cd4+ and cd8+ depletion cocktail  (STEMCELL Technologies Inc)

Journal: Journal for Immunotherapy of Cancer

Article Title: Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy

doi: 10.1136/jitc-2024-010684

Figure Lengend Snippet: Characterization of human DNTs by single-cell RNA sequencing. ( A ) Integrated UMAP plot of ex vivo expanded DNTs from three different donors. ( B ) Scaled average expression of CD3G , CD4, NCAM1 (CD56), CD8A , CD8B , CD19 , TRDC , TRDV1, and TRDV2 in DNT clusters shown by violin plot. ( C ) Integrated UMAP plot of αβTCR expression and clonotype frequencies in DNTs. (D–H) Top differential gene markers (log 2 FC>2) in DNT cluster 0 ( D ), cluster 4 ( E ), cluster 12 ( F ), cluster 9 ( G ), and clusters 1/2/3/5/6/7/10 ( H ). ( I ) Representative flow plot (left) and average relative abundance of different TCR subsets in ex vivo expanded DNTs (n=22). DNTs, double-negative T-cell; UMAP, uniform manifold approximation and projection.

Article Snippet: DNTs were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors through CD4 + and CD8 + depletion cocktail (StemCell Technologies) followed by density centrifugation (Lymphoprep; StemCell Technologies).

Techniques: RNA Sequencing, Ex Vivo, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy

doi: 10.1136/jitc-2024-010684

Figure Lengend Snippet: DNTs proliferate faster and transcriptionally differ from conventional Vδ2 T cells expanded with zoledronic acid. ( A ) Integrated UMAP plot of DNTs and donor-matched Vδ2 T cells expanded ex vivo (n=2 for each cell type). ( B ) Scaled average expression of CD3G , CD4, NCAM1 (CD56), CD8A , CD8B , TRDV1, and TRDV2 in DNT and Vδ2 T-cell clusters shown by violin plot. ( C ) Integrated UMAP plot showing the αβTCR repertoire profile in DNTs and Vδ2 T cells. ( D ) Expansion fold (left), yield (middle), and purity (right) of ex vivo expanded DNTs and donor-matched Vδ2 T cells 13 days postexpansion (n=6). Purity of cellular products (CD3 + CD4 − CD8 − for DNTs and CD3 + γδTCR + for Vδ2 T cells) was determined by flow cytometry. Paired dots indicate donor-matched samples. Paired Student’s t-test was used for statistics. ( E ) Volcano plot of top differential markers between bulk (top) and TRDV2 + (log 2 FC>1, bottom) cells in DNTs and Vδ2 T cells. Significance cutoffs denoted by the dotted lines are log 2 FC >0.5 and p<10 −10 . ( F ) Selected gene set enrichment analysis of differential markers between TRDV2 + (log 2 FC >1) cells in DNTs and Vδ2 T cells important for cellular immunity. Statistically significant gene sets (p<0.05) are shown. Fisher’s exact test was used for statistics. ( G ) Inferred number of interactions and interaction strength between TRDV2 + (log 2 FC>1) cells (left) or total cells (right) among DNTs and Vδ2 T cells. ( H ) Scatterplot comparing the incoming and outgoing interaction strength of inferred ligand-receptor networks between DNT (red) and Vδ2 T-cell (blue) clusters. Size of each dot represents the number of cells, and each dot indicates a cluster. *p<0.05, **p<0.01. DNT, double-negative T-cell; UMAP, uniform manifold approximation and projection.

Article Snippet: DNTs were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors through CD4 + and CD8 + depletion cocktail (StemCell Technologies) followed by density centrifugation (Lymphoprep; StemCell Technologies).

Techniques: Ex Vivo, Expressing, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Single-cell RNA sequencing of human double-negative T cells reveals a favorable cellular signature for cancer therapy

