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: 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: eLife

Article Title: NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

doi: 10.7554/eLife.98258

Figure Lengend Snippet: A549 lung metastases was developed in 6–8 wk post-humanized mice and lung met-bearing humanized mice were then treated with NPRL2 , pembrolizumab, and its combination. 3-to 5 d after the treatment, lung metastases tissues were analyzed for infiltrating human immune cells. Single-cell suspensions were prepared from fresh lung metastases and in-depth immune analyses were performed using multicolor flow cytometry for determining human (A) CD45 +leukocytes, ( B ) CD3 + T, ( C ) CD8 + T, ( D ) regulatory T cells, and ( E ) natural killer (NK), cells. The level of human immune cells is shown for different treatment groups. ( F ) The level of activating CD8 + T cells was determined by the expression of CD69 expressing markers on infiltrating T cells among different treatment groups. ( G ) Percentage of PD1 expressing CD8 + T cells and its alterations after treatment are shown. ( H–I ) NPRL2 mediated alteration on the level of effector memory and central memory of CD8 + T cells in the tumor microenvironment. ( J ) Percentages of tissue-resident T cells (T RM ) in tumors and their alteration by NPRL2 treatment. CD103 + expressing T cells were considered as T RM . ( K ) The effect of NPRL2 treatment on the myeloid populations was also investigated. The level of HLA-DR + DC cells was determined among the lineage-negative population. ( L ) Level of human myeloid-derived suppressor cells (MDSC) based on the expression of CD33 + HLA-DR-ve population and the changes of MDSC in different treatment groups shown in lung met and (M) the percentage of M1 macrophages and its alteration upon NPRL2 treatment in lung metastases. These populations were gated as Lin-ve >CD11b+ve > HLA-DR+ve > CD163-ve. Statistics are shown at a significance level of p<0.05 unless otherwise noted. Data is shown as mean percentage ± SD, n=5.p<0.05; **p<0.005; ***p<0.0005. Figure 3—source data 1. NPRL2 induces antitumor immune responses in anti-PD1 resistant KRAS/LKB1 mutant A549-Lung Met in humanized mice: Tumor microenvironment analysis in humanized mice.

Article Snippet: Fluorochrome–conjugated monoclonal antibodies to the following human antigens were used: CD45-Alexa Fluor 700 (clone 2D1, HI30), CD45-phycoerythrin (PE; clone 2D1, HI30), CD3-PerCp/cy5.5 (clone HIT3a), CD19-PE-cyanine 7 (clone HIB19), CD8-allophycocyanin-cyanine 7 (clone RPA-T8, HIT8a), CD4-Pacific blue (clone OKT4), CD56-PE/BV510 (clone HCD56), CD69-FITC/APC/PE-Alexa Flour 610 (clone FN50; Thermo fisher), HLA-DR-PerCp/cy5.5 (clone LN3), CD33-PE (clone WM-53) (Thermo Fisher), CD11b-PE-Cy7 (clone 1CRF-44) (Thermo Fisher), Granzyme B-FITC (clone GB11), and IFN-γ-APC (clone 4 S.B3), CD103-Super bright 600 (Colne B-LY7; Thermo fisher), CD279 (PD-1)-Super Bright 702 (Clone J105; Thermo Fisher), CCR7-FITC (Clone G043H7), CD45RA-PE (Clone HI100), CD25-APC (clone CD25-4E3), Lin-FITC (Biolegend), CD163-APC (clone ebioGH1/61; Thermo fisher), CD11c-Pacific blue (clone Bu15; Thermo Fisher).

