anti cd8β pe  (Bio-Rad)

Journal: bioRxiv

Article Title: Stage-specific CAR-mediated signaling generates naïve-like, TCR-null CAR T cells from induced pluripotent stem cells

doi: 10.1101/2024.11.25.624041

Figure Lengend Snippet: (a) Schematic of gene editing strategy for the insertion of a transgene into the 3’ UTR of an endogenous gene. CRISPR/Cas9 HDR template was designed to replace the endogenous stop codon (red box) with a 2A ribosome skip sequence, followed by the transgene cDNA in-frame for translation. The template included loxp -flanked (yellow triangles) puromycin resistance gene (PuroR) to enrich for edited iPSC clones. 5’ and 3’ homology arms (HA) complementary to the Cas9-mediated cut site were used to direct homologous recombination. (b) Bulk RNA-Seq measurements of GZMA expression at each stage of T cell development during ATO differentiation (from PSC and Cord Blood) and primary human thymus. Gating strategy for the isolation of each population is as follows: Pluripotent stem cell (PSC: TRA-1-60+SSE.A-4+), embryonic mesodermal progenitors (EMP: CD56+CD326-), embryonic mesodermal organoids (EMO: CD34+CD90+CD43+CD45±), double negative (DN: CD45+CD7+CD1a±), immature single positive CD4+ (ISP4: CD45+CD4+CD8-TCR-CD3-CD7+), double positive (DP: CD4+CD8+CD3-TCRɑβ-), single positive CD8+ (SP8: CD3+CD4-CD8+CD45RA+), and single positive CD4+ (SP4: CD3+CD4+CD8-CD45RA+). Sources for each population are as follows: undifferentiated PSC (left y-axis, white), PSC-derived EMP, EMO, DP, SP8, SP4 (left y-axis, white), CB-ATO derived DN, ISP4 (left y-axis, white), and primary thymus derived DN, ISP4, DP, SP8, SP4 (right y-axis, gray). RPKM = reads per kilo base per million mapped reads. Bulk RNA-seq original data sources derived from GSE116015 and PRJNA741323 (mean ± SD, n = 6 biological replicates for EMO and DN; n = 3 biological replicates for all other cell types). (c) Simplified schematic of iPSC differentiation using the Artificial Thymic Organoid (ATO) culture system. CRISPR/Cas9 edited T-iPSCs were differentiated into embryonic mesodermal progenitors (EMP) and then aggregated with MS5-hDLL4 into embryonic mesodermal organoids (EMOs). After 2 weeks of hematopoietic induction, EMOs were re-aggregated with MS5-hDLL4 to generate ATOs. (d) Analysis of reporter expression at each T cell stage during differentiation of GZMA -mCitrine T-iPSC in ATOs. T cell populations analyzed at weeks 1, 2 and 5 of ATO culture are indicated in the key to the left. mCitrine negative gating was based on unedited T-iPSC ATO control at the same stage of T cell development (data representative of n = 4 independent experiments). (e) Summary of reporter data at each stage of GZMA -mCitrine T-iPSC differentiation in ATOs (mean ± SD, n = 4 independent experiments). (f-g) Analysis of T cell development in ATOs differentiated from (f) CAR19LH Lenti and (g) GZMA -CAR19LH T-iPSCs at indicated time points (data representative of n = 3 independent experiments). (h) Kinetics of T cell development during 5 weeks of ATO differentiation using T-iPSC lines shown above each graph. Percent of T cell subsets are of total CD45+CD56-mCD29-DAPI-cells (mean ± SD, n = 3 independent experiments). (i) Phenotype and CAR19LH expression (anti-FMC63) in GZMA -CAR19LH SP8 T cells analyzed at week 5 of ATO culture (data representative of n = 5 independent experiments). (j) Expression of ILC2 markers CD25 and CD200R in CAR19LH Lenti- and GZMA -CAR19LH ATO-derived cells analyzed at week 5 (data representative of n = 3 independent experiments). (k) Analysis of T cell development in GZMA -CAR19LH ( TRAC -/- ) ATOs at week 6 of culture (data representative of n = 3 independent experiments). (l) Frequencies of T cell subsets at week 6 of ATO culture generated from unedited, GZMA -CAR19LH (i.e. TRAC intact), TRAC -disrupted only control ( TRAC -/- ), and GZMA -CAR19LH ( TRAC -/- ) T-iPSCs, gated on total DAPI-mCD29-cells (mean ± SD, n = 3 independent experiments). (m) Number of total cells generated per ATO at week 6 of culture based on manual trypan blue counting (mean ± SD, **** P < 0.0001 by two-tailed unpaired t -test, n = 3 independent experiments for GZMA -CAR19LH ( TRAC -/- ) T-iPSC; n = 4 independent experiments for GZMA -CAR19LH T-iPSC).

