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: British Journal of Cancer

Article Title: Gemcitabine enhances the efficacy of reovirus-based oncotherapy through anti-tumour immunological mechanisms

doi: 10.1038/bjc.2013.695

Figure Lengend Snippet: Effect of Reo+GEM combination on T-cell functionality and survival, and tumour antigen presentation capacity. Splenocytes isolated from naive C57BL/6 mice were stimulated with Con A (2.5 μ g ml −1 ) and treated with Reo (1 MOI) and GEM (1 μ M ) alone or in combination for 24 h. ( A ) Splenocytes were harvested and stained with anti-CD3, anti-CD4, and anti-CD8 antibodies along with annexin-V and analysed by flow cytometry. ( B – D ) Alternatively, cells were stained for anti-CD4 ( B ) and anti-CD8 ( C ) antibodies along with anti-CD69 and anti-CD71 antibodies and analysed by flow cytometry. Bar graph represents a representative data from one of four independent experiments ( D ). Asterisks shown immediately on top of the bars signify the P- values obtained by comparing the Con A-stimulated groups against their respective Con A-stimulated counterparts, whereas asterisks shown above the horizontal lines display the P- values obtained through comparison between Con A-stimulated PBS control and other Con A-stimulated experimental groups as indicated. Asterisks represent the P- values obtained by comparing the PBS control groups at indicated time points with that of various experimental conditions at respective time points. Statistical analysis was performed with two-tailed, Student's t -test; ns= P >0.05; * P ⩽0.05; ** P ⩽0.01; *** P ⩽0.001. Error bars are defined as mean+s.d. and data are representative of four independent experiments.

Article Snippet: We have previously demonstrated that inhibition of T-cell responses using anti-CD8 or anti-CD4 depletion antibodies enhances reovirus spread and direct tumour oncolysis ( Hirasawa et al , 2003 ).

Techniques: Immunopeptidomics, Isolation, Staining, Flow Cytometry, Comparison, Control, Two Tailed Test

Journal: eLife

Article Title: Antigen receptor control of methionine metabolism in T cells

doi: 10.7554/eLife.44210

Figure Lengend Snippet:

Article Snippet: To generate Th1s, murine CD8 + T cells were depleted from lymph node preparations using CD8 depletion kit (EasySep, STEMCELL Technologies, UK).

Techniques: Activation Assay, Staining, Blocking Assay, Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Methylation, Adjuvant, Incubation, Software

Journal: Nature Communications

Article Title: Cytotoxic CD8 + T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling

doi: 10.1038/s41467-021-21737-9

Figure Lengend Snippet: A Representative examples of CD4 + and CD8 + T cell staining in the heart of C57BL/6J mice following coronary ligation (MI) or sham surgery. B Kinetic of CD8 + T cell infiltration in the myocardium, performed at days 0, 1, 3, 7, and 14 after surgery in MI (white circle, n = 9/12/12/5, respectively, at day 1/3/7/14) and sham-operated mice (black box, n = 6/time point); data are presented as mean ± SEM. C Immunostaining in the ischemic myocardium at day 3 after MI or sham showing CD8 + T infiltration (green) in the ischemic heart tissue, scale bar 40 μm. D Kinetic of CD4 + T cell infiltration in the myocardium, performed at days 0, 1, 3, 7, and 14 after surgery in MI (white circle, n = 10/9/12/5, respectively, at day 1/3/7/14) and sham-operated mice (black box, n = 6/time point); data are presented as mean ± SEM. E Flow cytometry characterization of CD8 + T cell subsets in the ischemic heart at days 1, 3, and 7 after MI including naïve (CD3 + CD8 + CCR7 High CD44 − ), effector memory (CD3 + CD8 + CCR7 Low CD44 + ), and central memory (CD3 + CD8 + CCR7 High CD44 + ) subsets. F Representative examples and quantitative analysis of CD8 + T cells expressing CD69 or CD107a within the ischemic heart tissue in MI (white circle, n = 6/9/12, respectively, at day 1/3/7) and sham-operated mice (black box, n = 6/time point); data are presented as mean ± SEM. G Immunostaining in the ischemic myocardium at day 3 after MI showing CD8 + T cells (green), and Granzyme B (Red) and merged area (Yellow), Inf for infarct area; scale bar 40 μm. H mRNA levels of Granzyme B within the injured myocardium on days 1, 3, and 7 after coronary ligation (gray, n = 5/4/5 at day 1/3/7) or sham (white, n = 4/8/6 at day 1/3/7). I C57Bl6 WT mice received intraperitoneal injection of isotype control (CTR, blue borderline, n = 4/time point) or anti-CD4 depleting monoclonal antibody (blue filled, n = 5/time point) (150 μg/mice) 1 day before coronary occlusion. CD4 + T cell depletion was confirmed in the heart by flow cytometry at day 1 and day 3 following MI. J , K Quantification and representative example of CD8 + T cell count in the heart of control ( n = 4/time point) or CD4-depleted ( n = 5/time point) mice at day 1 and day 3 after MI. P values were calculated using two-tailed Mann-Whitney test ( B , D , F , H , I , J ). Inf, infarct; MI, myocardial infarction; FMO, fluorescent minus one.

