Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Splenocytes (A-D) and tumor-infiltrating lymphocytes (TIL) (E-H) were isolated from HIS mice 16-18 days after tumor implantation and analyzed by flow cytometry. A, frequency of splenic T cells in tumor-bearing or naïve HIS mice; parent population refers to frequency (%) of CD3 + T cells within human CD45 + cells and CD4 + and CD8 + T cells within CD3 + T cells. B , CD8 + T cell differentiation defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ) in tumor-bearing or naïve HIS mice. C-D , expression of indicated markers on CD8 + T cells from spleen of tumor-bearing or naïve HIS mice. E, frequency of CD4 + and CD8 + T cells within TILs, gated on total CD3 + T cells. F, CD8 + T cell differentiation within TILs. G-H, expression of indicated markers on CD8 + T cells within TILs. I, expression of CD137 on splenic CD8 + and CD4 + T cells of tumor-bearing or naïve HIS mice. J, expression of PD-1 and CD137 on splenic CD8 + T cells of tumor-bearing mice. K , expression of PD-1 on splenic CD8 + T cells based on CD137 in tumor-bearing HIS mice or bulk CD8 + T cells from naïve HIS mice. L, CD8 + T cell differentiation in spleen of tumor-bearing HIS mice. M-O , expression of indicated markers on splenocyte-derived CD8 + T cell populations based on expression of CD137 and PD-1. Data are pooled from at least 3 independent experiments, n=10-23 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by paired t-test, one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Isolation, Tumor Implantation, Flow Cytometry, Cell Differentiation, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation, expansion and characterization of T cells from HIS mice bearing autologous LCL tumors. B , fold expansion of FACS sorted splenic CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). C , CD8 + T cell differentiation after ex vivo expansion defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ). D, IFN-ψ ELISpot of expanded T cells in 5:1 (E:T) co-culture with autologous LCL tumor cells for 24 hours. Spot count is normalized to the spots produced by expanded CD8 + bulk T cells from naïve HIS mice. E , TNF⍺ ELISA of supernatant of expanded T cells in co-culture (5:1, E:T) with autologous LCL tumor cells for 24 hours. TNF⍺ concentration is normalized to the TNF⍺ secretion from expanded CD8 + bulk T cells derived from naïve mice. Data are pooled from at least 3 independent experiments, n=6-19 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by mixed-effects analysis (paired), RM one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Cell Differentiation, Ex Vivo, Enzyme-linked Immunospot, Co-Culture Assay, Produced, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Transcriptome analysis and pathway analysis of expanded CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). A, Upset plot (intersect) showing number of differentially expressed genes between groups in bulk RNAseq. p(FDR) < 0.05, log2 FC > 1.5. B , PCA plot of RNAseq showing PC1 and PC2. C, Top 50 upregulated and D, downregulated genes of T cell subsets based on the DEG between CD137 + vs. CD137 - PD1 - CD8 + T cells. p(FDR) < 0.05, log2 FC > 1.5. E, Volcano plot showing DEG between CD137 + and CD137 - PD1 - CD8 + T cells with genes of interest highlighted in yellow. F , differential expression of genes of interest between groups. G , Overrepresentation analysis (ORA) of upregulated pathways in CD137 + CD8 + T cells. H , Gene set enrichment analysis (GSEA) of signatures described on the y-axis. Gene ratio (# genes related to GO term / total number of sig genes) is displayed on the x-axis. Signatures with an adjusted p-value <0.05 are highlighted with a red box. Shown are data from 5 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , total number of individual clonotypes found per population. Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. B, TCR (CDR3 of TRA , TRB , TRG and TRD ) sequence sample diversity estimation using Hill numbers method, with Q=1 describing the Shannon diversity. C, rare clonal proportion showing the occupied repertoire space by clonotypes with defined counts (1, 2-3, 4-10, etc.). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. D, relative abundance of clonotypes with defined frequencies (size). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. E, repertoire overlap analysis cross-comparing every population from every experiment (each with different donor). F , repertoire overlap comparing the repertoire of bulk CD8 + T cells from naïve HIS mice to the populations from tumor-bearing HIS mice from individual experiments (each with different donor; data from individual experiments are indicated by different symbols). Mixed effects analysis with Tukey’s multiple comparisons test. G , tracking of clonotypes over populations. The top 10 most abundant clonotypes of the TCR repertoire of CD137 + CD8 + T cells from one representative experiment are shown. H , proportion of the top 10 most abundant clonotypes (from repertoires of CD137 + CD8 + T cells) in the repertoire of all populations, correlated with the spot count of IFN-ψ ELISpot. Shown are data from 4 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Sequencing, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation of tumor-reactive T cells and subsequent ACT. NSG mice were injected with 2 x 10 LCL s.c. in the flank and after three days, 10 x 10 ex vivo expanded T cells were adoptively transferred intravenously. Transferred T cells and LCL tumors were autologous to each other. B , tumor volume on the day of sacrifice in NSG recipient mice after ACT of the indicated cell populations. C, waterfall plot of tumor size in NSG recipient mice of ACT on the day of sacrifice relative to the tumor volume of control mice (no ACT). Bars depict individual mice. D , frequency of CD3 + T cells (% of human CD45 + cells) in TIL from NSG mice after ACT of the indicated cell populations, measured by flow cytometry. E , frequency of CD8 + T cells (% of total cells) in tumors of NSG mice after ACT of the indicated cell populations, measured by immunohistochemistry. F , differentiation of CD8 + T cells in TIL of NSG mice after ACT of the indicated cell populations defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ) and T EMRA (CD45RA + CD62L - ). G, correlation between tumor volume and infiltration of CD8 + T cells (measured by IHC) in tumors of NSG mice after adoptive transfer of CD137 + CD8 + T cells. H , schematic of ACT. CD137 + CD8 + T cells, CD137 - CD8 + T cells and CD137 - PD-1 - CD8 + T cells were isolated from spleen of tumor-bearing HIS mice or bulk CD8 + T cells from spleen of naïve HIS mice and expanded ex vivo . Recipient HIS mice were injected with 2 x 10 LCL s.c. in the flank and after three days, ex vivo expanded T cells were adoptively transferred intravenously. Tumor-bearing HIS recipient mice received 2 x 10 T cells without prior conditioning/lymphodepletion. Donor and recipient HIS mice as well as LCL were autologous to each other. I, tumor volume on the day of sacrifice in HIS recipient mice after ACT of the indicated cell populations. J, waterfall plot of tumor size on the day of sacrifice of HIS mice receiving ACT relative to the tumor volume of control HIS mice (no ACT). Bars depict individual mice. B-G: Data are pooled from 2-3 independent experiments, n=6-13 per group. I, data are pooled from 2-3 independent experiments (n=6-13 per group); J, data are from 1-3 independent experiments (n=3-13 per group). For each experiment, a with different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by one-way ANOVA. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Injection, Ex Vivo, Control, Flow Cytometry, Immunohistochemistry, Adoptive Transfer Assay, Isolation

