Journal: bioRxiv

Article Title: Replicating retroviral delivery of an IL-15 superagonist improves antitumor immunity and long-term survival in poorly immunogenic glioblastoma models

doi: 10.1101/2024.10.14.618095

Figure Lengend Snippet: ( A ) Schematic of the RRV RLI construct, illustrating the proposed mechanism of action and delivery of the therapeutic transgene. ( B ) in vivo spread of RRV-EMD in the SB28 GBM murine tumor model at endpoint. There was no significant difference between RRV-EMD injection (RRV-EMDinj) and RRV-EMD ex vivo pre-transduction (RRV-EMDpre) (85.5% vs. 95.3% EMD+ cells, p=0.14, Welch’s t-test). Both RRV-EMDinj (p<0.01) and RRV-EMDpre (p<0.001) demonstrated significantly higher infection rates compared to HBSS injection control. ( C ) in vitro RRV RLI replication in SB28 is efficient, reaching >80% tumor cell infection by day 15 regardless of starting multiplicity of infection (MOI) as measured by the expression of viral Gag protein on flow cytometry with no change in AZT control groups. ( D ) in vitro RRV RLI replication in Tu2449 is similarly effective, reaching >90% tumor cell infection by day 15 regardless of starting multiplicity of infection (MOI) as measured by the expression of viral Gag protein on flow cytometry with no change in AZT control groups. ( E ) RRV RLI demonstrates stability and lack of transgene drop out after 8 weeks in culture in the SB28 cell line ( fig. S1B ). ( F ) RRV RLI stimulates production of 16.72 pg/100K cells over 24 hours in the SB28 cell line with minimal production in RRV (no transgene, 0.2 pg/100K cells/24hr)) or uninfected control cells (0.02 pg/100K cells/24hr). ( G ) RLI produced by SB28 cells is functional and stimulates the growth of cytokine dependent CTLL-2 cells as compared to RRV (p<0.001, Welch’s t-test) and media control (p<0.001, Welch’s t-test). Nanomolar matched (0.361 nm) recombinant IL-15 (p=0.01, Welch’s t-test) and IL-2 (p<0.001, Welch’s t-test) were used as positive controls. ( H ) Co-culture of CD8 T cells with infected SB28 tumor cells demonstrates increased CD69 after 48 hours compared to RRV and uninfected cells (p<0.01, Welch’s t-test). ( H ) Co-culture of NK cells with infected SB28 tumor cells demonstrates increased change in NK cell frequency relative to all events at 48 hours when compared to RRV infected and uninfected (1.4 vs. 0.05, vs. 0.03, p<0.001, Welch’s t-test). ( J ) In NK cell: SB28 tumor cell co-culture, there was increased double positivity for GZMB and CD69 at 24 hours (p<.005, Welch’s t-test).

Article Snippet: A total of 400 μg of anti-CD8 antibody (200 μg YTS 169.4, BioXcell; 200 μg 53-6.7, BioXcell) and 200 μg of anti-CD4 antibody (GK1.5, BioXcell) were administered.

Techniques: Construct, In Vivo, Injection, Ex Vivo, Transduction, Infection, Control, In Vitro, Expressing, Flow Cytometry, Produced, Functional Assay, Recombinant, Co-Culture Assay

Journal: bioRxiv

Article Title: Replicating retroviral delivery of an IL-15 superagonist improves antitumor immunity and long-term survival in poorly immunogenic glioblastoma models

doi: 10.1101/2024.10.14.618095

Figure Lengend Snippet: ( A ) Volcano plot showing differentially expressed genes between RRV RLI and PBS treatment groups at day 14 post-tumor implantation timepoint. ( B ) Heat map demonstrating calculated immune infiltrations scores between RRV RLI, RRV, and PBS treatment groups at day 14 post-tumor implantation timepoint. ( C ) Directed global significance scores of the top 10 upregulated gene pathways in RRV RLI treatment vs. PBS treatment. ( D ) Heat map detailing differential gene expression for genes related to T cell function, NK cell function, and antigen processing between RRV RLI and PBS treatment groups. ( E ) Heat map detailing differential gene expression for genes related to cytokines and receptors and IL-15 signaling between RRV RLI and PBS treatment groups. ( F ) Flow cytometric analysis of tumor infiltrating immune cells revealed increased CD3 + cells in RRV RLI relative to PBS (8.7% vs. 1.1%, p<0.02, Welch’s t-test) and RRV (8.7% vs. 2.6%, p<0.02, Welch’s t-test). (G ) Flow cytometric analysis of tumor infiltrating immune cells revealed increased CD3 + CD8 + cells in RRV RLI relative to PBS (7.4% vs. 1.4%, p<0.01, Welch’s t-test) and RRV (7.4% vs. 0.67%, p<0.01, Welch’s t-test). (G ) Flow cytometric analysis of tumor infiltrating immune cells revealed no difference in CD3 + CD4 + cells in RRV RLI relative to PBS (1.3% vs. 0.37%, p=0.08, Welch’s t-test) and RRV (1.3% vs. 1.1%, p=0.66, Welch’s t-test). (H ) Flow cytometric analysis of tumor infiltrating immune cells revealed increased CD3 - NK1.1 + cells in RRV RLI relative to PBS (11.8% vs. 0.65%, p<0.05, Welch’s t-test) and RRV (11.8% vs. 0.59%, p<0.05, Welch’s t-test).

