Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: NK cell-triggered CCL5/IFNγ-CXCL9/10 axis underlies the clinical efficacy of neoadjuvant anti-HER2 antibodies in breast cancer

doi: 10.1186/s13046-023-02918-4

Figure Lengend Snippet: Specialized tumor-infiltrating NK cell subsets associate to distinct immune contextures in human breast tumors. Unsorted multicellular suspensions derived from fresh breast tumor specimens were cultured in complete medium with IL2 in the presence or absence of trastuzumab (Tz; 210 ng/ml) for 24 h. A Dot plots showing the NK cell gate (CD56 + CD3 - ) used for the analysis of the expression of CD16 and CD103 in TI-NK cells of three representative samples. B-C Proportions of CD137 + cells in CD16 + , CD16 - CD103 + and CD16 - CD103 - subpopulations in fresh breast tumor-derived multicellular cultures treated or not with trastuzumab. D-G Fresh breast tumor specimens ( n = 84) were processed and stained with a combination of antibodies specific for CD45, CD3, CD56, CD8, CD4, CD16, CD103, NKG2C and PD1 and analysed by flow cytometry. D tSNE of major alive lymphocyte subsets (DAPI - CD45 + ) in breast tumor immune infiltrates generated with data from 18 tumors. Red, blue and green circles indicate major NK, CD4 and CD8 T cell subsets, respectively. E Percentage of CD16 + , CD16 - CD103 + and CD16 - CD103 - TI-NK cell subpopulations in treatment naïve, fresh tumor samples. F , G Heat maps showing Spearman’s correlation coefficients between the indicated lymphocyte subsets in breast tumors ( n = 73–84) and HER2 tumors ( n = 20). Asterisks label significant correlations (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05

Article Snippet: After culture, cells were stained with a combination of directly labeled antibodies, which included: anti-CD45 (clone 2D1, 56–9459-42, eBiosciences), anti-CD56 (clone CMSSB, 17–0567-42, Invitrogen), anti-CD3 (clone SK7, 345766, BD), anti-CD16 (clone CB16, 47–0168-42, eBiosciences), anti-CD103 (clone B-Ly7, 11–1038-42, eBiosciences) and anti-CD137 (clone 4B4, 12–1379, eBiosciences).

Techniques: Derivative Assay, Cell Culture, Expressing, Staining, Flow Cytometry, Generated

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: Journal of Neuroinflammation

Article Title: CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species

doi: 10.1186/1742-2094-9-173

Figure Lengend Snippet: CD137L signaling activates microglia in vitro . Microglia cells were grown on plates that had been coated with PBS (grey bars) or 10 μg/mL of Fc control protein (white bars) or 10 μg/mL of CD137-Fc protein (black bars). ( A ) Attachment and morphological changes of primary microglia were documented by photography (40× magnification) 1 h after plating. Scale bar: 20 μm. ( B ) Attachment and morphological changes of BV-2 and N9 cells and primary microglia were documented by photography (63×) 24 h after plating. Cells exposed to immobilized CD137-Fc protein developed long spiky projections* and became amoeboid cells^ with shortened protrusions # . Scale bar: 20 μm. ( C ) The concentrations of cytokines in supernatants of primary microglia were determined by ELISA at indicated time points. Depicted are means ± standard deviations of triplicate measurements. ( D ) The phagocytic capacity of the cells was determined by adding FITC-labeled latex beads for 1 h before analysis by flow cytometry. Control: Autofluorescence of the cells. Numbers above the histograms state the percentages of positive cells and mean fluorescence intensities (MFI). These experiments were repeated three times with similar results. * P <0.05; ** P <0.01.

Article Snippet: Unspecific staining was blocked by 2% serum for 30 min. Endogenous peroxidases were inactivated by 3% hydrogen peroxide for 15 min. Anti-CD137 (goat polyclonal, R&D Systems) and anti-Iba-1 (rabbit polyclonal, Wako Chemicals) in PBS were used as primary antibodies and hybridized overnight.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species

