Journal: Oncoimmunology

Article Title: Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality

doi: 10.1080/2162402X.2022.2093426

Figure Lengend Snippet: Flow cytometry stainings per panel.

Article Snippet: Primary anti-human CD8a (Biolegend) and anti-human CD4 (R&D Systems) antibodies were added and incubated before DNAHoechst (Invitrogen), secondary rat-anti-mouse AF647 (Biolegend) and donkey-anti-goat AF488 (Thermo Scientific) antibodies were added and incubated for 24 h. For mesothelin staining, spheroids were incubated with PE-conjugated anti-human MSLN antibody (R&D Systems) for 24 h. All stainings were performed in PBS 1% BSA at 4°C.

Techniques: Flow Cytometry, Conjugation Assay, Functional Assay, Blocking Assay

Journal: Oncoimmunology

Article Title: Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality

doi: 10.1080/2162402X.2022.2093426

Figure Lengend Snippet: Phenotypic characterization of MSLN-CAR T cells after CAR production . A) Frequency of CD4 + and CD8 + cells within CD3 + EGFRt + M28z and MBBz CAR T cells and within CD3 + untransduced T cells (UT). N = 10 donors. B) Ratio of CD4 + /CD8 + within EGFRt + (CAR + ) and EGFRt- (CAR − ) M28z and MBBz transduced T cells. N = 10 donors. C) Maturation phenotype determined by CD45RA and CCR7 expression in CD3 + EGFRt + M28z and MBBz CAR T cells and within CD3 + UT cells. Gated on lymphocytes→ single cells→ viable cells (7AAD − )→ CD3 + EGFRt + (M28z and MBBz) or CD3 + EGFRt − (UT)→ CD4 + /CD8 + and ultimately CD45RA/CCR7. Median of n = 9 donors displayed. D) IFNy, TNF, IL2 cytokine production and CD107a degranulation by CD4 + and CD8 + M28z and MBBz transduced T cells following 6 hours of co-culture with OVCAR-3 MSLN + , determined by intracellular cytokine staining. Grey symbols represent M28z CAR T cells and green symbols represent MBBz CAR T cells. Wilcoxon tests were performed to detect differences between and within M28z and MBBz CAR constructs. For comparison of three T cell subsets (M28z vs MBBz vs UT) Friedman tests were used. * = P < .05 and ** = P < .01. Each dot represents 1 donor.

Article Snippet: Primary anti-human CD8a (Biolegend) and anti-human CD4 (R&D Systems) antibodies were added and incubated before DNAHoechst (Invitrogen), secondary rat-anti-mouse AF647 (Biolegend) and donkey-anti-goat AF488 (Thermo Scientific) antibodies were added and incubated for 24 h. For mesothelin staining, spheroids were incubated with PE-conjugated anti-human MSLN antibody (R&D Systems) for 24 h. All stainings were performed in PBS 1% BSA at 4°C.