Article Snippet: A total of 400 μg of anti-CD8 antibody (200 μg YTS 169.4, BioXcell; 200 μg 53-6.7, BioXcell) and 200 μg of anti-CD4 antibody (GK1.5, BioXcell) were administered.

Techniques: Tumor Implantation, Gene Expression, Cell Function Assay

Journal: bioRxiv

Article Title: Replicating retroviral delivery of an IL-15 superagonist improves antitumor immunity and long-term survival in poorly immunogenic glioblastoma models

doi: 10.1101/2024.10.14.618095

Figure Lengend Snippet: ( A ) Schematic illustration of RRV RLI in vivo assessment in T cell depletion experiments. ( B ) RRV RLI treatment reduces average bioluminescent signaling 9 days post-tumor implantation in SB28 tumors with isotype treatment. ( C ) In isotype antibody-treated mice bearing SB28 tumors, RRV RLI therapy significantly extended survival compared to PBS-treated controls (median survival 25 days vs. 15 days; p < 0.001, 37% long-term survivorship, Log-Rank Mantel-Cox test). ( D ) In anti-CD4 and anti-CD8 antibody-treated mice bearing SB28 tumors, RRV RLI therapy does not significantly change tumor growth on bioluminescent imaging (day 14, p=0.10, Welch’s t-test). ( E ) In anti-CD4 and anti-CD8 antibody-treated mice bearing SB28 tumors, RRV RLI does not significantly alter survival (median survival 16.5 days vs. 19 days, p=0.31, Log-Rank Mantel-Cox) when compared to PBS treated mice. ( F ) In anti-CD8 antibody-treated mice bearing SB28 tumors, RRV RLI therapy does not significantly change tumor growth on bioluminescent imaging (day 14, p=0.37, Welch’s t-test). ( G ) in anti-CD8 antibody-treated mice bearing SB28 tumors, RRV RLI does not significantly alter survival (median survival 34.5 days vs. 26.5 days, p=0.11, Log-Rank Mantel-Cox) when compared to PBS treated mice.

Article Snippet: A total of 400 μg of anti-CD8 antibody (200 μg YTS 169.4, BioXcell; 200 μg 53-6.7, BioXcell) and 200 μg of anti-CD4 antibody (GK1.5, BioXcell) were administered.

Techniques: In Vivo, Tumor Implantation, Imaging

Journal: bioRxiv

Article Title: Replicating retroviral delivery of an IL-15 superagonist improves antitumor immunity and long-term survival in poorly immunogenic glioblastoma models

doi: 10.1101/2024.10.14.618095

Figure Lengend Snippet: ( A ) Schematic illustration of RRV RLI in vivo assessment in temozolomide (TMZ) combination experiments. ( B ) RRV RLI + TMZ improves survival relative to RRV RLI + Vehicle (median survival undefined vs. 48 days; p<0.05, Log-Rank Mantel-Cox test) and PBS + TMZ (median survival undefined vs. 32 days; p<0.001, Log-Rank Mantel-Cox test). Mice reaching non-tumor endpoint were censored at that time. ( C ) Average bioluminescent tumor imaging. ( D-G ) Individual mouse bioluminescent imaging for all treatment groups. ( H ) Example flow cytometric gating for CD4 and CD8 T cells as well as NK cells at day 18 post-implantation timepoint. ( I ) Percent infiltrating CD3 + cells with significant differences between RRV RLI + TMZ and PBS + TMZ (p<0.01, Uncorrected Dunn’s test) as well as RRV RLI + Vehicle and PBS + Vehicle (p<0.02, Uncorrected Dunn’s test) ( J ) Percent infiltrating CD3 + CD4 + cells with significant differences between RRV RLI + TMZ and PBS + TMZ (p<0.01, Uncorrected Dunn’s test) as well as RRV RLI + Vehicle and PBS + Vehicle (p<0.01, Uncorrected Dunn’s test) ( K ) Percent infiltrating CD3 + CD8 + cells with significant differences between RRV RLI + TMZ and PBS + TMZ (p<0.01, Uncorrected Dunn’s test) as well as RRV RLI + Vehicle and PBS + Vehicle (p<0.05, Uncorrected Dunn’s test) ( L ) Percent infiltrating CD3 + NK1.1 + cells with significant differences between RRV RLI + TMZ and PBS + TMZ (p<0.001, Uncorrected Dunn’s test) as well as RRV RLI + Vehicle and PBS + Vehicle (p<0.05, Uncorrected Dunn’s test) ( M ) Percent infiltrating CD3 - NK1.1 + cells with significant differences between RRV RLI + TMZ and PBS + TMZ (p<0.001, Uncorrected Dunn’s test) as well as RRV RLI + Vehicle and PBS + Vehicle (p<0.05, Uncorrected Dunn’s test) ( N ) Percent infiltrating CD11b + cells with significant differences between RRV RLI + TMZ and PBS + TMZ (p<0.01, Uncorrected Dunn’s test) as well as RRV RLI + Vehicle and PBS + Vehicle (p<0.01, Uncorrected Dunn’s test). All flow cytometry samples were collected at day 18 post-implantation timepoint.

