Journal: Cancer Research

Article Title: A Novel Small Molecule Inhibitor of Signal Transducers and Activators of Transcription 3 Reverses Immune Tolerance in Malignant Glioma Patients

doi: 10.1158/0008-5472.can-07-1243

Figure Lengend Snippet: Figure 4. WP1066 enhances phosphorylation of intracellular signaling molecules. Monocytes and Tcells isolated from the PBMCs of GBM patients were incubated with either the medium or medium supplemented with 5 Amol/L WP1066. After 2 h, monocytes and T cells were stimulated for 5 min with 2 Ag/mL LPS and 5 Ag/mL anti-CD3 antibody, respectively. Subsequently, cells were lysed, electrophoretically fractionated in 8% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with anti-phosphotyrosine monoclonal antibody 4G10. A, for monocyte membranes, subsequently the same membrane was stripped and reblotted with antibodies to phosphorylated p72Syk (Tyr352), Syk, phosphorylated Hck, Hck, and Lyn. B, for the T-cell membranes, after stripping, the membrane was reblotted with antibodies to phosphorylated ZAP-70 (Tyr319), ZAP-70, p56-Lck, and h-actin.

Article Snippet: T cells from the same patients were isolated with human CD3 microbeads and a magnetic separation column (LS MACS column, Miltenyi Biotec) with the exception of one patient who lacked sufficient PBMCs for both monocyte and T-cell isolations.

Techniques: Phospho-proteomics, Isolation, Incubation, Membrane, Stripping Membranes

Journal: Journal for Immunotherapy of Cancer

Article Title: TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant

doi: 10.1136/jitc-2024-010239

Figure Lengend Snippet: 15–4P Stimulation increases NKG2D expression and murine NK cell proliferation compared with IL-15/IL-15Rα stimulation alone. ( A ) Schematic showing AgN2a-4P and NK cell receptor-ligand interactions. B6 NK cells are isolated and cultured with IL-15/IL-15Rα conjugate and with or without irradiated AgN2a-4P cells at a 1:1 ratio for 12 days. All expression markers were examined on CD3 − NK1.1 + NK cells. ( B ) Dot plots of NKG2D (top) and NKp46 (bottom) on IL-15 NK cells (left) and 15–4P stimulated NK cells (right) are shown with percentage of gated cells. ( C ) Per cent of NK cells positive for NKG2D (left) and NKp46 (right) and MFI below. ( D ) Fold change of IL-15 NK cells and 15–4P stimulated NK cell expansion at day 12 compared with day 0. ( E ) Dot plots of proliferation marker Ki-67 on IL-15 NK cells (left) and 15–4P stimulated NK cells (right) are shown with ( F ) percentage and MFI. ( G ) Percentage and MFI of TRAIL (top) and Fas-L (bottom) were assessed for each group. Data are representative of an experiment that was replicated two times. Representative dot plots examples of one replicate out of a minimum of three replicates. All bar graphs show individual experimental replicates plotted with mean and SEM (n=3–5). Two-sided two-sample t-tests were performed where indicated (*p<0.05, **p<0.01, ***p<0.001, ns=not significant). AgN2a, aggressive variant of the Neuro-2a murine neuroblastoma cell line; MFI, median fluorescence intensity; NK, natural killer.

Article Snippet: T cells were depleted using a CD3 MicroBead Kit (Miltenyi Biotec) by negative selection on the AutoMACs Pro Separator (Miltenyi Biotec) at day+0.

Techniques: Expressing, Isolation, Cell Culture, Irradiation, Marker, Variant Assay, Fluorescence