Article Snippet: In order to assess the role of CD4 + T cells in the recruitment of CD8 + T cells in ischemic cardiac muscle, C57bl6 WT mice were injected i.p. with a depleting rat anti-mouse CD4 monoclonal antibody (100 μg/mouse, Biotem Clone YTS 169.4) or with a rat IgG2b isotype control (R&D Systems; Abingdon, UK) 24 h before MI.

Techniques: Staining, Ligation, Immunostaining, Flow Cytometry, Expressing, Injection, Control, Cell Counting, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Cytotoxic CD8 + T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling

doi: 10.1038/s41467-021-21737-9

Figure Lengend Snippet: A Flow cytometry analysis of blood CD4 + T and CD8 + T subsets at day 1 after MI in control (PBS, CTR, n = 6), low (light gray, n = 5), and high (dark gray, n = 5) CD8-depleted groups. B Quantification of CD8 + T cell count in the blood at baseline, day 0, day 1, and day 3 after MI . C Representative picture and quantification of infarct size at day 14 in control, low, and high CD8-depleted groups. D Quantitative evaluation of left ventricle ejection fraction (Simpson) of CTR or CD8-depleted pigs. E , F Detection of CD8 + T cells (brown) in human heart biopsy of MI patients, using immunohistochemistry at day 3 ( E , upper) and day 8 ( F , lower) after MI, scale bar 50 μm. G Detection of GRANZYME B+ cells (brown) in human heart biopsy of MI patients, using immunohistochemistry, scale bar 20 μm. H Quantification of GRANZYME B+ cells (brown) in human heart biopsy of MI patients, using immunohistochemistry at different time points ( n = 9 day ≤ 7, n = 8 day ≥ 8). I Survival according to baseline circulating GRANZYME B level (< or > median value) in patients with acute MI ( n = 1046). High level of GRANZYME B at the admission for acute MI were independently predictive of death after 1 year of follow-up after multiple adjustments (see Methods and Supplementary Table ). HR = hazard ratio. P values were calculated using two-tailed Mann-Whitney test ( H ) or Kruskal - Wallis test ( B – D ).

Article Snippet: In order to assess the role of CD4 + T cells in the recruitment of CD8 + T cells in ischemic cardiac muscle, C57bl6 WT mice were injected i.p. with a depleting rat anti-mouse CD4 monoclonal antibody (100 μg/mouse, Biotem Clone YTS 169.4) or with a rat IgG2b isotype control (R&D Systems; Abingdon, UK) 24 h before MI.

Techniques: Flow Cytometry, Control, Cell Counting, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Proinflammatory allogeneic dendritic cells enhance the therapeutic efficacy of systemic anti-4-1BB treatment

doi: 10.3389/fimmu.2023.1146413

Figure Lengend Snippet: AlloDCs enhanced the therapeutic efficacy of α4-1BB antibody that dependent on CD8 + T-cells. (A) Schematic illustration of the experiment outline of the CT-26 model. (B) Mouse survival (Kaplan-Meier curve) in the CT-26 model after different treatments as indicated (PBS n=10, AlloDC n=14, α4-1BB n=15, α4-1BB/AlloDC n=10, α4-1BB/AlloDC/CD4 depletion n=6, α4-1BB/AlloDC/CD8 depletion n=6). (C, D) Tumor size in individual mouse and survival after re-challenge with CT-26 tumor. All mice that survived in (B) were re-challenged and none of them got tumor after re-challenge. The number of rechallenged mice from each group are labeled in figure panel. (E–J) Tumor size in individual mouse in different treatment groups as in (B) . (K) Schematic illustration of the experiment outline of the B16 model. (L) Mouse survival (Kaplan-Meier curve) in the B16 model after different treatments as indicated. (PBS n=10, AlloDC n=9, α4-1BB n=8, α4-1BB/AlloDC n=9). (M–P) Tumor size in individual mouse in different treatment groups as in (L) . The size of each group (n) is labeled in each panel below. All survival curves were compared using the Log-Rank test. (*P<0.05, **P<0.01, ****P<0.0001).