Techniques: Expressing, Co-Culture Assay, Staining, Construct, Comparison

Journal: Oncoimmunology

Article Title: Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality

doi: 10.1080/2162402X.2022.2093426

Figure Lengend Snippet: Superior lysis and infiltration of MSLN high SKOV-3 spheroids by M28z CAR T cells as compared to MBBz CAR T cells . A) Lysis of MSLN high OVCAR-3 cells as determined by LDH release following co-culture with CD4 + enriched, CD8 + enriched and unsorted CD4 + /CD8 + MSLN-CAR T cells at a 1:5 or 2:1 effector to target ratio for 24 hours. N = 6 donors, each dot represents one donor. B, C) Lysis of MSLN high SKOV-3 spheroids in response to co-culture with UT cells, M28z or MBBz transduced T cell was monitored for 24 hours by detection of Caspase3/7 activation using the IncuCyte S3 live cell imaging system. B) Caspase3/7 activation as depicted by the green signal in representative spheroid. C) Caspase3/7 activation illustrated by the integrated GCU over time. Dots represent the mean+SEM of 3 technical replicates of 6 donors. GCU = green calibrated unit. D) Non-linear regression revealed EC50 of Caspase3/7 activation. E) Frequency of total T cells (%CD4 + +CD8 + ) within SKOV-3 MSLN high spheroids as detected by confocal microscopy following 6 hours of co-culture. F) CD4 + or CD8 + T cells within MSLN high SKOV-3 spheroids as detected by confocal microscopy after 6 hours of co-culture. Each dot represents the mean of 3 technical replicates from 1 donor, n = 6 donors. G) Representative confocal image of T cell infiltration of one donor. Grey symbols represent M28z CAR T cells and green symbols represent MBBz CAR T cells. Friedman tests were performed to detect differences in target cell lysis by CD4 + , CD8 + or CD4 + /CD8 + T cells (OVCAR-3 vs SKOV-3). Wilcoxon was performed to compare OVCAR-3 vs SKOV-3 per T cell condition and M28z vs MBBz. One-way ANOVA was used to compare caspase3/7 signal over a 24 hour time course in response to UT, M28z or MBBz treatment. N = 6. * = P < .05, ** = P < .01, *** = P < .001.

Article Snippet: Primary anti-human CD8a (Biolegend) and anti-human CD4 (R&D Systems) antibodies were added and incubated before DNAHoechst (Invitrogen), secondary rat-anti-mouse AF647 (Biolegend) and donkey-anti-goat AF488 (Thermo Scientific) antibodies were added and incubated for 24 h. For mesothelin staining, spheroids were incubated with PE-conjugated anti-human MSLN antibody (R&D Systems) for 24 h. All stainings were performed in PBS 1% BSA at 4°C.

Techniques: Lysis, Co-Culture Assay, Activation Assay, Live Cell Imaging, Confocal Microscopy

Journal: Oncoimmunology

Article Title: Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality

doi: 10.1080/2162402X.2022.2093426

Figure Lengend Snippet: Dynamic expression of PD-1, LAG-3 and TIM-3 exhaustion markers by M28z and MBBz CAR T cells after co-culture with target cells . A) Prior to start of experiment (t = 0), CAR surface expression on M28z and MBBz transduced T cells was determined by hFAB staining. During co-culture with MSLN high OVCAR-3 and SKOV-3 hFAB expression was monitored. B) Kinetics of PD-1, LAG-3 and TIM-3 frequency (top) and MFI (bottom, logarithmic y-axis) within CD4 + or CD8 + CAR + M28z and MBBz transduced T cells prior (0 h) and during co-culture (4 h and 24 h) with MSLN high OVCAR-3 cells. Each dot represents one donor, n = 6 donors. C) Frequency of PD-1/LAG-3/TIM-3 triple positive (TP or Triple+) within CAR + M28z and MBBz transduced T cells overtime. Median of 6 donors is displayed. D) Comparison between CIM (co-)expression following 24 hours of co-culture with MSLN high OVCAR-3 and SKOV-3 cells. Each dot represents one donor, n = 6 donors. Grey symbols represent M28z CAR T cells and green symbols represent MBBz CAR T cells. Wilcoxon tests were performed to compare between two time points, CAR constructs and cell lines (delta hFAB 4 hours vs 24 hours, M28z vs MBBz, and OVCAR-3 vs SKOV-3). Friedman tests were used per CAR construct over time (≥3 time points). * = P < .05, ** = P < .01.

Article Snippet: Primary anti-human CD8a (Biolegend) and anti-human CD4 (R&D Systems) antibodies were added and incubated before DNAHoechst (Invitrogen), secondary rat-anti-mouse AF647 (Biolegend) and donkey-anti-goat AF488 (Thermo Scientific) antibodies were added and incubated for 24 h. For mesothelin staining, spheroids were incubated with PE-conjugated anti-human MSLN antibody (R&D Systems) for 24 h. All stainings were performed in PBS 1% BSA at 4°C.

