during whole-cell patch clamp experiment with Mg 2+ -free intracellular solution. TRPM7 WT (WT, gray) and KO2 Jurkat clone (KO2, orange), n (WT) = 9; n (KO2) = 10. (C) Cell counts and (D) viability of natively proliferating TRPM7 WT and KO2 Jurkat clone in RPMI medium with 10% FBS, with and without supplementation of 6 mM MgCl 2 , n = 3, measured in duplicates. (E) Cellular Mg 2+ contents quantified by ICP-MS. TRPM7 WT and KO2 Jurkat clone, cultured in regular (WT-)media without or with 6 mM MgCl 2 supplementation for 18 h ahead of sampling, n = 4. (F) Fura-2-based imaging of cytosolic Ca 2+ concentration of Jurkat T cells. Passive store release was induced with 5 μM thapsigargin at indicated time point (arrow). TRPM7 WT (WT, gray) and KO2 (KO2, orange) Jurkat clone, n (WT) = 111; n (KO2) = 59. (G) Quantification of the area under the curve (AUC) of respective curves shown in (F). (H) Representative immuno-fluorescent images of NFATc1 localization in TRPM7 WT and KO2 clone before (basal) and after 30 min stimulation (stim.) with 5 μM thapsigargin, scale bar = 2 μm. NFATc1 in red, DAPI in blue. (I, J) Representative intensity profiles of subcellular NFATc1 localization (NFATc1 in red, DAPI in blue) of Jurkat TRPM7 WT (WT, gray) and KO2 (KO2, orange) in basal state (I) and upon 30 min passive store depletion induced with 5 μM thapsigargin (J). (K) Quantification of nuclear NFATc1 levels (corresponding to AF647 signal intensity) upon stimulation of TRPM7 WT (WT, gray) and KO (KO2, orange) clone, n (WT) = 261; n (KO2) = 149. (L) Histograms and (M) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT (WT, gray) and KO2 (KO2, orange) cells after overnight α-CD3 stimulation, n = 4–6. (B, F, G, H, J) Statistics: One-way ANOVA (B), Two-way ANOVA (F, G), or t test (H, J). * P < 0.05; **** P < 0.0001, n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-human CD4-VioBlue (REA623; Miltenyi), α-human CD45RA-APC-Vio770 (REA562; Miltenyi), α-human CD69-APC (REA824; Miltenyi), α-human CD25-VioBright515 (REA570; Miltenyi).

Techniques: Patch Clamp, Cell Culture, Sampling, Imaging, Concentration Assay, Expressing

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A, B) TRPM7 current densities and (B) TRPM7 I/V relationship of Jurkat T cells during whole-cell patch clamp experiment with Mg 2+ -free intracellular solution. Control (Ctrl, gray) and cells treated with 1 μM Apamin (Apamin, blue), n (Ctrl) = 9; n (Apamin) = 6. (C) Cell counts and (D) viability of natively proliferating Jurkat T cells in RPMI medium with 10% FBS, treated with 1 μM Apamin (Apamin, blue) or control (Ctrl, gray), n = 4. (E) Cell counts and (F) viability of natively proliferating Jurkat TRPM7 WT and KO cells in RPMI medium with 10% FBS, treated with 30 μM NS8593, 30 μM NS8593 with additional 6 mM MgCl 2 or untreated controls, n = 3, measured in duplicates. (G) Fura-2 based imaging of cytosolic Ca 2+ concentrations of Jurkat TRPM7 WT and KO cells treated with 30 μM NS8593 or left untreated. Passive store depletion was induced with 5 μM thapsigargin at indicated time point (arrow). (H) Quantification of area under the curve (AUC) of traces shown in (G), n = 24–70. (I) Representative FACS plots and gating strategy for CD69 visualization, shown for Jurkat WT cells. (J) Histogram and (K) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT cells treated with 1 μM Apamin (Apamin, blue) compared with untreated controls (Ctrl, light gray), n = 3. (L) Histogram and (M) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT or KO cells treated with 30 μM NS8593 or left untreated, n = 4–6. (H, K, M) Statistics: One-way ANOVA (H, M) and t test (K). ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-human CD4-VioBlue (REA623; Miltenyi), α-human CD45RA-APC-Vio770 (REA562; Miltenyi), α-human CD69-APC (REA824; Miltenyi), α-human CD25-VioBright515 (REA570; Miltenyi).