Article Snippet: The anti-human antibody CD8β (REA715) was obtained from Miltenyi Biotec.

Techniques: CRISPR, Sequencing, Clone Assay, Homologous Recombination, RNA Sequencing, Expressing, Isolation, Derivative Assay, Control, Generated, Two Tailed Test

Journal: bioRxiv

Article Title: Stage-specific CAR-mediated signaling generates naïve-like, TCR-null CAR T cells from induced pluripotent stem cells

doi: 10.1101/2024.11.25.624041

Figure Lengend Snippet: (a) Bulk RNA-Seq measurements of CD8β expression at each stage of T cell development during PSC-ATO and CB-ATO differentiation measured as RPKM (left y-axis, white) and primary human thymus (right y-axis, gray). Gating strategy for each population was previously outlined in . RPKM = reads per kilo base per million mapped reads. Bulk RNA-seq original data sources derived from GSE116015 and PRJNA741323 (mean ± SD, n = 6 biological replicates for EMO and DN; n = 3 biological replicates for all other cell types). (b) Summary of reporter data at each stage of CD8β -mCitrine T-iPSC differentiation in ATOs (mean ± SD, n = 4 independent experiments). (c) Simplified schematic of CD8B- CAR19BB ( TRAC -/- ) T-iPSC differentiation in ATOs. After undergoing hematopoietic induction in EMOs, isolated progenitors were re-aggregated with MS5-hDLL4 or CD19-MS5-hDLL4 to generate ATOs. (d,e) Analysis of T cell development in CD8B- CAR19BB ( TRAC -/- ) ATOs using MS5-hDLL4 (top) or CD19-MS5-hDLL4 (bottom) at (d) week 2 and (e) week 6 (data representative of n = 6 independent experiments). (f) Kinetics of T cell development during 6 weeks of CD8B- CAR19BB ( TRAC -/- ) T-iPSC differentiation in ATOs generated with MS5-hDLL4 and CD19-MS5-hDLL4. Percent of T cell subsets were of total CD45+CD56-mCD29-DAPI-(mean ± SD, n = 4 independent experiments). (g) Mean number of total SP8 T cells generated per ATO at week 6 of culture, based on manual trypan blue counting (mean ± SD, **** P < 0.0001 by two-tailed unpaired t -test, n = 5 independent experiments for unedited T-iPSC, n = 6 independent experiments for CD8B- CAR19BB ( TRAC -/- ) T-iPSC). (h) Phenotype of CD8B- CAR19BB ( TRAC -/- ) SP8 T cells analyzed at week 6 of CD19-expressing ATO cultures (data representative of n = 6 independent experiments). (i) CAR19BB expression (Mean Fluorescence Intensity (MFI) of anti-FMC63) in CD8B- CAR19BB ( TRAC -/- ) SP8 T cells analyzed at week 6 of CD19-expressing ATO cultures (Day 0), and 5 and 7 days after isolation from ATOs (post-ATO) (mean ± SD, *** P = 0.0003, **** P < 0.0001 by two-tailed unpaired t -test, n = 5 independent experiments for post-ATO day 0 and n = 3 independent experiments for days 3 and 5).

Article Snippet: The anti-human antibody CD8β (REA715) was obtained from Miltenyi Biotec.