Techniques: Expressing, Co-Culture Assay, Staining, Comparison, Construct

Journal:

Article Title: Actin-Bundling Protein L-Plastin Regulates T Cell Activation

doi: 10.4049/jimmunol.1001424

Figure Lengend Snippet: A) Normal expression of surface markers on LPL−/− CD4+ cells. Surface markers were stained by specific antibodies conjugated with fluorescence and analyzed by FACS. a) CD4 staining of total splenocytes; CD3 (b), CD62L(c), CD44(d) and CD25(e) staining of CD4+ cells. Wt is in solid line and LPL−/− is dashed. Shown is a representative of 6 mice of each genotype. B) Primary CD4+ cells isolated from wt, LPL−/− or WASP−/− mice were stimulated with plate-bound anti-CD3. Proliferation was measured at 72 h by an MTT colorimetric assay. C) Proliferation of CD4+ cells activated by plate bound anti-CD3 with or without exogenous IL-2. D, E) CD4+ cells stimulated with plate-bound anti-CD3 alone (D) or in the presence of anti-CD28 (E) for 48 h. IL-2 secreted to the medium was measured by ELISA. Shown are means ± SD of triplicate samples.

Article Snippet: For IHC, formalin-fixed, paraffin-embedded sections were stained with the monoclonal antibody F4/80 (Serotec, Raleigh, NC)) to detect macrophages or with a goat anti-mouse CD4 (R&D Systems).

Techniques: Expressing, Staining, Fluorescence, Isolation, Colorimetric Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Actin-Bundling Protein L-Plastin Regulates T Cell Activation

doi: 10.4049/jimmunol.1001424

Figure Lengend Snippet: A) n3.L2 CD4+ cell responses to Hb peptide. Cells were stimulated with irradiated splenocytes in the presence of Hb peptide and IL-2 secreted was measured at 24 h. Shown are means of duplicates, a representative of 3 independent experiments. B) T cell responses to immunized OVA. On day 7 post immunization, cells isolated from draining lymph nodes were restimulated with OVA and proliferation was measured by incorporation of [3H]-thymidine. Shown are means ± SD (5 mice per group). C) Mixed lymphocyte reaction. CD3+ T cells in B6 background were stimulated with irradiated BALB/c splenocytes and proliferation was measured by incorporation of [3H]-thymidine. Shown are means ± SD of triplicates.

Article Snippet: For IHC, formalin-fixed, paraffin-embedded sections were stained with the monoclonal antibody F4/80 (Serotec, Raleigh, NC)) to detect macrophages or with a goat anti-mouse CD4 (R&D Systems).

Techniques: Irradiation, Isolation

Journal:

Article Title: Actin-Bundling Protein L-Plastin Regulates T Cell Activation

doi: 10.4049/jimmunol.1001424

Figure Lengend Snippet: A) IL-2 mRNA level. CD4+ cells were stimulated with plate-bound anti-CD3 at low (2 µg/ml) or high (10 µg/ml) doses. At 4 or 24 h, the IL-2 mRNA level was measured by quantitative RT-PCR and standardized to GAPDH levels. Error bars represent means±SD of triplicates, a representative of 3 independent experiments. B) Intracellular staining of IL-2 protein. CD4+ cells were stimulated by plate-bound anti-CD3 (10 µg/ml), or PMA plus ionomycin for 5.5 h in the presence of monensin. The IL-2 protein was measured by intracellular staining assayed by FACS. Quantitation shows percentage of IL-2 positive cells out of CD4+ cells after anti-CD3 stimulation and averaged 1.6%±0.3% in wt compared with 0.8%±0.03% in LPL−/− CD4+ cells from three independent experiments.

Article Snippet: For IHC, formalin-fixed, paraffin-embedded sections were stained with the monoclonal antibody F4/80 (Serotec, Raleigh, NC)) to detect macrophages or with a goat anti-mouse CD4 (R&D Systems).