doi: 10.1186/1742-2094-9-173

Figure Lengend Snippet: CD137L signaling activates microglia in vivo . ( A ) CD137L is required for activation of microglia in vivo . Cortex and spinal cord tissue sections of WT and CD137L -/- mice with EAE were immunohistochemically stained with an isotype control antibody or for Iba-1 (brown). Shown in the inset is a close-up of a single Iba-1-positive microglia cell in the cortex of a WT mouse with EAE. ( B ) Quantification of Iba-1 + microglia in the spinal cords and cortices of WT and CD137L -/- mice with EAE. Evaluated were three fields from two sections each using the Metamorph NX Software. Depicted are means ± standard deviations. ( C ) CD137 is expressed in the CNS during EAE. Spinal cord of naïve WT mice and WT mice with EAE was sectioned and stained with an isotype control antibody or for CD137 (brown). ( D ) The presence of CD137L is required for oligodendrocyte apoptosis in EAE. Tissue sections from the dorsal column of the spinal cord and the white matter of the cerebellum of WT and CD137L -/- mice with EAE were stained for oligodendrocytes using a Cy3-labeled anti-Nogo-A antibody (red). Apoptosis was detected by TUNEL staining (green). Nuclei were visualized by DAPI (blue). The yellow staining results from an overlay of red and green and indicates apoptotic oligodendrocytes. Magnification: 40×. ( E ) Quantification of apoptotic oligodendrocytes in the cerebellum of WT and CD137L -/- mice with EAE. Evaluated were three fields from two sections each using the Metamorph NX Software. Depicted are means ± standard deviations.

Article Snippet: Unspecific staining was blocked by 2% serum for 30 min. Endogenous peroxidases were inactivated by 3% hydrogen peroxide for 15 min. Anti-CD137 (goat polyclonal, R&D Systems) and anti-Iba-1 (rabbit polyclonal, Wako Chemicals) in PBS were used as primary antibodies and hybridized overnight.

Techniques: In Vivo, Activation Assay, Staining, Software, Labeling, TUNEL Assay

Journal: Journal of Neuroinflammation

Article Title: CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species

doi: 10.1186/1742-2094-9-173

Figure Lengend Snippet: Induction of oligodendrocyte death by CD137L-activated microglia. (A ) Expression of CD137L on the oligodendrocyte cell line OLN93 was determined by flow cytometry. Open histogram: Isotype control. Grey histogram: Anti-CD137L monoclonal antibody (clone TKS-1). ( B ) N9 and OLN93 cells were cultured for 24 h at a 1:1 ratio (1.5×10 5 each) on plates that had been coated with nothing (PBS) or 10 μg/mL of Fc control protein or 10 μg/mL of CD137-Fc protein. ( C ) The rates of OLN93 cell apoptosis in co-cultures with primary microglia from WT or CD137L -/- mice was determined 48 h after initiation of CD137L signaling by 7-AAD and Annexin V staining. Numbers in quadrants indicate percentages of cells. These experiments were repeated two to three times with similar results. * P <0.05; ** P <0.01.

Article Snippet: Unspecific staining was blocked by 2% serum for 30 min. Endogenous peroxidases were inactivated by 3% hydrogen peroxide for 15 min. Anti-CD137 (goat polyclonal, R&D Systems) and anti-Iba-1 (rabbit polyclonal, Wako Chemicals) in PBS were used as primary antibodies and hybridized overnight.

Techniques: Expressing, Flow Cytometry, Cell Culture, Staining

Journal: Journal of Neuroinflammation

Article Title: CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species

doi: 10.1186/1742-2094-9-173

Figure Lengend Snippet: CD137L-activated microglia induces oligodendrocyte apoptosis via ROS. ( A ) BV-2 cells were cultured on uncoated plates (PBS) or plates coated with Fc or CD137-Fc protein for 24 h, and were then stimulated with 0.4 μg of PMA for 1 h, stained with DHR123 before production of ROS was quantified by flow cytometry. White histogram: No DHR123. The number in the panel indicates the percentage of positive cells. ( B ) BV-2 cells were co-cultured with OLN93 cells at a 1:1 ratio with or without 10,000 U/mL catalase. The rate of apoptosis of cultures was determined after 24 h by 7-AAD and Annexin V staining. Numbers in quadrants indicate percentages of cells. ( C ) The percentages of 7-AAD + OLN93 cells with and without catalase treatment of B are presented as means ± standard deviations of triplicate measurements. * P < 0.05; ** P < 0.01.

Article Snippet: Unspecific staining was blocked by 2% serum for 30 min. Endogenous peroxidases were inactivated by 3% hydrogen peroxide for 15 min. Anti-CD137 (goat polyclonal, R&D Systems) and anti-Iba-1 (rabbit polyclonal, Wako Chemicals) in PBS were used as primary antibodies and hybridized overnight.

Techniques: Cell Culture, Staining, Flow Cytometry