Journal: Cancer Immunology Research

Article Title: A Bispecific γδ T-cell Engager Targeting EGFR Activates a Potent Vγ9Vδ2 T cell–Mediated Immune Response against EGFR-Expressing Tumors

doi: 10.1158/2326-6066.cir-23-0189

Figure Lengend Snippet: Figure 3. PB and tumor-infiltrating Vg9Vd2 T cells of cancer patients can be activated by the EGFR-Vd2 bsTCE to trigger autologous tumor cell lysis. A, Specific lysis of tumor cells (relative to tumor alone condition) after a 7-day coculture of dissociated tumor cells (peritoneal metastases of colorectal cancer: n ¼ 3) with autologous PBMCs (5:1 E:T ratio) and 50 nmol/L EGFR-Vd2 bsTCE. B, CD25 expression on lymphocyte subsets after a 7-day culture of patient-dissociated peritoneal colorectal cancer metastases and autologous PBMC (5:1 E:T ratio, n ¼ 5) 50 nmol/L EGFR-Vd2 bsTCE. C, CD107a expression on tumor-infiltrating Vg9Vd2 T cells (primary colorectal cancer: n ¼ 6, peritoneal colorectal cancer metastases: n ¼ 4 and liver colorectal cancer metastases: n ¼ 3) after a 4-hour incubation 50 nmol/L EGFR-Vd2 bsTCE. D, Specific lysis (relative to tumor alone condition) of tumor cells after a 24-hour culture of patient-dissociated tumor cell suspensions (primary colorectal cancer: n ¼ 4 and peritoneal colorectal cancer metastases: n ¼ 3) with autologous tumor-infiltrating total T-cell fractions (TI-T cells, 1:1 E:T ratio) 50 nmol/L EGFR-Vd2 bsTCE. Data are all generated through flow cytometry and represent mean and SEM , P < 0.05; , P < 0.01. Paired t test (A–D).

Article Snippet: Autologous patient tumor cell lysis (tumor cells were identified as EpCAMþ/dimCD45 cells, and lysis was quantified using 7AAD and 123counting eBeads) was assessed (using flow cytometry) by coculturing colorectal cancer patient PBMCs or MACS-purified (CD3 REAlease kit, 130-121-562; Miltenyi Biotec) tumor-infiltrating T-cell fractions (purity >80%) enriched from primary or peritoneal metastatic colorectal cancer single-cell suspensions with matched patient tumor cells 50 nmol/L EGFR-Vd2 bsTCE for 24 hours (tumor-infiltrating T cells; 1:1 E:T ratio) or 7 days (PBMC; 5:1 E:T ratio).

Techniques: Lysis, Expressing, Incubation, Generated, Cytometry

Journal: Cancer Immunology Research

Article Title: A Bispecific γδ T-cell Engager Targeting EGFR Activates a Potent Vγ9Vδ2 T cell–Mediated Immune Response against EGFR-Expressing Tumors

doi: 10.1158/2326-6066.cir-23-0189

Figure Lengend Snippet: Figure 4. EGFR-Vd2 bsTCE–activated Vg9Vd2 T cells exert tumor preferential activity. A, CD107a expression on Vg9Vd2 T cells present in nonmalignant colon (n ¼ 3), peritoneum (n ¼ 3), and liver tissue (n ¼ 3) after a 4-hour incubation 50 nmol/L EGFR-Vd2 bsTCE. B, Specific lysis (relative to tissue alone condition) of malignant and nonmalignant cells after a 4-hour culture of dissociated tumor cell suspensions (primary colorectal cancer, n ¼ 10; peritoneal colorectal cancer metastases, n ¼ 5; liver colorectal cancer metastases, n ¼ 3; primary HNSCC, n ¼ 5; and primary NSCLC, n ¼ 4) or dissociated nonmalignant cell suspensions (colon tissue, n ¼ 3; peritoneum tissue, n ¼ 4; and liver tissue, n ¼ 3) with healthy donor–derived Vg9Vd2 T cells (1:1 E:T ratio) 50 nmol/L EGFR-Vd2 bsTCE. C, Expression of EGFR and BTN3A on dissociated tumor [primary colorectal cancer, n ¼ 9 (EGFR) and n ¼ 8 (BTN3A); peritoneal colorectal cancer metastases; n ¼ 16; liver colorectal cancer metastases, n ¼ 3; and primary esophageal cancer, n ¼ 15] and nonmalignant tissue (colon, n ¼ 4; peritoneum, n ¼ 10; liver, n ¼ 3; and esophageal, n ¼ 8) derived from cancer patients. D, CD107a expression on expanded Vg9Vd2 T cells (n ¼ 6) after a 4-hour coculture with WiDr tumor cells (1:1 E:T ratio) or Vg9Vd2 T cells in PBMCs of patients with peritoneal colorectal cancer metastases (n ¼ 4) after a 4-hour coculture with matched autologous tumor cells suspensions (5:1 E:T ratio). Where indicated, Vg9Vd2 T cells were preincubated with 10 mg/mL anti-Fc receptor and/or NKG2D-specific blocking Ab, and tumor cells were preincubated 10 mg/mL BTN3A-specific blocking Ab or EGFR-specific blocking Ab 10 pmol/L EGFR-Vd2 bsTCE. Data are all generated through flow cytometry and represent mean and SEM (B), mean (C) and box and whisker plots indicate the median, 25th to 75th percentiles and minimum to maximum (D). , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. Paired t test (A, B) and one-way ANOVA with Dunnet multiple comparisons test (C, D).