Techniques: Quantitative RT-PCR, Staining, Quantitation Assay

Journal:

Article Title: Actin-Bundling Protein L-Plastin Regulates T Cell Activation

doi: 10.4049/jimmunol.1001424

Figure Lengend Snippet: A), B), D), Immunoblotting. CD4+ cells were mixed with anti-CD3 coated beads at 37°C for indicated time. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with Abs against phospho-tyrosine or GAPDH as loading control (A), phosphorylated forms of ZAP-70, LAT and AKT (B) and total or phospho-ERK (D). C) Calcium flux. Fura 2-AM labeled CD4+ cells were mixed with anti-CD3 coated beads. Time-lapse images were collected at Emission 510nm/Excitation 340 or 380nm, 10 sec/frame, for 10 min. The average ratio of emission 340nm/380nm (coded 0–255) of multiple cells (LPL−/−, n=18; wt, n=22) is plotted over time. E) IL-2 produced by CD4+ cells stimulated with hamster IgG (hIgG)- or anti-CD3- coated beads. Shown are average of duplicates, a representative of two experiments.

Article Snippet: For IHC, formalin-fixed, paraffin-embedded sections were stained with the monoclonal antibody F4/80 (Serotec, Raleigh, NC)) to detect macrophages or with a goat anti-mouse CD4 (R&D Systems).

Techniques: Western Blot, SDS Page, Control, Labeling, Produced

Journal:

Article Title: Actin-Bundling Protein L-Plastin Regulates T Cell Activation

doi: 10.4049/jimmunol.1001424

Figure Lengend Snippet: A) Primary wt (a) or LPL−/− (b) CD4+ cells were seeded onto anti-CD3 coated plates, incubated at 37°C for 5 min, fixed and analyzed by phase contrast microscopy. Spreading cells appear phase dark (indicated by arrows). Original magnification ×40. Scale bar, 5µm. B) Percentage of spreading cells quantitated after incubations of 5 and 30 min. Shown are means± SEM of 4 fields from two independent experiments (each field with >100 total cells). * P < 0.01. C) CD4+ cells were seeded onto glass chamber wells coated with anti-CD3. Phase contrast images were captured at 10 sec intervals for 30 min at 37°C. Shown are single frames at the indicated time points. Time zero is defined as the point when the cell started to attach to the glass. Original magnification ×60. D) Percentage of cells with >8 filopodia-like spikes in more than half of the images acquired in the first 5 minutes of spreading. Spreading cells (n=20 per group) were chosen randomly from 6 movies from two independent experiments. E) Actin cytoskeletal structure of spreading CD4+ cells. Cells were seeded onto anti-CD3 mAb coated cover glass for indicated times, fixed and permeabilized, stained for F-actin by phalloidin Alexa Fluor 488 and analyzed by widefield fluorescence microscopy. Shown are representative cells from two independent experiments. Original magnification ×60. F) and G), Change of cell area and shape of spreading cells. Cell area (F) and shape factor (G) were analyzed and shown as mean ± SEM of multiple cells (n>30 per group per time point) from different fields. *P <0. 05.

Article Snippet: For IHC, formalin-fixed, paraffin-embedded sections were stained with the monoclonal antibody F4/80 (Serotec, Raleigh, NC)) to detect macrophages or with a goat anti-mouse CD4 (R&D Systems).

Techniques: Incubation, Microscopy, Staining, Fluorescence

Journal:

Article Title: Actin-Bundling Protein L-Plastin Regulates T Cell Activation

doi: 10.4049/jimmunol.1001424

Figure Lengend Snippet: A) Conjugate formation. n3.L2 CD4+ cells and Hb peptide-pulsed CH27 cells were labeled with different fluorescent dyes as described in Methods, allowed to interact for 30 min at 37°C and analyzed by FACS. Double positive events were considered conjugates. B) Percentage of n3.L2 CD4+ cells incorporated into conjugates at a variety of antigen concentrations. Error bars represent means ± SEM of triplicate samples, representative of 3 independent experiments. C) LPL−/− CD4+ cells have smaller immunological synapses. CD4+-CH27 conjugates were formed as in A), stained for F-actin (green) and PKCθ (red), and analyzed by confocal microscopy. The arrows indicate the length across the synapse and define the synapse dimension. Original magnification ×63. Scale bar equals 5 µm. D) Quantitation of the maximal synapse (IS) dimension through z-dimension based on F-actin staining or differential interference contrast (DIC) image of LPL+/− (n=34) or LPL−/− CD4+ cells (n=43) from two independent experiments. *P <0.001. Error bars represent means ± SEM. E) Co-localization of MTOC and IL-2 vesicles. n3.L2 CD4+ cells were incubated with Hb peptide-pulsed CH27 cells for 4 hr before being stained for IL-2 (green) and pericentrin (red). Original magnification ×63. F) Percentage of IL-2 positive conjugates formed by wt (n= 309) or LPL−/− (n=278) CD4+ cells. G) Percentage of conjugates with co-localized IL-2 and MTOC. Results representative of two independent experiments.