Techniques: Patch Clamp, Control, Imaging, Expressing

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Fura-2 based imaging of cytosolic Ca 2+ concentration of Jurkat T cells. Passive store release was induced with 5 μM thapsigargin at the indicated time point (arrow) of WT (black) and TRPM7 KO (red) Jurkat T cells, n (WT) = 111; n (KO) = 113. (B) Quantification of the area under the curve (AUC) of respective curves shown in (A). (C) Representative immune-fluorescence images of the NFATc1 localization in TRPM7 WT and KO cells before (basal) and after 30 min stimulation (stim.) with 5 μM thapsigargin, scale bar = 2 μm. NFATc1 in red, DAPI in blue. (D, E) Representative intensity profiles of subcellular NFATc1 localization (NFATc1 in red, DAPI in blue) of Jurkat TRPM7 WT (black) and KO (red) cells in basal state (D) and upon 30 min passive store depletion induced with 5 μM thapsigargin (E). (F) Quantification of nuclear NFATc1 levels (corresponding to AF647 signal intensity) upon stimulation of TRPM7 WT (black) and KO (red) cells, n (WT) = 261; n (KO) = 279. (G) Relative IL-2 mRNA expression levels of Jurkat TRPM7 WT (black) and KO (red) cells, n = 4. (H) Histograms and (I) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT (black) and KO (red) cells after overnight stimulation with α-CD3, n = 4–6. (J) Quantification of Ca 2+ signals of TRPM7 WT Jurkat T cells, treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black). Passive store release was induced with 5 μM thapsigargin at indicated time point (arrow), n (Ctrl) = 95; n (NS) = 94. (K) Quantification of the area under the curve (AUC) of respective Ca 2+ signals shown in (G). (L) Representative immune-fluorescence images of NFATc1 localization of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black) before and after 30 min stimulation with 5 μM thapsigargin, scale bar = 2 μm. (M, N) Representative intensity profiles of subcellular NFATc1 localization (NFATc1 in red, DAPI in blue) of Jurkat TRPM7 WT (black) and KO (red) cells in basal state (M) and upon 30 min passive store depletion induced with 5 μM thapsigargin (N). (O) Quantification of nuclear NFATc1 levels upon stimulation of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n (Ctrl) = 196; n (NS) = 195. (P) Relative IL- 2 mRNA expression levels of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n = 7. (Q) Histograms and (R) quantification of up-regulated CD69 expression of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black) after α-CD3 stimulation, n = 6–7. (B, D, E, F, H, J, K, M) Statistics: t test (B, D, F, H, J, M) and Mann-Whitney U test (E, K). ** P < 0.005; *** P < 0.0005; **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-human CD4-VioBlue (REA623; Miltenyi), α-human CD45RA-APC-Vio770 (REA562; Miltenyi), α-human CD69-APC (REA824; Miltenyi), α-human CD25-VioBright515 (REA570; Miltenyi).