Article Snippet: Autologous patient tumor cell lysis (tumor cells were identified as EpCAMþ/dimCD45 cells, and lysis was quantified using 7AAD and 123counting eBeads) was assessed (using flow cytometry) by coculturing colorectal cancer patient PBMCs or MACS-purified (CD3 REAlease kit, 130-121-562; Miltenyi Biotec) tumor-infiltrating T-cell fractions (purity >80%) enriched from primary or peritoneal metastatic colorectal cancer single-cell suspensions with matched patient tumor cells 50 nmol/L EGFR-Vd2 bsTCE for 24 hours (tumor-infiltrating T cells; 1:1 E:T ratio) or 7 days (PBMC; 5:1 E:T ratio).

Techniques: Activity Assay, Expressing, Incubation, Lysis, Derivative Assay, Blocking Assay, Generated, Cytometry, Whisker Assay

Journal: Science Advances

Article Title: Laminin γ2–mediating T cell exclusion attenuates response to anti–PD-1 therapy

doi: 10.1126/sciadv.abc8346

Figure Lengend Snippet: ( A ) Representative images of computed tomography scans and IHC staining in patients with NSCLC treated with anti–PD-1 (nivolumab) therapy showed significantly higher Ln-γ2 expression in tumor tissues from progressors ( n = 36) than in tumor tissues from responders ( n = 35). In the left panel, NSCLC tumors are indicated by red dotted lines. H, heart; A, aorta; T, tumor; S, stroma. Scale bar, 100 μm. ( B ) IHC staining with an antibody against CD3 to detect T cells in NSCLC tissues. Blue arrows indicate CD3 + cells in the tumor. Scale bar, 100 μm. ( C ) Apoptotic cells in NSCLC tissues from progressors and responders were detected using TUNEL staining. Blue arrows indicate the TUNEL + cells. Scale bar, 100 μm. ( D ) Representative images of IHC staining for low or high Ln-γ2 expression in NSCLC tissues. Scale bar, 200 μm. ( E ) The correlation analysis revealed a positive correlation between high Ln-γ2 expression and the tumor size in patients with NSCLC (total n = 104). ( F ) Survival curves indicated that high expression of Ln-γ2 ( n = 46) predicted shorter survival of patients with NSCLC compared with patients with low expression of Ln-γ2 ( n = 58). The data presented in (A) and (E) were analyzed using Pearson’s chi-square (Fisher’s exact) test, statistical analyses of the data presented in (B) and (C) were performed using unpaired two-tailed t test with Welch’s correction, and the data presented in (F) were analyzed with the log-rank (Mantel-Cox) test. In all panels, *** P < 0.001.

Article Snippet: T cells were separated from PBMCs using immunomagnetic beads coated with an antibody against CD3 (#130-111-551, Miltenyi Biotec).