Article Snippet: For IHC, formalin-fixed, paraffin-embedded sections were stained with the monoclonal antibody F4/80 (Serotec, Raleigh, NC)) to detect macrophages or with a goat anti-mouse CD4 (R&D Systems).

Techniques: Labeling, Staining, Confocal Microscopy, Quantitation Assay, Incubation

Journal:

Article Title: Actin-Bundling Protein L-Plastin Regulates T Cell Activation

doi: 10.4049/jimmunol.1001424

Figure Lengend Snippet: A) Average clinical scores of MOG-immunized mice (wt, n=8; LPL−/−, n=12). * P <0.05. Error bars represent mean ± SEM. B) Time to disease development is significantly longer in LPL−/− mice than wt mice. The percentage of disease-free mice (Y-axis) is plotted against time (X-axis). P< 0.001. A) and B) are representative of four independent experiments with similar cohort size and equivalent results. C) Histology and immunohistochemistry of spinal cord sections of LPL−/− (a,c,e,g) or wt mice (b,d,f,h) 28 days post immunization demonstrating decreased inflammatory cellular infiltration and demyelination in LPL−/− mice. Sections were stained with H&E, (a, b); Luxol fast blue (c, d); F4/80 (e,f) and anti-CD4 (g, h). Shown are sections representative of multiple mice with clinical scores equal to the average for the genotype. Original magnification, ×4. Scale bar, 250µm. D) Mean histologic scores for inflammation and demyelination of spinal cord. Seven wt and 11 LPL−/− mice were evaluated and histologically scored (12 histologic sections per mouse). ** P< 0.001. E, F, G) Ex vivo re-stimulation. On day 11 post immunization, cells isolated from pooled draining lymph nodes from 3 mice were cultured in the presence of increasing doses of MOG-peptide. Proliferation was measured by [3H]-thymidine incorporation in the last 8 hours of a 3-day culture (E). IFN-γ (F) and IL-17 (G) in the medium of a 3-day culture were measured by ELISA. Shown are mean ± SD of triplicate samples.

Article Snippet: For IHC, formalin-fixed, paraffin-embedded sections were stained with the monoclonal antibody F4/80 (Serotec, Raleigh, NC)) to detect macrophages or with a goat anti-mouse CD4 (R&D Systems).

Techniques: Immunohistochemistry, Staining, Ex Vivo, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: BJC Reports

Article Title: Circulating extracellular vesicles containing S100A9 reflect histopathology, immunophenotype and therapeutic responses of liver metastasis in colorectal cancer patients

doi: 10.1038/s44276-023-00007-9

Figure Lengend Snippet: a Increased CD4 positive cell expression in interface region of the Desmoplastic Lesion. b Increased CD8 positive cell expression in interface region of the desmoplastic lesion.

Article Snippet: Briefly, slides were Incubated overnight at 4 °C with primary antibody anti-S100A9 (TFS, Catalogue PA5-82145, dilution 1:8000), anti-CD4 (R and D systems, Catalogue AF-379-NA, dilution 1:1200), anti-CD8 (Cell signalling, Catalogue 70306, dilution 1:200), anti-CD68 (Catalogue ab201340, dilution 1:4500) and Elastase (R&D Systems, Catalogue MAB91671, dilution 1:1000) and used secondary HRP Anti-Rabbit and mouse (Dako K400311-2 and K4003111-2).

Techniques: Expressing