Article Snippet: A total of 400 μg of anti-CD8 antibody (200 μg YTS 169.4, BioXcell; 200 μg 53-6.7, BioXcell) and 200 μg of anti-CD4 antibody (GK1.5, BioXcell) were administered.

Techniques: In Vivo, Imaging, Flow Cytometry

Journal: bioRxiv

Article Title: Replicating retroviral delivery of an IL-15 superagonist improves antitumor immunity and long-term survival in poorly immunogenic glioblastoma models

doi: 10.1101/2024.10.14.618095

Figure Lengend Snippet: ( A ) Single-cell RNA sequencing UMAP of all sequenced transcriptomes from CD45 + cells isolated from SB28 tumor bearing mice, demonstrating myeloid, T, and NK cell clusters. ( B ) T and NK cell focused UMAP including all samples. ( C ) Alluvial plot demonstrating changes in gross T and NK cell infiltration between samples. ( D ) Volcano plot demonstrating CD8 T cell differential gene expression in RRV RLI + TMZ vs. RRV RLI + Vehicle with additional GSEA. ( E ) X-Y graph showing increased recurrent T cell clones in RRV RLI + TMZ vs. RRV RLI + Vehicle. ( F ) Myeloid cell focused UMAP including all samples. ( G ) Alluvial plot demonstrating changes in gross myeloid cell infiltration between samples. ( H ) Heat map of MHC class I expression in myeloid populations across samples highlighting increased expression in RRV RLI + Vehicle and enhancement in RRV RLI + TMZ. ( I ) Cell chat analysis of incoming signaling in RRV RLI + Vehicle vs. PBS + Vehicle.

Article Snippet: A total of 400 μg of anti-CD8 antibody (200 μg YTS 169.4, BioXcell; 200 μg 53-6.7, BioXcell) and 200 μg of anti-CD4 antibody (GK1.5, BioXcell) were administered.

Techniques: RNA Sequencing, Isolation, Gene Expression, Clone Assay, Expressing

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 1 Pharmacological inhibition of BCL9 induces tumor regression and increases antigen presentation. a The BCL9 expression between tumors and normal tissues in TCGA COAD datasets (Normal, n = 41; Tumor, n = 462). b The antigen processing and presentation signature (left) and HLA-I signature (right) between low and high BCL9 expression (median value) in TCGA COAD datasets (BCL9Low , n = 209; BCL9High, n = 236). c Tumor growth of 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (n = 6). d Tumor growth of MC38 tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation (n = 6). e Heatmap of the genes included in the GO:0019882 from 30 mg/kg hsBCL9z96-treated CT26 tumors (Vehicle, n = 4; hsBCL9z96, n = 5). f, g The relative expression of Tap1, Tap2, B2m and Psmb9 of tumors from hsBCL9z96-treated CT26 tumor-bearing mice (f) and MC38 tumor- bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (g) analyzed by qPCR (n = 4–7). h–k Representative plot (h, j) and quantitative analysis (i, k) of OVA257-264-specific CD8+ T cells in TILs of tumors from MC38-OVA tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (h, i) and hsBCL9z96-treated MC38- OVA tumor-bearing mice (j, k) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation and analyzed by flow cytometry (n = 3). l Tumor growth of C57BL/6 WT (n = 6) and Batf3−/−mice (n = 5) that had been injected subcutaneously with MC38 tumor cells and were treated i.p. with vehicle or 40 mg/kg hsBCL9z96 every day for 2 weeks. These data are representative values expressed as the mean ± SD of each group; n indicates biological replicate; **p < 0.01; ***p < 0.001; ****p < 0.0001; Unpaired Student’s t test (a, b, i, k); Two-way ANOVA followed by Bonferroni test (c, d, f, g)

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Inhibition, Immunopeptidomics, Expressing, Cytometry, Injection

