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