doi: 10.1136/jitc-2024-010684

Figure Lengend Snippet: Memory and regulatory qualities in DNTs may contribute to longer in vivo persistence. (A–C) Volcano plot of top differential markers (cutoffs: log 2 FC>0.5 and p<10 −10 ) with different gene labels, and UMAP and violin plots of module scores based on exhaustion T (T EX )-cell ( A ), naïve T (T Naïve )-cell ( B ), and central memory T (T CM )-cell ( C ) signatures from Anderson et al between DNTs and Vδ2 T cells. Unpaired Student’s t-test was used for statistics. ( D ) Memory phenotype of DNTs and Vδ2 T cells based on CD62L and CD45RA expression by flow cytometry. Representative flow plots and individual T-cell memory phenotypes (n=6) are shown. Each paired dot represents a donor. Naïve/stem cell memory T (T Naïve/SCM ) cell=CD62L + CD45RA + , central memory T (T CM ) cell=CD62L + CD45RA − , effector memory T (T EM ) cell=CD62L − CD45RA − , effector T (T Effector ) cell=CD62L − CD45RA + . Paired Student’s t-test was used for statistics. ( E ) Volcano plot of top differential markers (cutoffs: log 2 FC>0.5 and p<10 −10 ), and UMAP and violin plots of module scores based on regulatory T (T reg ) cell signature from Anderson et al between DNTs and Vδ2 T cells. Unpaired Student’s t-test was used for statistics. ( F ) Allogeneic PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with or without anti-CD3/CD28 antibody in the presence or absence of ex vivo expanded DNTs or Vδ2 T cells (n=4) at a ratio of 1:1 or 2:1. Cells were harvested, and the CD4 + and CD8 + T conv -cell populations of the allogeneic PBMCs were assessed for inhibition of proliferation by CFSE dilution using flow cytometry. The data show the percentage of non-proliferating responder cells remaining after 5 days in culture. Red dotted line displays the average non-proliferating levels of allogeneic T conv cells alone with (stim) and without (no stim) anti-CD3/CD28 antibody stimulation. Each paired dot represents a donor. Paired Student’s t-test was used for statistics. ( G ) PBMCs were co-cultured with (stim) or without (non-stim) irradiated allogeneic DNTs as stimulators in the presence or absence of viable DNTs or Vδ2 T cells as suppressors for 6 days. Afterward, allogeneic CD8 + T cells were sorted from the co-culture and cultured with live DNT targets in an overnight assay. Percentage-specific killing of DNTs by allogenic CD8 + T cells under different co-culture conditions is shown. Schematic of the mixed lymphocyte reaction experiment (top) and percentage specific killing by allogenic CD8 + T cells against DNT targets following different co-culture conditions (bottom). Experiments were performed in triplicates, and data are representative of two independent experiments. Mean±SEM is shown. One-way ANOVA was used for statistics. ( H ) Sublethally irradiated (225 cGy) NSG mice were infused with 2×10 7 DNTs or Vδ2 T cells expanded from the same donor and 1×10 6 allogeneic PBMCs. Mice were bled, and circulating DNTs (n=5) or Vδ2 T cells (n=5) were counted throughflow cytometry for up to 42 days. Data shown are representative of two independent experiments. Error bars represent mean±SEM. Two-way ANOVA was used for statistics. ns=nonsignificant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; DNT, double-negative T-cell; PBMCs, peripheral blood mononuclear cells.

Article Snippet: DNTs were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors through CD4 + and CD8 + depletion cocktail (StemCell Technologies) followed by density centrifugation (Lymphoprep; StemCell Technologies).

Techniques: In Vivo, Expressing, Flow Cytometry, Labeling, Ex Vivo, Inhibition, Cell Culture, Irradiation, Co-Culture Assay, Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells

doi: 10.1136/jitc-2023-007277

Figure Lengend Snippet: CAR4-DNTs are able to target CD4 + T-ALL and PTCL in vitro. (A) Donor-derived DNTs were transduced with a retroviral vector expressing CAR4 or an EV control. Three days after transduction, the expression of CAR4 on DNTs was measured by protein L. Left: Representative histogram showing the protein L staining on EV-DNTs (black) and CAR4-DNTs (gray). Right: Summary of CAR4 expression level on CAR4-DNTs from 11 independent experiments. Each dot represents transduction efficiency of DNTs from an individual donor. Horizontal line represents the mean, and error bars represent SEM. (B) Expression of CAR4 measured on DNTs over the course of 18 days after transduction ( n =3) from three independent experiments. (C–D) DNTs and Tconv derived from the same donors (n=3) were transduced with EV and CAR4. Four days after transduction, cell counts and expression of CD4 and CD8 on transduced T cells were measured by flow cytometry. (C) Bar graphs showing cell counts of CAR4-DNTs and CAR4-Tconv relative to their EV-transduced counterparts. Error bars represent SEM. Two-way ANOVA Tukey’s multiple comparison test was used for statistical analysis. (D) Representative flow plots showing the frequencies of CD4 + and CD8 + T cells within each transduced T-cell population. (E) Ex vivo expanded EV-DNTs and CAR4-DNTs were stained with anti-CD3, anti-CD4, and anti-CD8 antibodies and analyzed by flow cytometry. Data shown are representative of three independent experiments. (F) EV-DNTs or CAR4-DNTs were expanded for 10 days (n=4) in four independent experiments. Mean fold expansion for EV-DNTs (circles) or CAR4-DNTs (squares). Error bars represent SEM. Two-way ANOVA was used for statistical analysis. (G) Memory status of EV-DNTs (left) and CAR4-DNTs (right) expanded from the same donor on day 10 after transduction. Cells were stained with CD45RA and CD62L. Flow plots are representative of three independent experiments. (H) Expression of activation and exhaustion markers, CD25, CD69, TIM3, LAG3, and PD1, on the surface of EV-DNTs or CAR4-DNTs on day 14 after transduction. Histograms are representative of three independent experiments, and numbers represent mean fluorescence intensity. (I) CAR4-DNTs from four different donors were cocultured with CCRF-CEM for 2 hours at indicated effector-to-target ratios. Per cent specific killing of the target is shown. Symbols represent mean per cent specific killing of triplicates, and error bars represent SD. (J and K) EV-DNTs (circles) or CAR4-DNTs (squares) were cocultured with T-ALL cell line, CCRF-CEM (J), or PTCL cell line, KARPAS-299 (K), for 2 hours at indicated effector-to-target ratios. Per cent specific killing of the target is shown. The experiment was independently performed seven (J) or three (K) times each with triplicates, and representative data are shown. Two-way ANOVA was used for statistical analysis. (L) EV-DNTs (white bars) and CAR4-DNTs (black bars) were cocultured with five primary T-ALL blasts for 2 hours at a 2:1 effector-to-target ratio. Per cent specific killing of T-ALL blasts is shown. Data represent mean per cent specific killing of the target using DNTs obtained from two different donors. Each experiment was done in technical duplicates. Error bars represent SEM. Numbers on the x-axis represent patient sample identifications. Two-way ANOVA Sidak’s multiple comparison test was used for statistical analysis. (M) Comparison of specific killing of the five primary T-ALL samples by EV-DNTs and CAR4-DNTs. Dots represent per cent specific killing, horizontal lines represent the mean, and the error bars represent SEM. Paired t-test was used for statistical analysis. *p<0.05; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; CAR4, CD4-CAR; DNTs, double-negative T cells; EV, empty-vector; PTCL, peripheral T-cell lymphoma; T-ALL, T-cell acute lymphoblastic leukemia; TCM, central memory T cell; Tconv, conventional T cells; TEM, memory T cells; TEMRA, terminally differentiated effector memory T cells; Tnaïve/SCM, naïve/stem cell memory T cell.

Article Snippet: Briefly, healthy donor-derived PBMCs were depleted of CD4 + and CD8 + cells using CD4-depletion and CD8-depletion cocktails (STEMCELL Technologies) and cultured on anti-CD3 antibody-coated plates (5 µg/mL; OKT3; Miltenyi Biotec) for 3–4 days in RPMI 1640 (Gibco) supplemented with 10% FBS and interleukin (IL)-2 (250 IU/mL; Proleukin, Novartis Pharmaceuticals).

Techniques: In Vitro, Derivative Assay, Transduction, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Comparison, Ex Vivo, Activation Assay, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells