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 2 Inhibition of BCL9/BCL9L enhances cDC1 activation and facilitates cross-priming of CD8+ T cells. a, b CD40 (left) and CD86 (right) expression by CD103+ cDC1 of TdLNs (a) and tumors (b) from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice analyzed by flow cytometry (n = 3-4). c, d CD40 (left) and CD86 (right) expression by CD103+ cDC1 of TdLNs (c) and tumors (d) from MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by flow cytometry (n = 3–4). e The representative plot of OT-I CD8+ T cells in TdLNs from hsBCL9z96-treated MC38-OVA tumor- bearing mice analyzed by flow cytometry. f and g Quantitative analysis of the percentage of OT-I CD8+ T cells (f) and CFSE dilution of OT-I CD8+ T cells (mean fluorescent intensity, MFI) (g) based on the result of (e) (n = 3). h The representative plot of OT-I CD8+ T cells in TdLNs from MC38-OVA tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, and +6 post inoculation analyzed by flow cytometry. i, j Quantitative analysis of the percentage of OT-I CD8+ T cells (i) and CFSE dilution of OT-I CD8+ T cells (j) based on the result of (h) (n = 3). These data are representative values expressed as the mean ± standard deviation (SD) for each group, derived from three independent experiments; “n” denotes the number of biological replicates. An unpaired Student’s t test was used for statistical analysis of the data in groups a–d, f, g, i, and j

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Inhibition, Activation Assay, Expressing, Cytometry, Standard Deviation, Derivative Assay

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 3 Single-cell transcriptional profiling of CD8+ T cells and cDC1 in tumors and TdLNs from B16-OVA tumor-bearing Bcl9/Bcl9l deficiency mice. a Illustration of experiment and analysis process of single-cell transcriptional analysis. b TSNE plots of clustering process and marker genes (Zbtb46 for DCs, Cd68 for myeloid cells, Mlana for B16-OVA tumor cells, Cd3e for T cells, Cd4 for CD4+ T cells and Cd8a for CD8+ T cells) in tumors from B16-OVA tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6 and +11 post inoculation. c–e TSNE plots of DC reclustering (c, e) and marker genes (Xcr1 for cDC1 and Clec10a for cDC2) (d) in tumors from B16-OVA tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Marker

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 4 Bcl9/Bcl9l deficient cDC1 are superior to WT cDC1 in activation, antigen presentation and cross-priming of CD8+ T cells. a Expression of genes related to cDC1 maturation and antigen presentation in tumors and TdLNs from B16-OVA tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/f

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Activation Assay, Immunopeptidomics, Expressing

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 6 Targeting of BCL9/BCL9L increases cDC1 accumulation in tumors through XCL1-XCR1 axis. a Gating strategy of XCR+ cDC1 (CD45+ CD11b−CD11c+ MHC-II+ CD103+ XCR1+) in TILs. b The XCR+ cDC1 in TILs of 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (left) and MC38 tumor-bearing Bcl9f/fBcl9lf/fCre-ERT2 mice (right) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation were analyzed by flow cytometry (n = 4). c iCD103+ DC migration toward XCL1 for 3 h by trans well assay (n = 3). d Heatmap of the genes included in GO:0070098 of 30 mg/kg hsBCL9z96-treated CT26 tumors (vehicle, n = 4; hsBCL9z96, n = 5). e, f Xcl1 mRNA (left) and XCL1 protein (right) levels in tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (e) and MC38 tumor-bearing Bcl9f/fBcl9lf/fCre-ERT2 mice (f) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation were analyzed by qPCR and ELISA, respectively (n = 4-5). g, h Representative plot (left) and quantitative analysis (right) of XCL1 expression of CD8+ T cells and NK cells in TILs from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice analyzed by flow cytometry (n = 4). g Representative plot (left) and quantitative analysis (right) of XCL1 expression among CD8+ T cells and NK cells in TILs from MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation were analyzed by flow cytometry (n = 4). h Results are presented as the mean ± standard deviation (SD) for each group, derived from three independent experiments; “n” denotes the number of biological replicates; Unpaired Student’s t test (b, c, e, f); Two-way ANOVA followed by Bonferroni test (g, h)

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Cytometry, Migration, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation, Derivative Assay

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 7 Targeting BCL9/BCL9L results in CD8+ T cells accumulation in tumors through CXCL9-CXCR3 axis. a Significantly upregulated GO terms related to IFN-γ response of 30 mg/kg hsBCL9z96-treated CT26 tumors are depicted (vehicle, n = 4; hsBCL9z96, n = 5). b and c Relative Ifng mRNA (left) and IFN-γ protein (right) levels in tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (b) and MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (c) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by qPCR and ELISA, respectively (n = 4–7). d, e Relative Cxcl9 mRNA (left) and CXCL9 protein (right) expression of tumors from 30 mg/ kg hsBCL9z96-treated CT26 tumor-bearing mice (d) and MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (e) treated i.p. with tamoxifen (1 mg/ 100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by qPCR and ELISA, respectively (n = 4-7). f Assessment of CD8+ T cell migration toward CXCL9 or with the indicated doses of antibodies or chemokine for 4 h by trans well assay (n = 3). g Representative plot (left) and quantitative analysis (right) of CXCL9 expression in cDC1 of tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice analyzed by flow cytometry (n = 3–4). h Representative plot (left) and quantitative analysis (right) of CXCL9 expression in cDC1 of tumors from MC38 tumor-bearing Bcl9/Bcl9l deficiency mice analyzed by flow cytometry (n = 4). i The expression of CXCR3 in CD8+ T cells of tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (left) and MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (right) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by flow cytometry (n = 4). Results are presented as the mean ± standard deviation (SD) for each group, derived from three independent experiments; “n” denotes the number of biological replicates; Unpaired Student’s t test (b–e, g–i); One-way ANOVA followed by Bonferroni test (f)