Techniques: RNA Sequencing, Expressing, Derivative Assay, Isolation, Generated, Two Tailed Test, Fluorescence

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Experimental scheme for characterizing the effect of BBM on primary CD3+ T cells. b Fold changes in cell number and c viability determined using the standard trypan blue method. Error bars indicate SD from six donors. d Representative FACS plots illustrating the expression of CCR7 and CD45RA in CD4+ cells (top) and CD8β+ cells (bottom) on day 14. e Estimated numbers of CD4+CCR7+CD45RA+ cells (left) and CD8β+CCR7+CD45RA+ cells (right) in cultured CD3+ T cells calculated from the total cell number and frequencies of the populations that are PI- on day 14. f , g Results of the CFSE assay in CD4+ ( f ) and CD8β+ ( g ) fractions within CD3+ cells on day 3. Error bars indicate SD from six ( b , c ) or four donors ( e – g ). Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (paired t -test).

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: Expressing, Cell Culture, CFSE Assay

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Experimental scheme designed to assess the in vivo persistence of BBM-treated primary CD8+ T cells. b Gating strategy employed to determine the expression of CCR7 and CD45RA in the CD45+CD8β+ fractions. c Frequencies of injected CD45+CD8β+ cells in the spleen (left), peripheral blood (center), and bone marrow (right) on day 14. Each data point represents one injected mouse and its corresponding donors: circle; donor A, triangle; donor B, square; donor C. All error bars indicate SD. Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (unpaired t -test). d Representative flow cytometry plots of CCR7 and CD45RA in CD45+CD8β+ cells in the spleen samples.

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: In Vivo, Expressing, Injection, Flow Cytometry

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Gating strategy for FACS to purify the naïve and memory T cell fractions from healthy donor-derived CD8+ cells. b , c Gene ontology analysis of DEGs in BBM-treated T SCM cells. Upregulated ( b ) or downregulated ( c ) genes from RNA-sequencing results on day 12. Genes were queried in the DAVID functional annotation database. d Transcripts per million (TPM) values of cell cycle-related and effector function-related genes. Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 ( n = 4, paired t -test). T SCM stem cell memory T, T CM central memory T, T EM effector memory T.

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: Derivative Assay, RNA Sequencing, Functional Assay

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Metabolomics of BBM-treated T SCM cells from healthy donor-derived CD8+ cells. Lipids commonly upregulated or downregulated in all three donors are shown. b Representative flow cytometry plots of Phosflow assay in BBM-treated CD3+ cells on day 10. Phosphorylated proteins in CD8+ T cells are shown. c Mean fluorescence intensity (MFI) of phosphorylated p38 in CD8+ T cells. Error bars indicate SD from four donors. d Oxygen consumption rate (OCR) across time for BBM-treated CD3+ T cells. e Spare respiratory capacity (SRC) calculated by OCRmax-OCRbasal. f Extracellular acidification rates (ECAR) across time for BBM-treated CD3+ T cells. Error bars indicate the standard error of the mean from six donors ( d – f ). Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (paired t -test) ( c , e ).

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: Derivative Assay, Flow Cytometry, Fluorescence

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Experimental scheme for characterizing the effect of BBM on primary CD3+ T cells. b Fold changes in cell number and c viability determined using the standard trypan blue method. Error bars indicate SD from six donors. d Representative FACS plots illustrating the expression of CCR7 and CD45RA in CD4+ cells (top) and CD8β+ cells (bottom) on day 14. e Estimated numbers of CD4+CCR7+CD45RA+ cells (left) and CD8β+CCR7+CD45RA+ cells (right) in cultured CD3+ T cells calculated from the total cell number and frequencies of the populations that are PI- on day 14. f , g Results of the CFSE assay in CD4+ ( f ) and CD8β+ ( g ) fractions within CD3+ cells on day 3. Error bars indicate SD from six ( b , c ) or four donors ( e – g ). Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (paired t -test).

Article Snippet: Briefly, CD8+ T cells purified from frozen human PBMCs were stained with anti-CD8β-PE (Beckman Coulter, IM2217U), anti-CD4-BV421 (BioLegend, 317434), anti-CD45RA-BV510 (BioLegend, 304142), anti-CD45RO-APC-Cy7 (BioLegend, 304228), anti-CCR7-APC (BioLegend, 353214), and anti-CD95-PE-Cy7 (BioLegend, 305622) antibodies.