doi: 10.1136/jitc-2023-007277

Figure Lengend Snippet: CAR4-DNTs can target CD4 + T-ALL and PTCL in xenograft models. (A) Schematic outline of in vivo experiments. NSG mice were sublethally irradiated (250 cGy) on day −1 and intravenously (i.v.) injected with 2–5×10 5 CCRF-CEM cells on day 0. On days 3, mice were i.v. treated with PBS or various dosages of EV-DNTs or CAR4-DNTs. All mice received intraperitoneal (i.p.) injections of IL-2 one time per week. Mice were sacrificed when they reached the end point of the survival experiment. (B) Kaplan-Meier curve showing the per cent survival of CCRF-CEM-engrafted mice treated with PBS (black, n=9), 0.5×10 6 (yellow, n=5), 2×10 6 (red, n=9) CAR + CAR4-DNTs, or 2×10 6 EV-DNTs (blue, n=12). Data are summary of three independently performed experiments. Log-rank test was used for statistical analysis. (C) CCRF-CEM engrafted mice were sacrificed on day 32 and bone marrow was examined. DNTs were identified via staining with anti-CD45 and anti-HLA-A2 antibody. Left: Representative flow plots for CD45 and HLA-A2 staining of bone marrow cells from untreated, EV-DNT-treated, and CAR4-DNT-treated mice on day 32. CCRF-CEM cells are HLA-A2 − ; an HLA-A2 + donor was used to manufacture EV-DNTs and CAR4-DNTs. Right: Number of DNTs detected by flow cytometry on day 32. Each dot represents the DNT count per million cells in the bone marrow of one mouse, horizontal line represents the mean, and error bar represents SEM. One-way ANOVA Tukey’s multiple comparison test was used for statistical analysis. (D) Number of DNTs in the spleen, liver, and lungs of CCRF-CEM engrafted mice on day 32 as detected by flow cytometry. Each dot represents the DNT count per million cells in the spleen (left), liver (middle), or lung (right) of one mouse, horizontal line represents the mean, and error bar represents SEM. One-way ANOVA Tukey’s multiple comparison test was used for statistical analysis. (E) Representative images (×20 magnification) of H&E-stained slides of liver sections taken from CCRF-CEM engrafted mice in PBS (left), EV-DNT (middle), or CAR4-DNT (right) treatment group (n=2 per group) at the endpoint of the experiment, showing tissue histology and lymphocytic blast infiltration (black arrow). (F) Tumor volume of KARPAS-299-engrafted mice treated with PBS (black, n=7), CAR4-DNTs (red, n=7), or EV-DNTs (blue, n=6). Dots represent mean tumor volume and error bars represent SEM. Two-way ANOVA Tukey’s multiple comparison test was used for statistical analysis. (G) Kaplan-Meier curve showing the per cent survival of KARPAS-299-engrafted mice treated with PBS (black, n=7), CAR4-DNTs (red, n=7), or EV-DNTs (blue, n=5). Log-rank test was used for statistical analysis. (H) KARPAS-299 engrafted mice were sacrificed on day 30. Tumors were excised and KARPAS-299 cells were identified via staining with anti-HLA-B7 antibody. Left: Representative flow plots for CD3 and HLA-B7 staining of tumor samples from untreated, EV-DNT-treated, and CAR4-DNT-treated mice on day 30. KARPAS-299 cells are HLA-B7 + ; an HLA-B7 − donor was used to manufacture EV-DNTs and CAR4-DNTs. Right: Number of DNTs detected by flow cytometry on day 30. Each dot represents the DNT count in the tumor sample of one mouse, horizontal line represents the mean, and error bar represents SEM. One-way ANOVA Tukey’s multiple comparison test was used for statistical analysis. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; CAR4, CD4-CAR; DNTs, double-negative T cells; EV, empty-vector; HLA, human leukocyte antigen; IL, interleukin; PBS, phosphate-buffered saline; PTCL, peripheral T-cell lymphoma; T-ALL, T-cell acute lymphoblastic leukemia.

Article Snippet: Briefly, healthy donor-derived PBMCs were depleted of CD4 + and CD8 + cells using CD4-depletion and CD8-depletion cocktails (STEMCELL Technologies) and cultured on anti-CD3 antibody-coated plates (5 µg/mL; OKT3; Miltenyi Biotec) for 3–4 days in RPMI 1640 (Gibco) supplemented with 10% FBS and interleukin (IL)-2 (250 IU/mL; Proleukin, Novartis Pharmaceuticals).

Techniques: In Vivo, Irradiation, Injection, Staining, Flow Cytometry, Comparison, Plasmid Preparation, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells

doi: 10.1136/jitc-2023-007277

Figure Lengend Snippet: PI3K inhibitor can further improve the persistence and function of CAR4-DNTs in vitro and in the T-ALL xenograft model. (A) Left: Representative flow plots showing the memory status of CAR4-DNTs and CAR4-Ide-DNTs obtained and expanded from the same donor over the course of 18 days after transduction. Cells were stained with CD45RA and CD62L. Right: Summary bar graph of memory phenotype of CAR4-DNTs and CAR4-Ide-DNTs on day 13 post transduction. Error bars represent SEM. Data are representative of two independent experiments. (B) CAR4-DNTs or CAR4-Ide-DNTs were cocultured with T-ALL cell line, CCRF-CEM, for 2 hours at a 1:1 effector-to-target ratio. Per cent specific killing of the target is shown. Bars represent mean of technical triplicates, and error bars represent SEM. The experiment was independently performed two times, and representative data are shown. Two-way ANOVA was used for statistical analysis. (C) CAR4-DNTs (circles) and CAR4-Ide-DNTs (squares) were cocultured with CCRF-CEM at a 4:1 effector-to-target ratio. Every 24 hours following coculture, per cent specific killing of the target was measured, and fresh target cells were added to the coculture at the original effector-to-target ratio. Mean per cent specific killing of triplicates are shown and error bars represent SEM. The experiment was independently performed two times, and representative data are shown. (D) Schematic outline of experiments. NSG mice were sublethally irradiated (250 cGy) on day −1 and intravenously (i.v.) injected with 2–5×10 5 CCRF-CEM cells on day 0. On days 3, 7, and 10, mice were intravenously treated with PBS, or 2×10 6 CAR + CAR4-DNTs or CAR4-Ide-DNTs. All mice received intraperitoneal (i.p.) injections of IL-2 once per week. Mice were sacrificed when they reached the end point of the survival experiment, and cells were harvested from the bone marrow, spleen, liver, and lungs. Human cells were identified via staining with anti-CD45 antibody. HLA-A2 antibody was used to distinguish between CCRF-CEM cells and donor DNTs; CCRF-CEM cells are HLA-A2 − and donor DNTs are HLA-A2 + . (E) Kaplan-Meier curve showing the per cent survival of CCRF-CEM-engrafted mice treated with PBS (black, n=5), CAR4-DNTs (red, n=6), CAR4-Ide-DNTs (green, n=5). Data are representative of two independently performed experiments. Log-rank test was used for statistical analysis. (F) DNT cell counts in the peripheral blood of mice 7 days after the last infusion with PBS, CAR4-DNTs and CAR4-Ide-DNTs (n = 3–4 for each treatment group). Column bars represent mean DNT cell count per milliliter of blood of mice, and error bars represent SEM. (G) Left: Representative flow plots for HLA-A2 and CD45 staining of blood cells from untreated, CAR4-DNT-treated, and CAR4-Ide-DNT-treated mice 26 days after CCRF-CEM engraftment. Right: CCRF-CEM cell counts in the peripheral blood of mice after treatment with PBS, CAR4-DNTs and Ide-CAR4-DNTs (n=3–4 for each treatment group). Each dot represents mean CCRF-CEM cell count per milliliter of blood of mice on each day after CCRF-CEM infusion and the error bars represent SEM. (H) Representative histograms showing CD4 staining of CCRF-CEM cells harvested from tissues from untreated, EV-DNT-treated, CAR4-DNT-treated, and CAR4-Ide-DNT-treated mice at the end point of the survival experiment. **p<0.01; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; CAR4, CD4-CAR; DNTs, double-negative T cells; HLA, human leukocyte antigen; Ide, idelalisib; IL, interleukin; PBS, phosphate-buffered saline; T-ALL, T-cell acute lymphoblastic leukemia; TCM, central memory T cell; TEM, memory T cells; TEMRA, terminally differentiated effector memory T cells; Tnaïve/SCM, naïve/stem cell memory T cell.

Article Snippet: Briefly, healthy donor-derived PBMCs were depleted of CD4 + and CD8 + cells using CD4-depletion and CD8-depletion cocktails (STEMCELL Technologies) and cultured on anti-CD3 antibody-coated plates (5 µg/mL; OKT3; Miltenyi Biotec) for 3–4 days in RPMI 1640 (Gibco) supplemented with 10% FBS and interleukin (IL)-2 (250 IU/mL; Proleukin, Novartis Pharmaceuticals).