Techniques: Flow Cytometry, Expressing, Derivative Assay, Mutagenesis

Journal: eLife

Article Title: NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

doi: 10.7554/eLife.98258

Figure Lengend Snippet: ( A ) Experimental strategy of H1299 subcutaneous tumor development in humanized mice. NSG mice were humanized for 7–8 wk and humanization was verified by blood screening followed by tumor cell injections. Subcutaneous tumors were developed for another 6 wk to obtain a tumor size of about 200 mm 3 . Tumors were treated with NPRL2 (i.v) and pembrolizumab (i.p.) treatment for 2 wk. ( B ) Humanization status was checked by detecting the human CD45 cells in the blood before tumor cell implantation. ( C ) At the end of the experiment, the humanization level was checked based on the number of human CD45 cells in the mouse. ( D ) H1299 tumors were treated with NPRL2 and pembrolizumab and tumor volume was measured twice a week. The tumor growth curve was generated based on tumor volumes and the antitumor effect was evaluated. ( E ) Growth curves showed the individual mouse response to treatment. ( F–K ) tumor microenvironment analysis was performed to evaluate the immune cell infiltration into tumors. Fresh tumors were harvested within 3–5 d after treatment and single-cell suspensions were prepared for in-depth immune analyses by using multicolor flow cytometry for determining the level of human (F) cytotoxic CD8 + T, ( G ) effector natural killer (NK) cells, and (H) antigen-presenting HLA-DR + DC. ( I ) Level of activating CD8 + T cells was determined by the expression of CD69 expressing markers on infiltrating T cells among different treatment groups. ( J ) Percentage of PD1 expressing CD8 + T cells and its alterations after treatment are shown. ( K ) Percentages of tissue-resident T cells (T RM ) in tumors and their alteration by NPRL2 treatment. CD103 +expressing T cells were considered as T RM . In-vivo experiment was repeated three times with at least 5 mice/group used in each experiment. Statistics are shown at a significance level of p<0.05 unless otherwise noted. Data is shown as mean percentage ± SD, n=5. *p<0.05; **p<0.005; ***p<0.0005. Figure 4—source data 1. NPRL2 induced synergistic antitumor immune response with pembrolizumab on anti-PD1 responsive H1299 tumors in humanized mice: NPRL2 antitumor effect on H1299 tumors and tumor microenvironment analysis.

Article Snippet: Fluorochrome–conjugated monoclonal antibodies to the following human antigens were used: CD45-Alexa Fluor 700 (clone 2D1, HI30), CD45-phycoerythrin (PE; clone 2D1, HI30), CD3-PerCp/cy5.5 (clone HIT3a), CD19-PE-cyanine 7 (clone HIB19), CD8-allophycocyanin-cyanine 7 (clone RPA-T8, HIT8a), CD4-Pacific blue (clone OKT4), CD56-PE/BV510 (clone HCD56), CD69-FITC/APC/PE-Alexa Flour 610 (clone FN50; Thermo fisher), HLA-DR-PerCp/cy5.5 (clone LN3), CD33-PE (clone WM-53) (Thermo Fisher), CD11b-PE-Cy7 (clone 1CRF-44) (Thermo Fisher), Granzyme B-FITC (clone GB11), and IFN-γ-APC (clone 4 S.B3), CD103-Super bright 600 (Colne B-LY7; Thermo fisher), CD279 (PD-1)-Super Bright 702 (Clone J105; Thermo Fisher), CCR7-FITC (Clone G043H7), CD45RA-PE (Clone HI100), CD25-APC (clone CD25-4E3), Lin-FITC (Biolegend), CD163-APC (clone ebioGH1/61; Thermo fisher), CD11c-Pacific blue (clone Bu15; Thermo Fisher).