Journal: Science Immunology

Article Title: Severely ill COVID-19 patients display impaired exhaustion features in SARS-CoV-2-reactive CD8 + T cells

doi: 10.1126/sciimmunol.abe4782

Figure Lengend Snippet: ( A ) Study design overview. ( B ) Representative FACS plots displaying surface staining of CD137 and CD69 in post-enriched CD8 + memory T cells, stimulated for 24 hours with SARS-CoV-2 peptide pools, from COVID-19 patients with mild and severe illness (left), and summary of the number of cells sorted per million PBMC (right); data are displayed as median with interquartile ranges for 17 and 22 patients with mild and severe COVID-19 disease, respectively. ( C) Representative FACS plots (left) showing surface expression of PD-1 in CD8 + memory T cells ex vivo (without in vitro stimulation) and in CD137 + CD69 + CD8 + memory T cells following stimulation, post-enrichment (CD137-based) and corresponding summary plots (right) showing proportion of PD-1 expressing cells in each study subject ( P = 0.26, unpaired t -test); data are displayed as median with interquartile ranges for 17 mild and 22 severe COVID-19 patients, respectively. *** P <0.001 by Mann-Whitney test ( B ).

Article Snippet: For subsequent magnetic-based enrichment of CD137 + cells, cells were sequentially stained with human serum IgG (Sigma Aldrich) for FcR block, cell viability dye (eFluor780/APC.Cy7, eBioscience), fluorescence-conjugated antibodies, Cell-hashtag TotalSeq-C antibody (0.5 μg/condition, clone: LNH-94;2M2, Biolegend), and a biotin-conjugated CD137 antibody (clone REA765; Miltenyi Biotec) followed by anti-biotin microbeads (Miltenyi Biotec).

Techniques: Staining, Expressing, Ex Vivo, In Vitro, MANN-WHITNEY

Journal: Science Immunology

Article Title: Severely ill COVID-19 patients display impaired exhaustion features in SARS-CoV-2-reactive CD8 + T cells

doi: 10.1126/sciimmunol.abe4782

Figure Lengend Snippet: ( A ) Uniform manifold approximation and projection (UMAP) analysis that displays single-cell transcriptomic landscape of sorted CD137 + CD69 + CD8 + memory T cells following 24 hours of stimulation with virus-specific peptide pools. Seurat-based clustering of 84,140 single cells colored based on cluster type. ( B ) Heatmap showing expression of the most significantly enriched transcripts in clusters 0-6 (see table S4, Seurat marker gene analysis – comparison of a cluster of interest versus all other cells). Shown are a subset of the top 200 transcripts that have an adjusted P < 0.05, log 2 fold change > 0.25, and >10% difference in the percentage of cells expressing the differentially expressed transcript between two groups compared. ( C ) Graph showing average expression (color scale) and percent of expressing cells (size scale) of selected marker transcripts in each cluster; cells in cluster 7 that comprise <1% of all cells are not shown ( B , C ). ( D ) UMAPs are illustrating exhaustion, interferon (IFN) response, cytotoxicity, ‘unhelped’, and glycolysis signature scores for each cell. ( E ) Gene Set Enrichment Analysis (GSEA) for the indicated gene signatures comparing each cluster with the rest of the cells. Heatmap shows summary of the normalized enrichment scores for each cluster. Gray color indicates that the signature does not reach statistical significance ( P >0.05) in a given cluster. ( F ) Violin plots showing normalized expression level (log 2 (CPM+1)) of representative exhaustion, IFN response and cytotoxicity marker transcripts ( LAG3 , MX1 and GZMB , respectively) in cluster 1 compared to an aggregation of remaining cells (Rest). Color indicates percentage of cells expressing indicated transcript. ( G ) UMAPs are depicting CD8 + memory T cells for individual virus-specific pool stimulation conditions (top panel). Each group of virus-reactive cells was randomly down-sampled to ensure equal representation; corresponding pie charts are displaying proportions of virus-reactive cells in individual clusters (bottom panel).

Article Snippet: For subsequent magnetic-based enrichment of CD137 + cells, cells were sequentially stained with human serum IgG (Sigma Aldrich) for FcR block, cell viability dye (eFluor780/APC.Cy7, eBioscience), fluorescence-conjugated antibodies, Cell-hashtag TotalSeq-C antibody (0.5 μg/condition, clone: LNH-94;2M2, Biolegend), and a biotin-conjugated CD137 antibody (clone REA765; Miltenyi Biotec) followed by anti-biotin microbeads (Miltenyi Biotec).

Techniques: Virus, Expressing, Marker, Comparison

Journal: Nature Communications

Article Title: Rapid protection induced by a single-shot Lassa vaccine in male cynomolgus monkeys

doi: 10.1038/s41467-023-37050-6

Figure Lengend Snippet: A Quantification of the percentage of CD4 + T cells expressing IFNγ (left panel) or CD154 (right panel) in response to stimulation with LASV-derived GP peptides. B Quantification of the percentage of CD4 + T cells expressing IFNγ (left panel) or CD154 (right panel) in response to stimulation with LASV-derived NP peptides (D-16 vs CT, p = 0.0382). C Quantification of the percentage of CD8 + T cells expressing IFNγ (left panel) or CD137 (right panel) in response to stimulation with LASV-derived GP peptides. D Quantification of the percentage of CD8 + T cells expressing IFNγ (left panel) or CD137 (right panel) in response to stimulation with LASV-derived NP peptides. The percentage of T cells is presented according to the time after challenge after subtraction of the respective value measured for unstimulated T cells. Each bar represents the mean ± SEM of three samples. A Kruskal–Wallis test was used after testing for normality with a Shapiro–Wilk normality test. Statistical significance: * p ≤ 0.05. Source data are provided as a Source Data file.

