Journal: Blood Advances

Article Title: Expression and treatment of ROR1 + cells with bispecific T-cell engagers in pediatric acute lymphoblastic leukemia

doi: 10.1182/bloodadvances.2024013814

Figure Lengend Snippet: ROR1 is expressed in both BCP-ALL and T-ALL. (A) Proportion of ROR1 + cells and (B) number of ROR1 binding sites in 19 BCP-ALL, 10 T-ALL cases, and 6 NBM samples. Each symbol can be distinguished by MRD status (low-, intermediate-, or high-risk). Solid horizontal lines represent medians, error bars represent the interquartile ranges, and interrupted lines represent the NBM median. ∗ P ≤ .05.

Article Snippet: On termination, femoral BM was assessed by flow cytometry for human cell engraftment using antibodies against CD34, CD45, CD7, and CD19 (all from BD Biosciences) and ROR1 (Miltenyi biotec).

Techniques: Binding Assay

Journal: Blood Advances

Article Title: Expression and treatment of ROR1 + cells with bispecific T-cell engagers in pediatric acute lymphoblastic leukemia

doi: 10.1182/bloodadvances.2024013814

Figure Lengend Snippet: ROR1 is coexpressed with BCP-ALL and T-ALL lineage markers. Proportion of ROR1 + cells in (A) BCP-ALL (Pts 1-19) or (B) T-ALL (Pts 20-29) and their respective LPC subpopulations. (C-D) Number of ROR1 binding sites in BCP-ALL and T-ALL and subpopulations. Each symbol represents an individual patient sample. Horizontal lines represent medians and error bars represent the interquartile ranges. ∗ P ≤ .05, ∗∗ P ≤ .01, and ∗∗∗ P ≤ .001 when directly compared to CD19 – and CD7 – subpopulations and analyzed by Kruskal-Wallis test.

Article Snippet: On termination, femoral BM was assessed by flow cytometry for human cell engraftment using antibodies against CD34, CD45, CD7, and CD19 (all from BD Biosciences) and ROR1 (Miltenyi biotec).

Techniques: Binding Assay

Journal: Blood Advances

Article Title: Expression and treatment of ROR1 + cells with bispecific T-cell engagers in pediatric acute lymphoblastic leukemia

doi: 10.1182/bloodadvances.2024013814

Figure Lengend Snippet: Proliferation of ROR1 + and ROR1 – cell populations. Cells from BCP-ALL (Pts 16, 17, and 19) (A), T-ALL (Pts 20, 22, and 27) (B), and 3 NBM cases (C) were stained with proliferation marker, CTV before being cultured for 7 days. CTV MFI of ungated, ROR1 + and ROR1 – cells and subpopulations was compared between day 0 (undivided) and day 7 cells and analyzed by Kruskal-Wallis test. ∗ P = .04. Data are expressed as mean ± standard deviation (SD). MFI, median fluorescence intensity.

Article Snippet: On termination, femoral BM was assessed by flow cytometry for human cell engraftment using antibodies against CD34, CD45, CD7, and CD19 (all from BD Biosciences) and ROR1 (Miltenyi biotec).

Techniques: Staining, Marker, Cell Culture, Standard Deviation, Fluorescence

Journal: Blood Advances

Article Title: Expression and treatment of ROR1 + cells with bispecific T-cell engagers in pediatric acute lymphoblastic leukemia

doi: 10.1182/bloodadvances.2024013814

Figure Lengend Snippet: ROR1 + LPC are resistant to induction therapy. Proportion of ROR1 positive cells (A-B) and number of binding sites (C-D) detected on samples at presentation (day 0), day 29, and wk 14 from BCP-ALL Pts 3, 12, and 17 (A,C) and T-ALL Pts 20 to 24 and 27 (B,D). Interrupted outlines indicate survival of ROR1 + LPC populations after induction therapy. Arrows indicate the emergence of ROR1 + in LPC. HSC, hematopoietic stem cell. Wk14, week 14.

Article Snippet: On termination, femoral BM was assessed by flow cytometry for human cell engraftment using antibodies against CD34, CD45, CD7, and CD19 (all from BD Biosciences) and ROR1 (Miltenyi biotec).