Techniques: Generated, Flow Cytometry, Expressing, In Vivo

Journal: eLife

Article Title: NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

doi: 10.7554/eLife.98258

Figure Lengend Snippet: NPRL2 stable clones in A549 and H1299 NSCLC cells were generated and developed tumors in humanized mice for tumor microenvironment analysis. The in-vitro assays were performed using these stable clones to elucidate cell death and molecular signaling. ( A ) Scheme showing the experimental strategy where NSG mice were humanized for 6–8 wk followed by H1299- NPRL2 ++/++ tumor cell implantation to develop the tumors for TME analysis. ( B ) Humanization status was checked before tumor implantation based on the level of human CD45 cells. Mice containing 25% or more human CD45 + cells were considered as humanized mice and only the verified mice were used for the tumor implantation. The status of CD3 + T, NK, and B cells was evaluated prior to tumor implantation. ( C ) Tumor growth curves showed the rate of tumor growth and differences in tumor growth between parental H1299 tumors and H1299- NPRL2 ++/++ tumors in humanized mice. ( D–N ) At day 43, fresh tumors were harvested, single cells were prepared, and multicolor flow cytometry was run for multiple innate and adaptive panels to identify the human immune cell populations in the tumor microenvironment. ( D–H ) The percentage of major human immune cells including (D) human CD45 + , ( E ) human CD19 + B, ( F ) human CD3 + T, ( G ) CD4 + T, and CD8 + T, and ( H ) natural killer (NK) cells in parental tumors and their alterations in NPRL2 -stably expressing tumors in humanized mice. ( I–K ) The status of ( I ) regulatory T cells, ( J ) PD1 expressing T cells (CD274 +CD8 + T), and ( K ) activating T cells (CD69 +CD8 + T) in both parental and NPRL2 -stably expressing tumors. ( L–N ) The presence of innate cells was also investigated and analyzed after gating out the lineage-positive population to identify the level of ( L ) MDSC (CD33 +HLA-DR- MDSC), ( M ) TAM (CD11b+HLA-DR-CD163+TAM), and (N) HLA-DR +DC in tumors in humanized mice. ( O–S ) A549- NPRL2 ++/++ and H1299- NPRL2 ++/++ cells were used for in-vitro assays compared with their respective parental counterparts. ( O ) Stable expression level of NPRL2 was verified by western blot, ( P ) Colony forming assay in H1299 and H1299- NPRL2 ++/++ cells showed the differences in colony formation inhibition in the absence or presence of different concentrations of carboplatin, ( Q ) Apoptosis assay was also performed in both pairs of cell lines to detect the annexin V positive apoptotic cells by flow cytometry following carboplatin treatment. The difference in the level of apoptosis was estimated and compared between parental and NPRL2 stably expressing counterparts. ( R ) The level of apoptosis was verified by PARP cleavage by detecting the cleavage of PARP by western blots in NRPL2 stable cells after carboplatin treatment. ( S ) Western blots were performed to detect a list of signaling molecules involved in downstream and upstream of the PI3K/AKT/mTOR signaling pathway, which included p-AKT, p-mTOR, p-S6, p-4E-BP, p-PRAS40, p-GSK-3b. The MAPK pathway was investigated based on the level of expression of pERK1/2. The in-vivo experiment was repeated three times with at least N=5 mice/group used in each experiment. Statistics were shown at a significance level of p<0.05 unless otherwise noted. Data is shown as mean percentage ± SD, n=5. *p<0.05; **p<0.005; ***p<0.0005. Figure 8—source data 1. Restoration of NPRL2 expression altered tumor microenvironment (TME), induced apoptosis, inhibited cell growth and signaling: Tumor microenvironment analysis in NPRL2 stable expressing tumors. Figure 8—source data 2. PDF file containing original western blots for , indicating relevant bands, treatments and samples. Figure 8—source data 3. Original files for western blot images displayed in .

Article Snippet: Fluorochrome–conjugated monoclonal antibodies to the following human antigens were used: CD45-Alexa Fluor 700 (clone 2D1, HI30), CD45-phycoerythrin (PE; clone 2D1, HI30), CD3-PerCp/cy5.5 (clone HIT3a), CD19-PE-cyanine 7 (clone HIB19), CD8-allophycocyanin-cyanine 7 (clone RPA-T8, HIT8a), CD4-Pacific blue (clone OKT4), CD56-PE/BV510 (clone HCD56), CD69-FITC/APC/PE-Alexa Flour 610 (clone FN50; Thermo fisher), HLA-DR-PerCp/cy5.5 (clone LN3), CD33-PE (clone WM-53) (Thermo Fisher), CD11b-PE-Cy7 (clone 1CRF-44) (Thermo Fisher), Granzyme B-FITC (clone GB11), and IFN-γ-APC (clone 4 S.B3), CD103-Super bright 600 (Colne B-LY7; Thermo fisher), CD279 (PD-1)-Super Bright 702 (Clone J105; Thermo Fisher), CCR7-FITC (Clone G043H7), CD45RA-PE (Clone HI100), CD25-APC (clone CD25-4E3), Lin-FITC (Biolegend), CD163-APC (clone ebioGH1/61; Thermo fisher), CD11c-Pacific blue (clone Bu15; Thermo Fisher).