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Migration, Cytometry, Standard Deviation, Derivative Assay

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A Double staining of CD8 and αSMA in mouse various subcutaneous tumors. Scale bar, 200 μm. The quantitative evaluation of sections from each tumor stained for B αSMA ( n = 12 fields from four mice per group) and C CD8 ( n = 12 fields from four mice per group). D The correlation between the number of CD8 + T cells and αSMA + area in each mouse tumor tissue. E Double staining of collagen 1 and CD45 to detect fibrocytes in mouse various subcutaneous tumors. Scale bar, 100 μm. F The quantitative evaluation of sections from each tumor stained for fibrocytes ( n = 12 fields from four mice per group). G The correlation between the number of fibrocytes and αSMA + area in each mouse tumor tissue. The correlation was estimated by Spearman’s correlation and a linear regression analysis (the best-fit line is indicated). Representative images of tumor sections resected from non-small cell lung carcinoma (NSCLC) patients stained to detect αSMA + cancer-associated fibroblasts ( H , αSMA + FAP + ) and fibrocytes ( I , CD45 + FSP-1 + ). Scale bar, 100 μm. J Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in NSCLC tumor tissue stained in Fig. 1H and I ( n = 50 patients). K Representative images of resected NSCLC tissue stained to detect CD8 + T cells and EpCAM + tumor cells. Scale bar, 100 μm. L Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in each NSCLC group divided by the immune phenotypes of tumor-infiltrating CD8 + T cells (inflamed, desert, and exclusion). * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Double Staining, Staining

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume of AB1-HA-bearing balb/c nude mice treated with KL001 from 5 days after tumor cell injection ( n = 6 per group). B Double staining of CD8 and αSMA in AB1-HA tumors from balb/c mice treated with or without KL001 (studied in Fig. ). Scale bar, 200 μm. C Representative images and D quantitative evaluation of sections from KL001-treated AB1-HA tumors stained for CD8, CD4, Foxp3, and F4/80 ( n = 15 fields from five mice per group). Scale bar, 200 μm. E Quantitative evaluation of sections from CLK8-treated AB1-HA tumors stained for CD8, CD4, Foxp3 ( n = 15 fields from five mice per group). * P < 0.05 by the Mann-Whitney U test. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Injection, Double Staining, Staining, MANN-WHITNEY

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume and B the representative image of tumor tissue of LLC-bearing mice treated with KL001 and/or αPD-L1 Ab (n = 7 per group). * P < 0.05 by a one-way ANOVA. C Representative images and D the quantitative evaluation of sections from LLC tumors stained for αSMA ( n = 15 fields from five mice per group). The tumors were harvested at day 21 from each group studied in Fig. 6A. Scale bar, 200 μm. Representative images and the quantitative evaluation of sections from LLC tumors stained for collagen 1 + CD45 + fibrocytes ( E, F ), CD8 + T cells ( G, H ) and CD4 + T cells ( I, J ). Scale bar, 100 μm. * P < 0.05 by a one-way ANOVA. K The evaluation of the tumor volume of AB1-HA-bearing mice treated with KL001 and/or αCTLA-4 Ab ( n = 6 per group). * P < 0.05 by a one-way ANOVA. The quantitative evaluation of sections from AB1-HA tumors ( n = 15 fields from five mice per group) stained for CD8 ( L ), CD4 ( M ), and Foxp3 ( N ). The tumors were harvested at day 18 from each group studied in Fig. 6K. * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects

doi: 10.1007/s00262-024-03777-4

Figure Lengend Snippet: TU2218 inhibits immunosuppression induced by TGFβ or coculture with cancer cells or deficient costimulatory signal. a CD4 + IFNγ + or CD8 + IFNγ + of CD3 (%) in PBMCs with indicated treatment condition. Human PBMCs were treated with indicated condition for 24 h and analyzed by flow cytometry. (One-way ANOVA, Dunnett vs. anti-CD3 + TGFβ) b IFNγ secretion by indicated treatment condition. The amount of IFNγ in PBMC culture supernatant was measured with ELISA. Human PBMCs were treated with indicated condition for 72 h. c The population of NKG2D + cells on CD56 dim or CD56 bright NK cells in human PBMCs compared to vehicle. NK2GD + cells (%) were assessed by flow cytometry. d The population of NKG2D + cells on NK92 cell lines (left) compared to vehicle and cellular cytotoxicity of K562 in NK92/K562 (E/T ratio = 2:1) coculture system (right). e Recovery of IFNγ secretion by indicated treatment condition. The amount of IFNγ in PBMC culture supernatant was measured with ELISA and calculated as relative ratio to anti-CD3/CD28 group. f Population of IFNγ-producing CD4 or CD8 T lymphocyte subsets in PBMC with indicated treatment condition analyzed by flow cytometry. Noted aCD3 indicates anti-CD3 stimulation, TU indicates TU2218, and V indicates vactosertib. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (One-way ANOVA, Dunnett or Tukey, vs. anti-CD3 + TGFβ or anti-CD3/CD28 + TGFβ or TGFβ)