Techniques: Computed Tomography, Immunohistochemistry, Expressing, TUNEL Assay, Staining, Two Tailed Test

Journal: Science Advances

Article Title: Laminin γ2–mediating T cell exclusion attenuates response to anti–PD-1 therapy

doi: 10.1126/sciadv.abc8346

Figure Lengend Snippet: ( A ) Double IF staining for Ln-γ2 (green) and CD3 (T cells, red) in NSCLC and ESCC tissues. Cell nuclei were counterstained with DAPI (blue). The boundary of tumor and stroma was marked with white dotted lines. Scale bar, 100 μm. The proportions of these T cells in tumor nests were counted in tumor tissues with high or low expression of Ln-γ2. ( B ) Schematic of the T cell chemotaxis assay. ( C ) Representative images of migratory T cells (green) labeled with CFSE. Scale bar, 20 μm. The number of migratory T cells was counted in the right panel. ( D ) RNA-seq was conducted to explore the gene expression in T cells regulated by Ln-γ2. ( E ) Heat map showing that Ln-γ2 significantly regulated the expression of T cell receptor (TCR) family genes. −, downregulation; +, upregulation. ( F ) These TCR genes were located in the TCR α chain (TRA). ( G ) Quantitative real-time PCR (qRT-PCR) confirmed the regulation of TCR gene expression by Ln-γ2 and conditioned media from A549 cells alone or cocultured with CAFs. In (A) and (C), statistical analyses were performed using the unpaired two-tailed t test with Welch’s correction. Data are presented as means ± SEM; *** P < 0.001. ns, no significant difference.

Article Snippet: T cells were separated from PBMCs using immunomagnetic beads coated with an antibody against CD3 (#130-111-551, Miltenyi Biotec).

Techniques: Staining, Expressing, Chemotaxis Assay, Labeling, RNA Sequencing, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

Journal: Science Advances

Article Title: Laminin γ2–mediating T cell exclusion attenuates response to anti–PD-1 therapy

doi: 10.1126/sciadv.abc8346

Figure Lengend Snippet: ( A ) The mouse Lewis lung cancer cell line LLC (2 × 10 5 cells per mouse) or primary ESCC cell line MEC2 (4 × 10 6 cells per mouse) in which Ln-γ2 was silenced by a short hairpin RNA was subcutaneously (s.c.) transplanted into C57BL/6 mice ( n = 5 mice per group), and tumor volumes were measured weekly for 4 weeks. ( B ) Double IF staining with antibodies against Ln-γ2 (green) and CD3 (red) was performed on transplanted tumors, and the proportions of CD3 + T cells in the tumor and stroma were summarized in the right panel. Scale bar, 100 μm. ( C ) Combination therapies, including galunisertib (Gal; 100 mg/kg, i.g.), anti–PD-1 (5 mg/kg, i.p.), and chemotherapeutic drugs, paclitaxel (PTX; 6 mg/kg, i.p.) and carboplatin (CBP; 6 mg/kg, i.p.), were administered to tumor-bearing mice every other day for five treatments. ( D ) Tumor volumes were measured every week for 8 weeks. ( E ) The expression of the Ln- γ 2 mRNA in tumors from mice subjected to different treatments was analyzed using qRT-PCR. ( F ) The proportions of tumor- and stroma-infiltrating CD3 + T cells were summarized on the basis of the results of double IF staining. In (A), (D), and (E), statistical analyses were performed using unpaired two-tailed t tests with Welch’s correction, and the data presented in (B) and (F) were analyzed with Pearson’s chi-square (Fisher’s exact) tests. In all panels, data are presented as means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, no significant difference.

Article Snippet: T cells were separated from PBMCs using immunomagnetic beads coated with an antibody against CD3 (#130-111-551, Miltenyi Biotec).

Techniques: shRNA, Staining, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Cell Metabolism

Article Title: The mitochondrial pyruvate carrier regulates memory T cell differentiation and antitumor function

doi: 10.1016/j.cmet.2022.03.013

Figure Lengend Snippet:

Article Snippet: StraightFrom BuffyCoat REALease CD3 microbeads , Miltenyi , Cat# 130-127-142.