Techniques: Expressing, Cell Culture, CFSE Assay

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Experimental scheme designed to assess the in vivo persistence of BBM-treated primary CD8+ T cells. b Gating strategy employed to determine the expression of CCR7 and CD45RA in the CD45+CD8β+ fractions. c Frequencies of injected CD45+CD8β+ cells in the spleen (left), peripheral blood (center), and bone marrow (right) on day 14. Each data point represents one injected mouse and its corresponding donors: circle; donor A, triangle; donor B, square; donor C. All error bars indicate SD. Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (unpaired t -test). d Representative flow cytometry plots of CCR7 and CD45RA in CD45+CD8β+ cells in the spleen samples.

Article Snippet: Briefly, CD8+ T cells purified from frozen human PBMCs were stained with anti-CD8β-PE (Beckman Coulter, IM2217U), anti-CD4-BV421 (BioLegend, 317434), anti-CD45RA-BV510 (BioLegend, 304142), anti-CD45RO-APC-Cy7 (BioLegend, 304228), anti-CCR7-APC (BioLegend, 353214), and anti-CD95-PE-Cy7 (BioLegend, 305622) antibodies.

Techniques: In Vivo, Expressing, Injection, Flow Cytometry

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Gating strategy for FACS to purify the naïve and memory T cell fractions from healthy donor-derived CD8+ cells. b , c Gene ontology analysis of DEGs in BBM-treated T SCM cells. Upregulated ( b ) or downregulated ( c ) genes from RNA-sequencing results on day 12. Genes were queried in the DAVID functional annotation database. d Transcripts per million (TPM) values of cell cycle-related and effector function-related genes. Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 ( n = 4, paired t -test). T SCM stem cell memory T, T CM central memory T, T EM effector memory T.

Article Snippet: Briefly, CD8+ T cells purified from frozen human PBMCs were stained with anti-CD8β-PE (Beckman Coulter, IM2217U), anti-CD4-BV421 (BioLegend, 317434), anti-CD45RA-BV510 (BioLegend, 304142), anti-CD45RO-APC-Cy7 (BioLegend, 304228), anti-CCR7-APC (BioLegend, 353214), and anti-CD95-PE-Cy7 (BioLegend, 305622) antibodies.

Techniques: Derivative Assay, RNA Sequencing Assay, Functional Assay

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Metabolomics of BBM-treated T SCM cells from healthy donor-derived CD8+ cells. Lipids commonly upregulated or downregulated in all three donors are shown. b Representative flow cytometry plots of Phosflow assay in BBM-treated CD3+ cells on day 10. Phosphorylated proteins in CD8+ T cells are shown. c Mean fluorescence intensity (MFI) of phosphorylated p38 in CD8+ T cells. Error bars indicate SD from four donors. d Oxygen consumption rate (OCR) across time for BBM-treated CD3+ T cells. e Spare respiratory capacity (SRC) calculated by OCRmax-OCRbasal. f Extracellular acidification rates (ECAR) across time for BBM-treated CD3+ T cells. Error bars indicate the standard error of the mean from six donors ( d – f ). Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (paired t -test) ( c , e ).

Article Snippet: Briefly, CD8+ T cells purified from frozen human PBMCs were stained with anti-CD8β-PE (Beckman Coulter, IM2217U), anti-CD4-BV421 (BioLegend, 317434), anti-CD45RA-BV510 (BioLegend, 304142), anti-CD45RO-APC-Cy7 (BioLegend, 304228), anti-CCR7-APC (BioLegend, 353214), and anti-CD95-PE-Cy7 (BioLegend, 305622) antibodies.