Article Snippet: Erythrocytes were lysed and single-cell suspensions were incubated with Fc block and stained with the following antibodies: anti-mouse CD31-FITC (clone 390, eBioscience), anti-mouse VCAM-1-APC (clone 429, Biolegend) anti-mouse CD45-BV510 (clone 30-F11, BD Bioscience), anti-mouse CD3-APC-Cy7 (clone 145-2C11, Invitrogen), anti-mouse CD8-BB515 (clone 53–6.7, BD Bioscience), and anti-mouse IFNγ-APC (clone XMG1.2, BD Bioscience).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects

doi: 10.1007/s00262-024-03777-4

Figure Lengend Snippet: TU2218 induces antitumor activity in combination with anti-PD1 in B16F10 syngeneic model. a Experimental design of B16F10-bearing C57BL/6 mouse model. b Tumor volume at indicated time points. *** p ≤ 0.001 vs. TU2218, * p ≤ 0.05 vs. anti-PD1 (Two-way ANOVA) (left). Mean body weight at indicated time points (right). (c) MFI of VCAM-1 in CD31 + populations in tumor tissue. * p ≤ 0.05, ** p ≤ 0.01 vs. vehicle (One-way ANOVA, Tukey) (left). Percent of CD8 + IFNγ + T cells in tumors. * p ≤ 0.05 vs. vehicle (One-way ANOVA, Tukey) (right). Data are shown as mean ± SEM

Article Snippet: Erythrocytes were lysed and single-cell suspensions were incubated with Fc block and stained with the following antibodies: anti-mouse CD31-FITC (clone 390, eBioscience), anti-mouse VCAM-1-APC (clone 429, Biolegend) anti-mouse CD45-BV510 (clone 30-F11, BD Bioscience), anti-mouse CD3-APC-Cy7 (clone 145-2C11, Invitrogen), anti-mouse CD8-BB515 (clone 53–6.7, BD Bioscience), and anti-mouse IFNγ-APC (clone XMG1.2, BD Bioscience).

Techniques: Activity Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects

doi: 10.1007/s00262-024-03777-4

Figure Lengend Snippet: The combination with TU2218 and anti-CTLA4 leads to the formation of immunological memory. a Tumor volume during drug treatment and additional cessation of drug treatment (left). Tumor volume after CT26-rechallenge in tumor-eradicated mice showing CR by anti-CTLA4 + TU2218 administration and age-matched naïve mice at indicated timepoint (middle) or endpoint (right). After tumor eradication or CT26-rechallenge, mice were observed without drug administration to evaluate the formation of antitumor immunological memory. Data are shown as mean ± SEM. *** p ≤ 0.001 vs. age-matched naïve mice group (Two-way ANOVA). b Representative images of mice with CT26-rechallenge at endpoint. c The relative population of CD4 + and CD8 + T effector memory subsets in spleen of mice with CT26-rechallenge at endpoint. The data were obtained in six mice per group * p ≤ 0.05, ** p ≤ 0.01 ( t -test, Two-tailed)

Article Snippet: Erythrocytes were lysed and single-cell suspensions were incubated with Fc block and stained with the following antibodies: anti-mouse CD31-FITC (clone 390, eBioscience), anti-mouse VCAM-1-APC (clone 429, Biolegend) anti-mouse CD45-BV510 (clone 30-F11, BD Bioscience), anti-mouse CD3-APC-Cy7 (clone 145-2C11, Invitrogen), anti-mouse CD8-BB515 (clone 53–6.7, BD Bioscience), and anti-mouse IFNγ-APC (clone XMG1.2, BD Bioscience).

Techniques: Two Tailed Test

Journal: Oncotarget

Article Title: The TRPV1 ion channel regulates thymocyte differentiation by modulating autophagy and proteasome activity

doi: 10.18632/oncotarget.21798

Figure Lengend Snippet: A. Western blot analysis of LC3 and p62 protein in WT thymocytes pre-treated with 4-PBA for 60 minutes and then treated for 120 minutes with CPS. The ratio of LC3 II/I was calculated from densitometric data. p62 densitometric values were normalized to GAPDH used as loading control. Blots are representative of one of three separate experiments. * p < 0.01 vs untreated cells, ** p < 0.01 vs CPS-treated cells. B. Flow cytometric analysis was performed by Annexin V-FITC and PI double-staining of WT thymocytes, pre-treated with 4-PBA for 60 minutes and then treated for 120 minutes with CPS. Data represent the percentage of PI and/or Annexin V positive cells. One representative out of three independent experiments is shown. C. Thymocytes, pre-treated with 4-PBA, CQ or NAC for 60 minutes and then treated for 120 minutes with CPS, were stained with anti-CD4-PE and anti-CD8-Cy5 mAbs and analyzed by FACS. The black gate indicates the DP dull subpopulation. One representative out of three independent experiments is shown.