Techniques: Imaging, Concentration Assay, Fluorescence, Expressing, Control, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A, B) Representative FACS plots and gating strategy to confirm identity of isolated naïve CD4 T cells and (B) total CD4 T cells. (C) Representative traces of Fura-2-based imaging of cytosolic Ca 2+ concentrations following anti-CD3/CD28 stimulation in CD4 T cells. Antibodies bound to microscopy chamber bottom with cells sinking down in saline containing 2 mM Ca 2+ during running measurement, coming to rest in focus plane with contact to stimulation antibodies. Cells treated with 1 μM Apamin (Apamin, blue) or control (Ctrl, gray). (C, D, E) Quantification of area under the curve (D) and oscillation frequency (E) of data shown in (C), n = 29–37. (F) Representative FACS plots and gating strategy for CD69 and CD25 in total CD4 T cells. (G, H) Quantification of flow cytometry data of NS8593 dose-dependent up-regulation of CD69 (G) and CD25 (H) expression on total CD4 T cells, 48 h after anti-CD3/CD28 stimulation or PMA/ionomycin stimulation, respectively, n = 3–4. (I, J) Quantification of flow cytometry data of up-regulation of CD69 (I) and CD25 (J) expression on total CD4 T cells treated with 1 μM Apamin (Apamin, blue) or control (Ctrl, gray), 48 h after anti-CD3/CD28 stimulation or PMA/ionomycin stimulation, respectively, n = 3. (K) Respective FACS plots and gating strategy for CD4 T cell proliferation, shown for control cells. (L) Respective quantification of NS8593 dose-dependent proliferation of total CD4 T cells, with and without supplementation of 6 mM MgCl 2 , corresponding to , n = 4–7. (M) Representative histograms of dose-dependent proliferation (CSFE dye dilution) of total CD4 T cells in presence 1 μM Apamin in comparison to control, with (right) and without (left) supplementation of 6 mM MgCl 2 . Cells gated on T cell population, single cells and CD4 + T cells. Color code as in (N). (N) Respective quantification of proliferation of total CD4 T cells treated with 1 μM Apamin (Apamin, blue) or control (Ctrl, gray), with and without supplementation of 6 mM MgCl 2 , n = 5–8. (O) Respective quantification of Waixenicin A dose-dependent proliferation of total CD4 T cells, with and without supplementation of 6 mM MgCl 2 , corresponding to , n = 4–7. (D, E, G, H, I, J, L, N, O) Statistics: t test (D, E, I, J) and one-way ANOVA (G, H, L, N, O). * P < 0.05; ** P < 0.005, **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-human CD4-VioBlue (REA623; Miltenyi), α-human CD45RA-APC-Vio770 (REA562; Miltenyi), α-human CD69-APC (REA824; Miltenyi), α-human CD25-VioBright515 (REA570; Miltenyi).

Techniques: Isolation, Imaging, Microscopy, Saline, Control, Flow Cytometry, Expressing, Comparison

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) IL-2 quantification in supernatant of naïve CD4 T cells 48 h after α-CD3/α-CD28 stimulation, n = 4–5. (B, C, D, E) Histograms and quantification of up-regulated activation markers CD69 (B, C) and CD25 (D, E) in naïve CD4 T lymphocytes 48 h after stimulation. Cells were treated with 30 μM NS8593 or DMSO control, both with (Ctrl, blue; NS, orange) and without (Ctrl, black; NS, red) supplementation of 6 mM MgCl 2 . (F) IL-2 quantification in supernatant of total CD4 T cells 48 h after α-CD3/α-CD28 stimulation or cells treated with 30 μM NS8593 or DMSO control, both with (Ctrl, blue; NS, orange) and without (Ctrl, black; NS, red) supplementation of 6 mM MgCl 2 , n = 4–5. (G, H, I, J) Histograms and quantification of up-regulated activation markers CD69 (G, H) and CD25 (I, J) in total CD4 T lymphocytes 48 h after stimulation. Cells treated with either 30 μM NS8593 or DMSO control, both with (Ctrl, blue; NS, orange) and without (Ctrl, black; NS, red) supplementation of 6 mM MgCl 2 . (K) Representative TRPM7 I/V relationships of total CD4 T cells obtained via whole-cell patch clamp with Mg 2+ -free intracellular solution. Cells were treated with 10 μM Waixenicin A (WxA, green) or EtOH control (Ctrl, black). (L, M, N, O) Histograms and quantification of up-regulated activation markers CD69 (L, M) and CD25 (N, O) in total CD4 T lymphocytes 48 h after stimulation. Cells treated with 10 μM Waixenicin A or EtOH control, both with (Ctrl, blue; WxA, light green) and without (Ctrl, black; WxA, green) supplementation of 6 mM MgCl 2 , n = 7. (P) Representative histograms of dose-dependent proliferation (CSFE dye dilution) of total CD4 T cells in presence of various NS8593 concentrations, with (right) and without (left) supplementation of 6 mM MgCl 2 . Cells gated on T cell population, single cells and CD4 + T cells. Color code as in (Q). (Q) Respective quantification of NS8593 dose-dependent proliferation of total CD4 T cells, with and without supplementation of 6 mM MgCl 2 , corresponding to (P), n = 4–7. (R) Representative histograms of dose-dependent proliferation (CSFE dye dilution) of total CD4 T cells in presence of various Waixenicin A concentrations, with (right) and without (left) supplementation of 6 mM MgCl 2 . Cells gated on T cell population, single cells and CD4 + T cells. Color code as in (S). (S) Respective quantification of Waixenicin A dose-dependent proliferation of total CD4 T cells, with and without supplementation of 6 mM MgCl 2 , corresponding to (S), n = 4–8. (A, C, E, F, H, J, M, O, Q, S) Statistics: one-way ANOVA (A, C, E, F, H, J, M, O, Q, S). * P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-human CD4-VioBlue (REA623; Miltenyi), α-human CD45RA-APC-Vio770 (REA562; Miltenyi), α-human CD69-APC (REA824; Miltenyi), α-human CD25-VioBright515 (REA570; Miltenyi).