Techniques: Derivative Assay, Flow Cytometry, Fluorescence

Journal: Cell Reports Medicine

Article Title: IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T cell engager immunotherapy

doi: 10.1016/j.xcrm.2024.101567

Figure Lengend Snippet: rhIL-7-hyFc treatment increases PD-1 − bystander CD8 TILs (A‒E) scRNA-seq analysis of CD8 TILs. Mice bearing palpable MC38 tumors treated subcutaneously (s.c.) with rhIL-7-hyFc (10 mg kg −1 ). Tumors were collected 7 days after rhIL-7-hyFc treatment. Collected tumor tissues were pooled for analysis ( n = 5–7 per group). Unless specified otherwise, the data include TILs from both buffer- and rhIL-7-hyFc-treated mice. (A) UMAP plots of six distinct CD8 TIL clusters from MC38-bearing mice, numbered and colored according to the transcriptional clusters. (B) Dot plot showing the expression of various T cell-related genes in the six different clusters. (C) UMAP plot of top five expanded clones. (D) UMAP showing the distribution of expression of Pdcd1 transcript. (E) UMAP showing CD8 TIL clusters from buffer- or rhIL-7-hyFc-treated mice (left) and bar graph depicting the proportion of six clusters in each treatment condition (right). (F and G) rhIL-7-hyFc (10 mg kg −1 ) were treated s.c. in mice bearing various palpable tumors. Tumors were collected 7 days after rhIL-7-hyFc treatment ( n = 5–11 per group). (F) Frequency of PD-1 − CD8 T cells among total CD8 TILs. (G) Number of PD-1 + CD8 T cells (left) and PD-1 − CD8 T cells (right). Numbers on the bar indicate fold changes between buffer- and rhIL-7-hyFc-treated groups. Data are shown as mean ± SEM and representative of two independent experiments. (H) Schematic of clinical study design. Patients with metastatic colorectal and ovarian cancer were treated with rhIL-7-hyFc. Pre- and post-treatment tumor samples were collected during the screening period and at indicated time points. (I) Percentage of total CD8 T cells (left), PD-1 + CD8 T cells (middle), and PD-1 − CD8 T cells (right) among the total cells. Each dot represents a single region of interest (ROI) in each patient’s sample. The ROIs were manually designated by pathologists. (J) Representative immunofluorescence images of tumor biopsies from patient SB13. Purple, CD8; yellow, PD-1; gray, DAPI. Magnification, ×200, scale bars, 50 μm ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by unpaired (F, G, and I) two-tailed Student’s t test. See also Figures S1 ; Table S1 .

Article Snippet: Anti-mouse CD8β PE (clone: H35–17.2) , eBioscience , Cat# 12-0083-83; RRID: AB_657767.

Techniques: Expressing, Clone Assay, Immunofluorescence, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T cell engager immunotherapy

doi: 10.1016/j.xcrm.2024.101567

Figure Lengend Snippet: rhIL-7-hyFc treatment changes the transcriptome profiles of tumor-reactive and bystander CD8 TILs (A) UMAP of scTCR-seq data colored according to the clone size of expanded CD8 TILs from mice bearing MC38 tumors between groups (left) and a bar graph showing the proportion of CD8 TILs with each clone size between groups (right). (B) Number of DEGs in tumor-reactive and bystander CD8 TILs from tumor-bearing mice with rhIL-7-hyFc treatment compared with buffer treatment. (C) Top 5 enriched GO terms for up- or downregulated genes in tumor-reactive cells by rhIL-7-hyFc treatment. (D) Violin plots with an expression of genes related to T cell exhaustion, function, and TFs in tumor-reactive cells. (E) GSEA analysis with the gene set of exhausted vs. naive CD8 T cells (GEO: GSE9650 ), top TFs correlated with the dysfunctional program (Li et al. ), glycolysis (MSigDB: hallmark), and IFN-alpha response (MSigDB: hallmark) in tumor-reactive CD8 TILs. (F) Top 5 enriched GO terms for up- or downregulated genes in bystander cells by rhIL-7-hyFc treatment. (G) Violin plots with expression of genes related to ribosomal proteins, CM T cell, and T cell regulation in bystander cells. (H) GSEA analysis with the gene set of memory vs. exhausted CD8 T cells (GEO: GSE9650 ), positive regulation of TCR pathway (GO: 0050862 ), oxidative phosphorylation (MSigDB: hallmark), and IFN-alpha response (MSigDB: hallmark) in bystander CD8 TILs. Tumor-reactive cells are defined as cells with a clone size of 3 or greater, and bystander cells are defined as cells with a clone size of 1 or 2 and belonging to one of clusters 0, 2, 3, and 5. BIR., break-induced replication; DSBs., double-strand breaks; neg., negative; reg., regulation; sys., system; Pos., positive. See also Figures S2 and ; Table S2 .