Article Snippet: Thymocytes from WT or TRPV1 KO mice were stained with anti-CD4-PE (clone: RM4-5) and/or anti-CD8-Cy5 (clone 53-6.7) mAb (1μl/1x10 6 cells; BD Biosciences, Milan, Italy) and analyzed by a FACScan cytofluorimeter (BD Biosciences) using the CellQuest software.

Techniques: Western Blot, Double Staining, Staining

Journal: Oncotarget

Article Title: The TRPV1 ion channel regulates thymocyte differentiation by modulating autophagy and proteasome activity

doi: 10.18632/oncotarget.21798

Figure Lengend Snippet: Freshly purified thymocytes from WT and KO mice were counted using trypan blue solution A. , or stained with Annexin-V FITC and analyzed by FACS analysis B. Data are the mean ± SEM of three separate experiments, * p < 0.01 vs WT thymocytes. C. From the total number of cells per thymus, the relative number of cells in each CD4/CD8 subset was determined staining TRPV1 KO and WT thymocytes with anti-CD4-PE and anti-CD8-Cy5 mAbs and using FACS analysis. Data are the mean ± SEM of three separate experiments * p < 0.01 TRPV1 KO vs WT thymocytes.

Article Snippet: Thymocytes from WT or TRPV1 KO mice were stained with anti-CD4-PE (clone: RM4-5) and/or anti-CD8-Cy5 (clone 53-6.7) mAb (1μl/1x10 6 cells; BD Biosciences, Milan, Italy) and analyzed by a FACScan cytofluorimeter (BD Biosciences) using the CellQuest software.

Techniques: Purification, Staining

Journal: Oncotarget

Article Title: The TRPV1 ion channel regulates thymocyte differentiation by modulating autophagy and proteasome activity

doi: 10.18632/oncotarget.21798

Figure Lengend Snippet: A. Thymocytes from WT and KO mice, stimulated with anti-TCRβ plus anti-CD2 mAbs after 18h of culture and 12h of recovery, were analyzed by FACS analysis. One representative out of three independent experiments is shown. B. Purified DP thymocytes from TRPV1 KO and WT mice stimulated with TCRβ plus CD2 mAbs for 18h, followed by 12h recovery, were stained with anti-CD4-PE and anti-CD8-Cy5 mAbs and analyzed by FACS. One representative out of three independent experiments is shown. C. Blood and spleen PBMC from WT and KO thymocytes were stained with anti-CD4-PE and anti-CD8-Cy5 mAbs and analyzed by FACS. One representative out of three independent experiments is shown in each panel.

Article Snippet: Thymocytes from WT or TRPV1 KO mice were stained with anti-CD4-PE (clone: RM4-5) and/or anti-CD8-Cy5 (clone 53-6.7) mAb (1μl/1x10 6 cells; BD Biosciences, Milan, Italy) and analyzed by a FACScan cytofluorimeter (BD Biosciences) using the CellQuest software.

Techniques: Purification, Staining

Journal: Cell Research

Article Title: LKB1 orchestrates dendritic cell metabolic quiescence and anti-tumor immunity

doi: 10.1038/s41422-019-0157-4

Figure Lengend Snippet: LKB1 functions in DCs to restrain excessive Treg expansion and thymic differentiation. a Flow cytometry analysis (left) and statistics (right) of BrdU incorporation in CD4+TCRβ+Foxp3+ Tregs in the spleen, PLN and thymus of WT and LKB1ΔDC mice. b CellTrace-labelled CD45.1+ Tregs were transferred into WT and LKB1ΔDC mice and the CellTrace dilution was analyzed seven days later (left). Right, the relative percentage of CellTracelo cells. The percentage of CellTracelo cells in WT mice was set as 1. c Relative thymidine incorporation of WT Tregs cultured with splenic DCs from WT and LKB1ΔDC mice (values cultured with WT splenic DCs was set as 1). d Flow cytometry analysis (left) and statistics (right) of p-S6 expression in CD4+TCRβ+Foxp3+ Tregs in the spleen and PLN of WT and LKB1ΔDC mice. e Real-time PCR analysis of Il2, Il7 and Il15 mRNA expression in untreated splenic DCs from WT and LKB1ΔDC mice. f Thymidine incorporation of WT Tregs cultured with splenic DCs from WT and LKB1ΔDC mice in the presence or absence of anti-IL-2 + anti-CD25 blocking antibodies. g Relative thymidine incorporation of WT thymic Tregs cultured with thymic DCs from WT and LKB1ΔDC mice (values cultured with WT thymic DCs was set as 1). h CD4+CD8–CD25–YFP-Foxp3– thymocytes (tTreg precursors) were cultured with thymic DCs from WT and LKB1ΔDC mice in the presence of 10 ng/mL IL-7 for 4 days and analyzed for Foxp3+ Treg induction. Data in plots indicate the means ± s.e.m; each symbol represents an individual mouse. Numbers in gates indicate percentage of cells. NS not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-tailed Mann–Whitney test (a, b, d) or two-tailed unpaired Student’s t-test (c, e–h). Data are from at least three (a–f) or two (g, h) independent experiments