Techniques: Activation Assay, Control, Patch Clamp

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Schematic description of naïve CD4 T-cell differentiation towards FOXP3-expressing regulatory T cells and RORƔt-expressing T H 17 cells, including respective cytokine polarization milieus. (B, C) Percentages of CD45RA − cells and (C) CD25 + CD127 lo cells upon polarization of naïve CD4 T cells toward iT reg cells in various NS8593 concentrations (red) compared with DMSO control (Ctrl, black), n = 6–7. (D, E) Representative FACS histograms and (E) quantification of FOXP3 expression levels of CD25 + CD127 lo iT reg cells upon 6 d polarization of naïve CD4 T cells in presence of various NS8593 concentrations (red) or DMSO control (Ctrl, black), n = 6–7. (F, G) Percentages of CD45RA − cells and (G) CD25 + CD127 lo cells upon polarization of naïve CD4 T cells toward iT reg cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 7. (H, I) Representative FACS histograms and (I) quantification of FOXP3 expression levels of CD25 + CD127 lo iT reg cells upon 6 d polarization of naïve CD4 T cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 6. (J) Percentages of CCR6 + cells upon polarization of naïve CD4 T cells towards iT H 17 cells in presence of various NS8593 concentrations (red) compared with DMSO control (Ctrl, black), n = 6. (K, L) Representative FACS histograms and (L) quantification of RORƔt expression levels of CCR6 + iT H 17 cells upon 6 d polarization of naïve CD4 T cells in presence of various NS8593 concentrations (red) or DMSO control (Ctrl, black), n = 4–6. (M) Percentages of CCR6 + cells upon polarization of naïve CD4 T cells towards iT H 17 cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 6. (N, O) Representative FACS histograms and (O) quantification of RORƔt expression levels of CCR6 + iT H 17 cells upon 6 d polarization of naïve CD4 T cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 5. (P) Graphical summary of TRPM7-(in)dependent T-cell activation and differentiation towards iT reg and iT H 17 cells. Pharmacological blockade of TRPM7 reduces intracellular Mg 2+ levels, leads to reduced Ca 2+ signaling and results in reduced IL-2 secretion, impaired up-regulation of T-cell activation markers CD69 and CD25, and diminished proliferation upon TCR stimulus (left). TRPM7 inhibition during polarization of naïve CD4 T cells into iT reg cells preserves FOXP3 + signals of CD25 + CD127 lo iT reg cells. Polarization of naïve CD4 T cells into iT H 17 cells results in augmented RORƔt expression in the presence of 6 mM Mg 2+ , which is reduced upon TRPM7 inhibition, highlighting the need for Mg 2+ uptake and related TRPM7-dependent intracellular signaling for iT H 17 cell polarization (right). (B, C, E, F, G, I, J, L, M, O) Statistics: one-way ANOVA (B, C, E, J, L) and t test (F, G, I, M, O). * P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-human CD4-VioBlue (REA623; Miltenyi), α-human CD45RA-APC-Vio770 (REA562; Miltenyi), α-human CD69-APC (REA824; Miltenyi), α-human CD25-VioBright515 (REA570; Miltenyi).