Article Snippet: Anti-mouse CD8β PE (clone: H35–17.2) , eBioscience , Cat# 12-0083-83; RRID: AB_657767.

Techniques: Expressing, Phospho-proteomics

Journal: Cell Reports Medicine

Article Title: IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T cell engager immunotherapy

doi: 10.1016/j.xcrm.2024.101567

Figure Lengend Snippet: TCE stimulation elicits tumoricidal activity of rhIL-7-hyFc-induced bystander CD8 TILs (A) Schematic protein structure of PD-L1×CD3 TCE. This figure was created with BioRender.com . (B) The cytotoxicity of PD-L1 −/− splenocytes from naive PD-L1-deficient mice was evaluated. These splenocytes were co-cultured with CTV-labeled MC38 WT or MC38 ΔPD−L1 tumor cells in the presence of TCE at indicated concentrations for 48 h ( n = 3 per group). (C‒F) Functional assay of PD-L1×CD3 TCE on rhIL-7-hyFc-induced tumor-reactive and bystander CD8 TILs ( n = 3 per group). (C) Experimental scheme. (D) Expression of PD-1 and GzmB in PD-1 + or PD-1 − CD8 T cells co-cultured with MC38 in the presence of TCE at indicated concentration (left) and frequencies of PD-1 + GzmB + cells among CD8 T cells. Black and blue dots indicate PD-1 + and PD-1 − CD8 TILs before co-culture, respectively (right). (E) Frequencies of PD-1 + Prf + cells among CD8 T cells. (F) Cytotoxicity of PD-1 + and PD-1 − CD8 T cells in the presence of TCE. The expression of ghost dye in tumor cells was measured by flow cytometry. CTV + Ghost dye + cells are considered dead tumor cells. (G‒K) The functional changes and antitumor effects of rhIL-7-hyFc-expanded PD-1 − bystander CD8 T cells were investigated ( n = 5–7 per group). (G) Experimental scheme. MC38-bearing RAG1 −/− mice were injected i.t. with 4 × 10 6 CD8 + CD44 + CD62L + PD-1 − T cells sourced from the spleen and lymph nodes of C57BL/6 mice treated with rhIL-7-hyFc (10 mg kg −1 ). PD-L1×CD3 TCE (2 μg) or PBS was administered i.t. 5 times daily from the next day after T cell transfer. For flow cytometry analysis, tumors were collected 24 h after the second TCE treatment. (H) Average (left) and individual (right) tumor growth curves of MC38-bearing RAG1 −/− mice. The timing of TCE or PBS administration is indicated by blue or gray columns, respectively. (I) Representative plots showing the expression of GzmB in CD8 T cells (left) and frequency of GzmB + cells among CD8 T cells (right). (J) Representative plots showing the expression of Prf in CD8 T cells (left) and frequency of Prf + cells among CD8 T cells (right). (K) Representative plots showing the expression of Ki-67 in CD8 T cells (left) and frequency of Ki-67 + cells among CD8 T cells (right). Data are shown as mean ± SEM and representative of two or three independent experiments (B, D-F, and H) or a summary of two independent experiments (I–K). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by unpaired two-tailed Student’s t test (B, I, J, and K), by one-way ANOVA and Tukey’s multiple comparisons test (D–F), and by two-way ANOVA and Tukey’s multiple comparisons test (H). ns, not significant. See also Figures S3 and .

Article Snippet: Anti-mouse CD8β PE (clone: H35–17.2) , eBioscience , Cat# 12-0083-83; RRID: AB_657767.