Techniques: Staining, Recombinant, Virus, Bacteria, Expressing, Red Blood Cell Lysis, Selection, Purification, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Magnetic Beads, Western Blot, Sequencing, Software

Journal: Cell reports

Article Title: Mass Cytometry Analysis Reveals that Specific Intratumoral CD4 + T Cell Subsets Correlate with Patient Survival in Follicular Lymphoma

doi: 10.1016/j.celrep.2019.01.085

Figure Lengend Snippet: (A) The tSNE maps of CD3 + , CD4 + , and CD8 + subsets from tonsil, reactive lymph node (rLN) and spleen (rSP) tissues, and FL biopsy specimens. The t-SNE analysis was performed on a concatenated file (31 FL, 6 tonsils, 3 rLNs, or 4 rSPs) using escalated parameter (surface makers). (B) Plots of principal-component analysis of CD3 + T cells from tonsil, rLN, rSP tissues, and FL biopsy specimens. (C) Plots from a representative sample of tonsil, rLN, rSP, or FL showing expression of CD45RO and CCR7. Graphs show the percentages of naive, central memory, effector memory, and terminally differentiated T cells from CD3 + T cells in tonsil, rLN, rSP, and FL. p value indicates a comparison between Tonsil and FL. See also .

Article Snippet: CD3 + or CD4 + T cells were isolated using positive selection with CD3 or CD4 microbeads kit (StemCell Technologies, Vancouver, Canada).

Techniques: Expressing, Comparison

Journal: Cell reports

Article Title: Mass Cytometry Analysis Reveals that Specific Intratumoral CD4 + T Cell Subsets Correlate with Patient Survival in Follicular Lymphoma

doi: 10.1016/j.celrep.2019.01.085

Figure Lengend Snippet: (A) Plots from a representative sample of FL showing expression of PD-1 on CD3 + , CD4 + , or CD8 + T cells. (B) The tSNE map of PD-1 + cells from CD3 + concatenated file (31 FL). (C) Graphs showing the percentages of PD-1 high or PD-1 low cells from CD4 + or CD8 + T cells from 31 FL specimens. (D) The tSNE maps of CD4 + T cells from a representative FL specimen showing the gating strategy to identify PD-1 + subsets . PD-1 expression on subsets S3–S6 in red circles was shown. (E) Graphs showing the percentages of surface markers from S3, S4, S5, or S6 in FL. (F) The tSNE maps of S3–S6 from the concatenated file of 31 FL specimens. (G) Kaplan-Meier curves for overall survival of FL patients (n = 31) by the number of total PD-1 + cells, S3, S4, S5, or S6 with a cutoff of 65.4%, 5.92%, 2.86%, 3.73%, or 14%, respectively. See also .

Article Snippet: CD3 + or CD4 + T cells were isolated using positive selection with CD3 or CD4 microbeads kit (StemCell Technologies, Vancouver, Canada).

Techniques: Expressing

Journal: Cell reports

Article Title: Mass Cytometry Analysis Reveals that Specific Intratumoral CD4 + T Cell Subsets Correlate with Patient Survival in Follicular Lymphoma

doi: 10.1016/j.celrep.2019.01.085

Figure Lengend Snippet: (A) The tSNE map of CD27 or CD28 + cells from CD3 + concatenated file (31 FL and 6 tonsils). CD4 + or CD8 + T cells were gated based on expression levels from CD4 or CD8 staining. (B) Graphs showing the percentages of CD27 − or CD28 − or CD27 − CD28 − cells from CD3 + T cells from 31 FL specimens and 6 tonsil tissues. (C) Graphs showing the percentages of CD27 − , CD28 − , or CD27 − CD28 − cells from CD25 − , CD25 + , PD-1 high , PD-1 low , PD-1 neg , T N , T CM , T EM , or T EMRA cells from 31 FL specimens. (D) Graphs showing the percentages of surface markers from CD27 − , CD27 + , CD28 − , or CD28 + cells from 31 FL specimens. (E and F) Kaplan-Meier curves for overall survival of FL patients (n = 31) by the number of CD3 + CD27 − , CD3 + CD28 − , CD3 + CD27 − CD28 − (E), CD4 + CD25 + , CD4 + CD25 + CD27 − , CD4 + PD-1 + , or CD4 + PD-1 + CD27 − (F) T cells. Bottom: p value indicates a comparison between T N and T EMRA . See also .

Article Snippet: CD3 + or CD4 + T cells were isolated using positive selection with CD3 or CD4 microbeads kit (StemCell Technologies, Vancouver, Canada).