Article Snippet: After fixation and permeabilization, cells were stained with IFNγ (559327, PE mouse anti-human, clone B27, 20 μL) and TNFα (557647, PE-Cy TM 7 mouse anti-human, clone Mab11, 5 μL) antibodies from BD Biosciences, CD137 (130-119-886, VioBright FITC mouse anti-human, clone 4B4-1, 2 μL), and CD154 (130-113-609, VioBlue mouse anti-human, clone 5C8, 2 μL) antibodies from Miltenyi Biotec.

Techniques: Expressing, Derivative Assay

Journal: Molecular Therapy Oncology

Article Title: Enhanced solid tumor cell targeting by a neoepitope-encoding oncolytic measles virus combined with CAR therapy

doi: 10.1016/j.omton.2025.201043

Figure Lengend Snippet: CAR-T cells targeting the MICB-derived octapeptide show CAR-dependent activation and efficiently kill tumor cells (A) Schematic representation of scFv of both 8-VHVL CAR and 8-VLVH CAR targeting the octapeptide VLQSQRTD used in this study. (B) Expression of CAR molecules on T cells. Left panel shows representative plots of flow cytometry data of primary T cells, 8-VHVL CAR-T and 8-VLVH CAR-T. Right panel shows expression of both experimental CARs (8-VHVL and 8-VLVH) as well as CD4 and CD19 CAR on T cells. Black bars represent CAR expression detected via anti-mouse F(abʹ) 2 antibody, and green bars represent EGFP signal; data from n = 18 independent experiments are depicted as mean (SEM). (C) CAR-T cells were cocultured with Mac-1 cells stably transduced to express indicated transgenes. Tumor cells were labeled with CellTrace Violet, and killing was assessed via PI staining. Killing is depicted as ratio of PI+ cells in cocultures and independently cultured Mac-1 cells. Data from n ≥ 6 experiments are depicted as mean (SEM). (D) CD137 expression was measured on CAR-T cells after incubation with Mac-1 for 24 h. Data are shown from EGFP+ CAR-T cells and are depicted from n = 6 independent experiments as mean (SEM). (E) CAR-T cells were cocultured with T cells from the same donor transduced to express indicated transgenes. T cells used as target cells were labeled with CellTrace Violet, and killing was assessed via PI staining. Only EGFP+ target T cells were analyzed for PI staining. Killing is depicted as the ratio of PI+ cells in cocultures and independently cultured target T cells. Data from n = 9 independent experiments are shown as mean (SEM). (F) CD137 expression was measured on CAR-T cells after incubation with transduced T cells as target cells for 24 h. Data are shown from EGFP+ CAR-T cells and are depicted from n = 6 independent experiments as mean (SEM). (G) LAG-3 expression was measured on CAR-T cells after incubation with transduced T cells as target cells for 72 h. Data are shown from EGFP+ CAR-T cells from n = 4 experiments as mean (SEM); statistical analysis of (C) through (G) was performed by two-way ANOVA followed by Dunnett’s multiple-comparisons test, and experimental groups were compared to CD19 CAR. ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, and ∗∗∗∗ = p ≤ 0.0001.

Article Snippet: For evaluation of CAR-T cell activation, staining with CD137 (CD137 Antibody, PE-Vio770, anti-human, REAfinity) (Miltenyi Biotech, Bergisch Gladbach, Germany) was carried out after 24 h of co-incubation.

Techniques: Derivative Assay, Activation Assay, Expressing, Flow Cytometry, Stable Transfection, Labeling, Staining, Cell Culture, Incubation