Techniques: Activity Assay, Cell Culture, Labeling, Functional Assay, Expressing, Concentration Assay, Co-Culture Assay, Flow Cytometry, Injection, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T cell engager immunotherapy

doi: 10.1016/j.xcrm.2024.101567

Figure Lengend Snippet: TCE combination promotes the cytotoxic activity of rhIL-7-hyFc-induced bystander CD8 TILs (A) Experimental scheme. Mice bearing palpable MC38 tumors were injected s.c. with rhIL-7-hyFc (1.25 mg kg −1 ). Three days after rhIL-7-hyFc treatment, mice were treated i.v. with PD-L1×CD3 TCE (0.4 μg) 2 times daily. Twenty-four hours after the last treatment, mice were analyzed for TILs ( n = 4–6 per group). (B) Frequencies (left) and numbers (right) of CD8 T, CD4 T reg , CD4 non-T reg , and NK cells. (C) Frequencies of PD-1 − cells (left) and numbers of PD-1 + and PD-1 − cells among the total CD8 T cells (right). (D) Representative plots showing the expression of CD44 and CD62L in PD-1 − CD8 T cells. (E) Frequencies of CD44 + CD62L + (left) and CD44 + CD62L − (right) cells among PD-1 − CD8 T cells. (F, H, and I) Frequencies (left) and numbers (right) of GzmB + cells (F), Prf + cells (H), and Ki-67 + cells (I) among PD-1 − CD8 T cells. (G) Histogram of GzmB expression in PD-1 − CD8 T cells (left) and fold change of GzmB geometric mean fluorescence intensity (gMFI) compared with the buffer group in PD-1 − CD8 T cells. Data are shown as mean ± SEM and summary of two independent experiments (B, C, F, G, and I) or representative of two independent experiments (E and H). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by one-way ANOVA and Tukey’s multiple comparisons test. ns, not significant. See also Figures S7 and .

Article Snippet: Anti-mouse CD8β PE (clone: H35–17.2) , eBioscience , Cat# 12-0083-83; RRID: AB_657767.

Techniques: Activity Assay, Injection, Expressing, Fluorescence

Journal: Cell Reports Medicine

Article Title: IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T cell engager immunotherapy

doi: 10.1016/j.xcrm.2024.101567

Figure Lengend Snippet: Transcriptome analysis of CD8 TIL subsets in combination therapy scRNA-seq results of CD8 TILs from mice bearing MC38 tumors treated with rhIL-7-hyFc alone or a combination of rhIL-7-hyFc and TCE. C57BL/6 mice bearing palpable MC38 tumors were injected s.c. with rhIL-7-hyFc (1.25 mg kg −1 ). Three days after rhIL-7-hyFc treatment, mice were treated i.v. with PD-L1×CD3 TCE (0.4 μg) three times daily. Tumors were collected 24 h after the last treatment. Collected tumor tissues were pooled for analysis ( n = 12 rhIL-7-hyFc and n = 24 combination). (A) UMAP plot showing each CD8 TIL cluster. (B) Supervised clustering of CD8 TILs according to gene-expression characteristics (left) and bar graph depicting the proportion of three CD8 TIL subclusters in each treatment condition (right). (C) Heatmap showing the DEGs between supervised groups. (D) Featured plots showing the expression of Pdcd1 (left) and Tcf7 (right) genes. (E) UMAP plot of top six most expanded clones. (F) Number of DEGs in each supervised CD8 TIL subcluster from mice with combination therapy compared with the rhIL-7-hyFc-treated group. (G) Dot plots of GO enrichment analysis of upregulated DEGs by combination therapy in each CD8 TIL subcluster. (H) Violin plots showing the expression of genes related to exhaustion, CM-associated, cytotoxicity, T cell activation, glycolysis, and cell motility. See also Figures S9 and ; Table S3 .

Article Snippet: Anti-mouse CD8β PE (clone: H35–17.2) , eBioscience , Cat# 12-0083-83; RRID: AB_657767.

Techniques: Injection, Gene Expression, Expressing, Clone Assay, Activation Assay

Journal: Cell Reports Medicine

Article Title: IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T cell engager immunotherapy

doi: 10.1016/j.xcrm.2024.101567

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD8β PE (clone: H35–17.2) , eBioscience , Cat# 12-0083-83; RRID: AB_657767.

Techniques: Purification, Recombinant, Formulation, Flow Cytometry, Amplification, Software, Staining