Article Snippet: CD4 depletion antibody (In Vivo Mab αmouse CD4, clone GK1.5, BioCell) and CD8 depletion antibody (In Vivo Mab αmouse CD8a, clone 2.43, BioCell) were injected (i.p) on day 13, 14, 15, 20, 21 and 22.

Techniques: Drug discovery, Labeling

Journal: Frontiers in Immunology

Article Title: Proinflammatory allogeneic dendritic cells enhance the therapeutic efficacy of systemic anti-4-1BB treatment

doi: 10.3389/fimmu.2023.1146413

Figure Lengend Snippet: AlloDCs boost the lymphoid compartment signature for α4-1BB/AlloDC therapies. (A) The experimental setup is depicted in a schematic illustration. (B) The comparison between the PBS-treated group and the grouped samples based on up- or down-regulation is presented as the sum of pathway signature scores determined from NanoString mRNA profiling. (C) The radar map displays the pathway scores from different treatment groups. (D, E) The heatmap showcases the top 15 differentially expressed genes from lymphoid compartment pathways and cell proliferation. (F) The abundance of tumor-infiltrating immune cells in each mouse from different treatment groups is presented as cell-type scores from NanoString mRNA profiling. Error bars represent SEM and the mean values were compared using one-way ANOVA nonparametric test. (*P<0.05, **P<0.01).

Article Snippet: CD4 depletion antibody (In Vivo Mab αmouse CD4, clone GK1.5, BioCell) and CD8 depletion antibody (In Vivo Mab αmouse CD8a, clone 2.43, BioCell) were injected (i.p) on day 13, 14, 15, 20, 21 and 22.

Techniques: Comparison

Journal: Frontiers in Immunology

Article Title: Proinflammatory allogeneic dendritic cells enhance the therapeutic efficacy of systemic anti-4-1BB treatment

doi: 10.3389/fimmu.2023.1146413

Figure Lengend Snippet: AlloDCs combined with α4-1BB treatment attract CD8 + T-cell populations with activated phenotype by enhanced DC activation. (A) The ratio of CD8+ and CD4+ T-cells in the tumor-infiltrating T-cell population (gated as CD3+ T-cells) is analyzed using flow cytometry. (B) The percentage of IFNγ + CD8 + T-cells in the tumor is measured. (C) The percentage of CD107a + CD8 + T-cells in the tumor is determined. (D–G) The percentage of phenotypically exhausted CD8+ T-cells in the tumor is assessed, represented as triple, double, and single positive for PD-1 + , Tim3 + and LAG3 + markers on CD8 + T cells. (H) Percentage of tumor infiltrating tissue-resident memory (T RM ) CD8 + T-cells (gated as CD49a + CD103 + T-cells out of CD69 + CD8 + T-cells) in each treatment group, analyzed by flow cytometry. (I) Representative density plot showing tumor infiltrating T RM in each treatment group. (J) Percentage of tumor-reactive CD8 + T-cells (CD39 + CD103 + out of CD8 + T-cells) in tumor samples from different treatment groups. (K) IFN-γ expression level in the supernatant of in vitro cultured splenocytes, harvested from each treatment group and re-stimulated with either gp70 peptides or non-relevant peptides. (L) Percentage of the tumor-infiltrating DCs (CD11C + F4/80 - out of CD11b + CD45 + cells) in each treatment group. (M, N) The expression of CD103a and CD8a on the tumor infiltrating DCs. (O, P) Percentage of the CCL5 + and IL12 + tumor-infiltrating DCs in each treatment group. Error bars represent SEM and the mean values were compared using one-way ANOVA nonparametric test. (*P<0.05, **P<0.01).

Article Snippet: CD4 depletion antibody (In Vivo Mab αmouse CD4, clone GK1.5, BioCell) and CD8 depletion antibody (In Vivo Mab αmouse CD8a, clone 2.43, BioCell) were injected (i.p) on day 13, 14, 15, 20, 21 and 22.

Techniques: Activation Assay, Flow Cytometry, Expressing, In Vitro, Cell Culture