Techniques: Expressing, Staining, Comparison

Journal: Cell reports

Article Title: Mass Cytometry Analysis Reveals that Specific Intratumoral CD4 + T Cell Subsets Correlate with Patient Survival in Follicular Lymphoma

doi: 10.1016/j.celrep.2019.01.085

Figure Lengend Snippet: (A) Heatmap showing clustering results for differentially expressed markers on CD3 + T cells from 31 FL specimens. Clustering was performed by Cluster 3.0 software. Each vertical column represents a patient sample. (B) Plot showing clustering results from FL patients divided by 2 groups (alive versus dead). Circles in red represent clusters that differed between two groups. Number in circles indicates a cluster ID. Clustering was performed by CITRUS from Cytobank. (C) Histogram plots in corners showing expression of selected markers by cells from 4 parent clusters overlapped to background. Expression level of each selected marker was expressed by Cluster (red) over Background (light blue). Graphs on the right showed quantitative results of abundance from 4 parent clusters between groups of Alive and Dead. The histograms and graphs were generated by CITRUS from Cytobank. (D) Kaplan-Meier curves for overall survival of FL patients (n = 31) by the clusters 21536 and 21523 using a cutoff point of abundance. See also .

Article Snippet: CD3 + or CD4 + T cells were isolated using positive selection with CD3 or CD4 microbeads kit (StemCell Technologies, Vancouver, Canada).

Techniques: Software, Expressing, Marker, Generated

Journal: Cell reports

Article Title: Mass Cytometry Analysis Reveals that Specific Intratumoral CD4 + T Cell Subsets Correlate with Patient Survival in Follicular Lymphoma

doi: 10.1016/j.celrep.2019.01.085

Figure Lengend Snippet: (A) Histograms showing CD27 or CD28 expression of CD27 + CD28 + T cells cultured in anti-CD3-coated plate plus anti-CD28 Ab for 2 or 5 days (activated). Cells cultured in uncoated plate were used as control (resting). (B) CD27 or CD28 expression from CD4 + T cells cultured in anti-CD3-coated plate plus anti-CD28 Ab in the presence or absence of TGF-β for 3 days. (C) Immunohistochemistry showing CD70 expression from FL (n = 8) and tonsil tissue. (D) Plots showing CD70 expression on CD19 + cells from representative lymphoma (n = 11) and tonsil tissues. (E) Histograms showing (i) CD70 expression on DoHH2 cells, (ii) expression of CD27 and CD28 on T cells co-cultured with DoHH2 cells for 3 days, (iii) CD70 expression of DoHH2 cells treated with either anti-CD70 blocking Ab or mIgG for 2 h, and (iv) expression of CD27 on CD4 + T cells co-cultured with DoHH2 cells pretreated with either anti-CD70 blocking Ab or mIgG for 3 days. (F) Histograms showing CFSE staining of CD27 + CD28 + , CD27 + CD28 − , CD27 − CD28 + , or CD27 − CD28 − T cells cultured in anti-CD3-coated plate plus anti-CD28 Ab for 3 days. Graph shows the numbers of CFSE dim cells of these subsets; n = 4. (G) Plots showing CD45RO and CCR7 expression from CD4 + CD27 − or CD4 + CD27 + T cells. Cells in box represent terminally differentiated T cells (CD45RO − CCR7 − ; T EMRA ). See also .

Article Snippet: CD3 + or CD4 + T cells were isolated using positive selection with CD3 or CD4 microbeads kit (StemCell Technologies, Vancouver, Canada).

Techniques: Expressing, Cell Culture, Control, Immunohistochemistry, Blocking Assay, Staining

Journal: Cell reports

Article Title: Mass Cytometry Analysis Reveals that Specific Intratumoral CD4 + T Cell Subsets Correlate with Patient Survival in Follicular Lymphoma

doi: 10.1016/j.celrep.2019.01.085

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD3 + or CD4 + T cells were isolated using positive selection with CD3 or CD4 microbeads kit (StemCell Technologies, Vancouver, Canada).

Techniques: Selection, Recombinant, Blocking Assay, Software