Techniques: Cell Differentiation, Expressing, Control, Activation Assay, Inhibition

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) Flow cytometric analysis (FCA) of CD25 (left) and CD69 (right) staining and quantification in CTL, n = 6/11 individual patients. ( B ) FCA of lymphocyte-activation gene 3 (LAG3), programmed cell death protein 1 (PD1), and T-cell immunoglobulin and mucin-domain containing-3 (TIM3) staining and quantification as indicated, n = 6/4 (not activated/+CD3/CD28) individual patients. P values (left to right): 0.2842, 0.2188, 0.6875, 0.5494, 0.1250, and >0.9999. ( C ) Quantification of relative cytotoxicity using a flow cytometric killing assay, n = 5 individual patients. ( D ) Quantification of caspase-3 activity reporter using a CTL-K562 killing assay, n = 4 individual patients. Data were represented as mean ± SEM with dots indicating individual values ( A , B ) or as individual data points ( C , D ). P values: as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test, as appropriate.

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Staining, Activation Assay, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of MitoTracker green and quantification in isolated mitochondria from CTL, n = 7 individual patients. ( B ) FCA of JC1 green and quantification of red/green fluorescence in isolated mitochondria from CTL, n = 4 individual patients. ( C ) Immunoblot of OXPHOS complexes using isolated mitochondria of CTL and normalized densitometric quantification of complex V, n = 5 individual patients. I–V complex I–V; H heat shock protein 60 (HSP60). Subunits detected by the antibody cocktail: complex I: subunit NDUFB8; complex II: subunit 30 kDa (SDHB); complex III: subunit Core 2 (UQCRC2); complex IV: subunit II (COXII); complex V: ATP synthase subunit alpha (ATP5). ( D , E ) Representative immunoblot of mitochondrial fusion and fission proteins of isolated mitochondria of CTL ( D ) and normalized densitometric quantification ( E ) of the respective proteins, n = 5/4/3 individual patients. ( F ) Representative projections of 3D image stacks of CTL stained with MitoTracker (MT, green, left) and the respective skeleton images (white, right), generated using mitochondrial analyzer, time points as indicated, representative of six individual patients. ( G ) Quantification of mitochondrial count per cell, n = 29 cells from three individual patients. ( H ) Quantification of mitochondrial length (left) and mean mitochondrial area using mitochondrial analyzer (right), n = 180/170 mitochondria from 14/22 cells from three individual patients (length) and n = 50 cells from three individual patients (area). ( I ) Representative projection of 3D image stacks (two per time point) of translocated mitochondria to the IS in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, and quantification of mitochondrial translocation to the CTL IS, time points as indicated, n = 14 from 5 individual patients. If not stated otherwise, data were represented as mean ± SEM. P values as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test ( A – E ), and unpaired t -test or Mann–Whitney test ( G – I ), as appropriate. .

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Isolation, Fluorescence, Western Blot, Staining, Generated, Translocation Assay, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of MitoTracker green and quantification in CD8 + CTL, n = 7 individual patients. ( B ) Representative Seahorse OCR plot. ( C ) Fold change of basal OCR, maximum OCR, spare respiratory capacity (SRC) and ATP production, n = 6 individual patients. ( D ) Quantification of mitochondrial length, n = 92 mitochondria from 28 cells from three individual patients. ( E ) Quantification of mitochondrial count per cell, n = 30/26 cells from three individual patients. ( F ) Representative confocal microscopy image of mitochondria translocated to the TCR in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, T2 image as depicted in Fig. , upper right image. ( G ) Quantification of mitochondrial translocation in proximity to the CTL IS, n = 12 from four individual patients. ( H ) Representative impedance plot and ( I ) quantification of relative CTL cytotoxicity using ECIS, n = 6 individual patients. If not stated otherwise, data are represented as mean ± SEM. P values as indicated, one-sample t -test ( C ), paired t -test or Wilcoxon matched-pairs signed-rank test ( A – C, H , I ), and unpaired t -test or Mann–Whitney test ( D – G ), as appropriate. .