Techniques: In Vitro, Transduction, Staining, Irradiation, Injection, Cell Counting, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells

doi: 10.1136/jitc-2023-007277

Figure Lengend Snippet: Molecular mechanism involved in EV-DNT-mediated and CAR4-DNT-mediated killing of T-cell malignancies. (A) An in vitro transwell assay was conducted with a 0.4 µm pore membrane that separates the top and bottom compartments. The target cells, CCRF-CEM and KARPAS-299, were cocultured with EV-DNTs (white bars) and CAR4-DNTs (black bars) in the top compartment, while only target cells were alone in the bottom compartment. Per cent specific killing of the target in each compartment is shown, and error bars represent SEM. The experiment was independently performed two times, and representative data are shown. Two-way ANOVA Sidak’s multiple comparison test was used for statistical analysis. (B) Representative flow plots showing expression of LFA-1 subunits, CD11a and CD18, NKG2D, DNAM-1, and granzyme B on EV-DNTs (blue) and CAR4-DNTs (red). Histograms are representative of two independent experiments. (C) An in vitro cytotoxicity assay was conducted using EV-DNTs (white bars) and CAR4-DNTs (black bars) against CCRF-CEM, primary T-ALL blasts, and KARPAS-299, in the presence of LFA-1, NKG2D, DNAM-1, or TNF-α blocking antibody, or isotype controls. EV-DNTs and CAR4-DNTs were cocultured with the targets for 24 hours. The effector-to-target ratio was 2:1 or 4:1 for EV-DNT cocultures and 0.2:1 for CAR4-DNT cocultures. Per cent specific killing of the target in the presence of each blocking antibody or the isotype control is shown. Error bars represent SEM. Each experiment was done in triplicates, and data are a combination of three independently performed experiments. One-way ANOVA Dunnett’s multiple comparisons test was used for statistical analysis. (D) EV-DNTs (white bars) and CAR4-DNTs (black bars) were pretreated with DMSO or 100 nM CMA for 30 min and cocultured with CCRF-CEM (left), KARPAS-299 (middle), or primary T-ALL blasts (right) for 2 hours at a 2:1 effector-to-target ratio. Per cent inhibition of specific killing of targets by CMA pretreated effector cells relative to the DMSO control is shown, and error bars represent SEM. Data are a combination of three independently performed experiments. Student’s t-test was used for statistical analysis. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; CAR4, CD4-CAR; CMA, concanamycin A; DMSO, dimethyl sulfoxide; DNAM-1, DNAX accessory molecule-1; DNTs, double-negative T cells; EV, empty-vector; LFA-1, lymphocyte function-associated antigen-1; NKG2D, natural killer group 2D; PTCL, peripheral T-cell lymphoma; T-ALL, T-cell acute lymphoblastic leukemia; TNF-α, tumor necrosis factor-α.

Article Snippet: Briefly, healthy donor-derived PBMCs were depleted of CD4 + and CD8 + cells using CD4-depletion and CD8-depletion cocktails (STEMCELL Technologies) and cultured on anti-CD3 antibody-coated plates (5 µg/mL; OKT3; Miltenyi Biotec) for 3–4 days in RPMI 1640 (Gibco) supplemented with 10% FBS and interleukin (IL)-2 (250 IU/mL; Proleukin, Novartis Pharmaceuticals).

Techniques: In Vitro, Transwell Assay, Membrane, Comparison, Expressing, Cytotoxicity Assay, Blocking Assay, Inhibition, Plasmid Preparation

Journal: bioRxiv

Article Title: In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer

doi: 10.1101/2022.03.13.484176

Figure Lengend Snippet: a , The effect of Adam2 overexpression in LLC cells on CD8 + OT-I T cell mediated killing. CTRL or Adam2 O/E cells labeled with CFSE were used as targets (T) and cocultured with day 4 activated and expanded OT-I CD8 T-cells (E) at different E:T ratios. Flow cytometry analysis was used to calculate the ratio of killed cells at different E:T ratios. Data presents mean ± s.e.m. of three replicates and analysed by two-sided student’s t-test. b , Tumor growth of transplanted CTLR or Adam2 O/E in B6 mice, untreated (n=5) or treated with OT-I T cells (n=5). c , Volcano plot showing differentially expressed genes between OT-I treated CTLR and Adam2 O/E tumors isolated from B6 mice. d , GSEA plots showing Adipogenesis and Peroxisomes pathways in CTLR tumors versus Adam2 O/E tumors. e and f , Bar graph showing changes of Gene Ontology of Biological Process (e) and Molecular Function (f) between OT-I treated CTLR versus Adam2 O/E tumors.

Article Snippet: DNTs were enriched by depleting CD4 + and CD8 + cells using RosetteSep™ human CD4- and CD8-depletion cocktails (Stemcell Technologies).

Techniques: Over Expression, Labeling, Flow Cytometry, Isolation