Techniques: Clone Assay, Generated, In Vitro, Tumor Implantation, Flow Cytometry, Stable Transfection, Expressing, Western Blot, Inhibition, Apoptosis Assay, In Vivo

Journal: NPJ Precision Oncology

Article Title: RevCAR-mediated T-cell response against PD-L1-expressing cells turns suppression into activation

doi: 10.1038/s41698-025-00828-6

Figure Lengend Snippet: a Target cells (5 × 10 3 ) were co-cultured with RevCAR T-cells (E:T = 5:1) in the presence or absence of PD-L1 RevTM for 24 h. The supernatant was harvested, and the secretion of pro-inflammatory cytokines was determined. Statistical significance was assessed by Student’s independent t test. P ≤ 0.05 (*), P ≤ 0.01 (**). b RevCAR T-cells were seeded in the absence of target cells, in the presence of target cells alone, or in the presence of target cells and PD-L1 RevTM for 24 h. Then, the RevCAR T-cells were stained with CD69 antibody. Statistical significance was assessed by two-way ANOVA followed by Tukey’s multiple comparisons test; P ≤ 0.0001 (****). c Co-cultures were set as described in b , but in this case, after incubation, the T-cells were stained with CD62L and CD45RO antibodies, and according to the expression levels of these markers, RevCAR T-cells were divided into four groups of increasing differentiation: Naive (T N ): CD62L high and CD45RO low ; Central memory (T CM ): high expression of both markers. Effector memory (T EM ): CD62L low and CD45RO high ; and Terminal effector (T TE ): low expression of both markers. a – c Data for three individual T-cell donors are shown as mean ± SD.

Article Snippet: After the co-culture, the T-cells of each condition were collected and incubated for 20 min at 4 °C with either anti-CD69-APC mAb (Miltenyi Biotec GmbH) in the case of activation staining or anti-CD45RO-PE mAb (Miltenyi Biotec GmbH) and anti-CD62L-Pacific BlueTM mAb (Biolegend) in the case of memory staining.

Techniques: Cell Culture, Staining, Incubation, Expressing

Journal: NPJ Precision Oncology

Article Title: RevCAR-mediated T-cell response against PD-L1-expressing cells turns suppression into activation

doi: 10.1038/s41698-025-00828-6

Figure Lengend Snippet: a Dual RevCAR T-cells express two separate receptors: the signaling (SIG) RevCAR-E7B6 triggering the CD3z signal and the costimulatory (COS) RevCAR-E5B9 triggering the CD28 co-stimulatory signal. The SIG contains intracellular CD3z signaling domain (purple), transmembrane domain (gray), hinge domain (yellow), and peptide epitope E7B6 (dark yellow). The COS comprises intracellular CD28 domain (brown), transmembrane domain (gray), hinge domain (pink), and peptide epitope E5B9 (red). Dual RevCAR T-cells are redirected to PSCA + PD-L1 + cancer cells via PSCA RevTM and PD-L1 RevTM. The simultaneous recognition of PSCA and PD-L1 by SIG and COS RevCARs via the respective RevTMs triggers both CD3z and CD28 signals resulting in complete activation of Dual RevCAR T-cells. b Comparative expression of PD-L1 and PSCA on the surface of PC-3 PSCA/PD-L1. c , d 5 × 10 3 PC-3 PSCA/PD-L1 cells were co-cultured for 24 h with Dual RevCAR T-cells (E:T = 5:1) in the absence of RevTM, in the presence of only PD-L1 or PSCA RevTM, or in the presence of both RevTMs. c Specific lysis and d cytokine secretion were determined. e , f Dual RevCAR T-cells were seeded in the absence of target cells, in the presence of target cells alone, in the presence of target cells and either PD-L1 RevTM or PSCA RevTM, or in the presence of target cells and both RevTMs, for 24 h (E:T = 5:1). Then, the T-cells were stained with e anti-CD69 antibody or f anti-CD62L and anti-CD45RO antibodies. Dual RevCAR T-cells were divided into four groups of increasing differentiation: Naive (T N ): CD62L high and CD45RO low ; Central memory (T CM ): high expression of both markers; Effector memory (T EM ): CD62L low and CD45RO high ; and Terminal effector (T TE ): low expression of both markers. c – f Data for three individual T-cell donors are shown as mean ± SD. c – e , statistical significance was assessed by ordinary one-way ANOVA ( c , e ) or two-way ANOVA ( d ) followed by Tukey’s multiple comparisons test; P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.0001 (****).

Article Snippet: After the co-culture, the T-cells of each condition were collected and incubated for 20 min at 4 °C with either anti-CD69-APC mAb (Miltenyi Biotec GmbH) in the case of activation staining or anti-CD45RO-PE mAb (Miltenyi Biotec GmbH) and anti-CD62L-Pacific BlueTM mAb (Biolegend) in the case of memory staining.

Techniques: Activation Assay, Expressing, Cell Culture, Lysis, Staining