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Confocal Microscopy, Staining, Translocation Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Inhibition of CD38 enzyme activity on engrafted human immune cells enhances NAD+ metabolism and inhibits inflammation in an in-vivo model of xeno-GvHD

doi: 10.3389/fimmu.2025.1640611

Figure Lengend Snippet: TNB-738 reduces inflammation in tissues at day 15 by limiting the expansion of human CD45 cells with relatively high Treg cell numbers. (A) NSG mice were irradiated and 24h later received hPBMCs intravenous (i.v) (day 0). Animals were then treated with intraperitoneal (i.p) injections of PBS (black line) or TNB-738 at 130µg (gray line) twice a week. All mice were sacrificed when mice from PBS group lost 20% of IBW, (datas from 7 independents experiments). Graph represents mean of body weight in each group as a percentage of IBW. (B) Protein was extracted from frozen spleen and liver tissues followed by protein estimation and analysis of NAD+ concentration (left 2 graphs) or sirtuin1 activity (right 2 graphs). (C) Left graph shows total number of spleen cells in PBS (black point) or TNB-738 (gray scare), right graph shows absolute number of hCD45+ spleen cells in PBS (black point) or TNB-738 (gray scare). (D) Graph shows total number of hCD3- (left graph) or hCD19+ (right graph) spleen cells in PBS (black point) or TNB-738 (gray scare). (E) Left graph shows total number of hCD4+ spleen cells in PBS (black point) or TNB-738 (gray scare), middle left graph shows absolute number of hCD8+ spleen cells in PBS (black point) or TNB-738 (gray scare), middle right graph shows total number of hCD4+CD69+ spleen cells in PBS (black point) or TNB-738 (gray scare), right graph shows absolute number of hCD8+CD69+ spleen cells in PBS (black point) or TNB-738 (gray scare). (F) Left graph shows percentage of hCD4+FOXP3+spleen cells in PBS (black point) or TNB-738 (gray scare), middle graph shows absolute number of hCD4+FOXP3+hCD69+ spleen cells in PBS (black point) or TNB-738 (gray scare), right graph shows hCD4+FOXP3-/hCD4+FOXP3+ ratio on spleen cells in PBS (black point) or TNB-738 (gray scare). (G) Cytokine levels in sera of from left to right: hIFNg, hIL-10, hTNF-a, hIL-2 or hTGFb levels in sera of PBS (black point), TNB-738 (gray scare) or NSG that did not receive hPBMC (black triangle). *p<0.05 **p<0.01 ***p< 0.001 ****p<0.0001.

Article Snippet: Cells were then collected, stained with anti-human CD3 PeCy7 (BD biosciences), anti-human CD4 PercPCy5.5 (BD biosciences), anti-human CD25 APCCy7 (BD biosciences), anti-human CD127 PE (BD biosciences), anti-human CD69 APC (Myltenyi Biotec), and Viability Dye eFluor 506 (eBiosciences).