Article Snippet: 73 Flow cytometry was performed as described previously, 72 with the following antibodies: anti-TCRβ (H57–597), anti-CD44 (IM7), anti-CD103 (2E7), anti-ICOS (C398.4A), anti-CD25 (PC61.5), anti-CTLA-4, anti-PDCA-1 (eBio927), anti-CD19 (eBio1D3 (1D3)), anti-CD11c (N418), anti-CD40 (1C10), anti-MHC-II (M5/114.15.2), anti-CD80 (16-10A1), anti-CD86 (GL1), anti-CCR7 (4B12) (all from eBioscience); anti-neuropilin-1 (3E12), anti-CD11b (M1/70) (both from BioLegend); and anti-CD8 (53-6.7, Sony Biotechnology Inc).

Techniques: Flow Cytometry, BrdU Incorporation Assay, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Blocking Assay, Two Tailed Test, MANN-WHITNEY

Journal: Cell Research

Article Title: LKB1 orchestrates dendritic cell metabolic quiescence and anti-tumor immunity

doi: 10.1038/s41422-019-0157-4

Figure Lengend Snippet: LKB1 deficiency in DCs promotes Th17 cell differentiation in an IL-6-dependent manner. a Statistics of IL-17 expression in CD4+ and CD8+ T cells in tumor-infiltrating lymphocytes and tumor draining lymph nodes (DLN) of WT and LKB1ΔDC mice. b Tumor growth curve in WT (n = 16), LKB1ΔDC (n = 8), Il17ra−/− (n = 10) and LKB1ΔDCIl17ra−/− (n = 13) mice following inoculation of MC38 tumor cells. c Flow cytometry analysis (upper) and statistics (lower) of IFNγ and IL-17 expression in splenic CD4+ and CD8+ T cells from WT and LKB1ΔDC mice. d Analysis of IL-17 and IFN-γ levels in the culture supernatant of popliteal DLN cells from KLH immunized WT and LKB1ΔDC mice after ex vivo stimulation with KLH for 72 h. e WT and LKB1ΔDC mice were transferred with naïve OT-II CD4+ T cells, followed by footpad immunization with OVA/CFA. IL-17 expression in donor-derived CD4+ T cells in the popliteal DLN was analyzed seven days later. f Flow cytometry analysis (left) and statistics (right) of IL-17 expression in naïve OT-II CD4+ T cells cultured with splenic DCs from WT and LKB1ΔDC mice for 5 days. g Real-time PCR analysis of Il6, Tgfb1, Tgfb2, Tgfb3, Il12a, Il12b, and IL23a mRNA expression in untreated splenic DCs from WT and LKB1ΔDC mice. h Flow cytometry analysis (left) and statistics (right) of IL-17 expression in naïve OT-II CD4+ T cells cultured with splenic DCs from WT and LKB1ΔDC mice for 5 days in the presence or absence of anti-IL-6 or anti-TGFβ blocking antibodies. i Flow cytometry analysis (left) and statistics (right) of IL-17 expression in splenic CD4+ T cells from WT, LKB1ΔDC, Il6−/−, or LKB1ΔDCIl6−/− mice. Numbers in the statistics graph indicate fold changes of IL-17 expression in LKB1ΔDC vs WT mice, and LKB1ΔDCIl6−/− vs Il6−/− mice, respectively. Data in plots indicate the means ± s.e.m; each symbol represents an individual mouse. Numbers in quadrants or gates indicate percentage of cells. NS not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-tailed Mann–Whitney test (a, c, e, f, h), two-tailed unpaired Student’s t-test (d, g), or two-way ANOVA (b, i). Data are from at least three (a, c, e–g, i) or two (b, d, h) independent experiments

Article Snippet: 73 Flow cytometry was performed as described previously, 72 with the following antibodies: anti-TCRβ (H57–597), anti-CD44 (IM7), anti-CD103 (2E7), anti-ICOS (C398.4A), anti-CD25 (PC61.5), anti-CTLA-4, anti-PDCA-1 (eBio927), anti-CD19 (eBio1D3 (1D3)), anti-CD11c (N418), anti-CD40 (1C10), anti-MHC-II (M5/114.15.2), anti-CD80 (16-10A1), anti-CD86 (GL1), anti-CCR7 (4B12) (all from eBioscience); anti-neuropilin-1 (3E12), anti-CD11b (M1/70) (both from BioLegend); and anti-CD8 (53-6.7, Sony Biotechnology Inc).

Techniques: Cell Differentiation, Expressing, Flow Cytometry, Ex Vivo, Derivative Assay, Cell Culture, Real-time Polymerase Chain Reaction, Blocking Assay, Two Tailed Test, MANN-WHITNEY