Techniques: Irradiation, Concentration Assay, Activity Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of CD38 enzyme activity on engrafted human immune cells enhances NAD+ metabolism and inhibits inflammation in an in-vivo model of xeno-GvHD

doi: 10.3389/fimmu.2025.1640611

Figure Lengend Snippet: Treatment of GVHD induced NSG mice with TNB-738 causes reduction in T cell proliferation and inflammatory cytokines in serum. (A) NSG mice were irradiated and 24h later received CPD-labeled hPBMCs intravenous (i.v) (day 0). Animals were then treated with intraperitoneal (i.p) injections of PBS (Upper graph, black histograms) or TNB-738 at 130µg (Lower graph, gray histogram) twice a week. Mice were sacrificed at D7. Histograms represent percentages of each CPD dividing hCD3+ populations. (B) Significative dot plots of hCD3/CPD staining in PBS group (upper graphs) and TNB-738 group (lower graphs) at D4 (left graphs) or D7 (right graph). (C) Spleen cells of D7 treated mice were analyzed by cytometry. Graphs show PBS (black circle) or TNB-738 (grey square) from left to right: absolute number of hCD3+; absolute number of CD3+CD69+ cells and percentage of CD3+CD69+ cells. (D) Cytokine level in sera: IFNγ (left graph), IL-2 (middle graph) or IL-17 (right graph), in PBS (black round) or TNB-738 (grey square) groups at D7. (E) hCD45 from spleen cells of D15 treated mice were sorted and cultured with or without IL-2. Graph represents percentage of proliferation of hCD4+ or hCD8+ in each condition. (F) In vitro inhibition of proliferation by TNB-738 in CD4+ or CD8+ populations. hPBMCs were cultured with or without NSG splenocytes in presence or not of TNB-738 for 7 days. At D7 proliferation of hCD4+ (left histograms) (CPD staining) or hCD8+ (right histograms) were analyzed in each group. Graphs represent means of percentage of proliferation (mean of 7 experiments); * p<0.05 ** p<0.01, ***p< 0.001, ****p<0.0001.

Article Snippet: Cells were then collected, stained with anti-human CD3 PeCy7 (BD biosciences), anti-human CD4 PercPCy5.5 (BD biosciences), anti-human CD25 APCCy7 (BD biosciences), anti-human CD127 PE (BD biosciences), anti-human CD69 APC (Myltenyi Biotec), and Viability Dye eFluor 506 (eBiosciences).

Techniques: Irradiation, Labeling, Staining, Cytometry, Cell Culture, In Vitro, Inhibition

Journal: iScience

Article Title: Impact of 3D cell culture hydrogels derived from basement membrane extracts or nanofibrillar cellulose on CAR-T cell activation

doi: 10.1016/j.isci.2025.113234

Figure Lengend Snippet: CAR-T cell cytotoxicity in short term cultures is comparable between NFC, Matrigel, and BME CD20 CAR-T cells were co-cultured with Daudi (Burkitt lymphoma cell line) cells labeled with CellTrace Violet (CTV) for 24 h at the different effector to target (E:T) ratios specified, in standard 2D suspension (control) or embedded in the specified hydrogels. (A) Representative gating strategy for analyzing the viability of Daudi and CD20 CAR-T cells by flow cytometry. (B) Number of viable CAR-T cells recovered after culture in the 1:1 E:T condition (50,000 Daudi +50,000 CAR-T cells/well) as measured by flow cytometry using counting beads (Flow-Count fluorospheres). Data were analyzed using a one-way ANOVA with Tukey’s multiple comparison test. (C) Specific lysis (%) of Daudi cells induced by CD20 CAR-T cells at the specified E:T ratios. Specific apoptosis was calculated by applying the following formula: [(%viable untreated − %viable treated)/%viable untreated] × 100. (D and E) Bar plot showing median fluorescence intensity (MFI) of (D) IFNγ and (E) CD69 in CAR-T cells cultured for 24 h in the described conditions. Data from two or three independent experiments are presented as means ± SD. Data were analyzed using a two-way ANOVA with Tukey’s multiple comparison test (∗ p < 0.05, ∗∗ p < 0.005). NFC, nanofibrillar cellulose; MG, Matrigel; BME, basement membrane extract.

Article Snippet: Anti-Human CD69 FITC , Miltenyi , Cat# 130-113-523; RRID: AB_2733656.

Techniques: Cell Culture, Labeling, Suspension, Control, Flow Cytometry, Comparison, Lysis, Fluorescence, Membrane