Techniques: Binding Assay

Journal: Blood Advances

Article Title: Expression and treatment of ROR1 + cells with bispecific T-cell engagers in pediatric acute lymphoblastic leukemia

doi: 10.1182/bloodadvances.2024013814

Figure Lengend Snippet: ROR1-bispecific T eng reduce ALL viability. (A) BCP-ALL cells (Pts 16 and 19), T-ALL cells (Pts 22-24 and 26), and NBM cells (n = 4) were incubated 1:1 with T cells ± ROR1 T eng or CD19 T eng for 24 hours and cell viability assessed by flow cytometry. (B) IFN-γ release was measured in supernatants of cytotoxicity assays. Data represent mean ± SD. ∗ P ≤ .05, ∗∗ P ≤ .01, and ∗∗∗ P ≤ .001.

Article Snippet: On termination, femoral BM was assessed by flow cytometry for human cell engraftment using antibodies against CD34, CD45, CD7, and CD19 (all from BD Biosciences) and ROR1 (Miltenyi biotec).

Techniques: Incubation, Flow Cytometry

Journal: Blood Advances

Article Title: Expression and treatment of ROR1 + cells with bispecific T-cell engagers in pediatric acute lymphoblastic leukemia

doi: 10.1182/bloodadvances.2024013814

Figure Lengend Snippet: T eng reduce leukemia burden in vivo. NSG mice were inoculated with BCP-ALL (Pts 16, 18, and 19) and T-ALL (Pt 22) samples, before being treated with T eng and T cells. (A) Engraftment levels in PB, monitored weekly by flow cytometry. Gray boxes represent duration of treatment. Data in PB graphs represent mean ± SD (n = 3-4 mice per treatment condition). (B) Proportion of ROR1 + and CD19 + cells in BM at termination. (C) IFN-γ levels in murine plasma at termination. Bars represent mean ± SD (n = 3-4).

Article Snippet: On termination, femoral BM was assessed by flow cytometry for human cell engraftment using antibodies against CD34, CD45, CD7, and CD19 (all from BD Biosciences) and ROR1 (Miltenyi biotec).

Techniques: In Vivo, Flow Cytometry, Clinical Proteomics

Journal: bioRxiv

Article Title: Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1 + Tcf1 + stem-like reservoir and overcomes resistance to PD-1 axis blockade

doi: 10.1101/2025.04.10.648245

Figure Lengend Snippet: A) Schematic of retroviral constructs used to engineer murine ROR1 CAR-T and control T cells. TM = transmembrane. tCD19 = truncated murine CD19. B) Frequency (left), PD-1 and TIM-3 expression (middle), and IFNψ production upon ex vivo restimulation with PMA and ionomycin (right) by infused CD8 + CD19 + T cells in lungs 8 days post-infusion into KP ROR1 mice. N=3-6 mice per group. Unpaired Student’s two-way t-test. C) Representative flow plots (left) and summary (right) of Tcf1 and PD-1 expression by CD8 + CD19 + CAR-T cell over time in vivo . N=5 mice per group. One-way ANOVA with Tukey’s post-test. D) PD-L1 expression on CD11b + Ly6G + neutrophils and CD11c + F4/80 + macrophages within KP ROR1 lung tumors 8 days post-treatment with control T cells or ROR1 CAR-T cells. N=3-6 mice per group. Unpaired Student’s two-way t-test. E) ROR1 CAR-T cell number (left) and PD-1 expression (right) 14 and 35 days post-infusion into KP ROR1 mice and treatment with vehicle or anti-PD-L1. N=5-6 mice per group. Unpaired Student’s two-way t-test. F) Representative IHC staining for ROR1 on lungs of KP ROR1 mice 45 days post-infusion with ROR1 CAR-T cells +/- anti-PD-L1. G) Quantification of ROR1 + tumor from IHC images of KP ROR1 lungs treated as indicated 45 days post-infusion. N=6-7 mice per group. Unpaired Student’s two-way t-test. Data are representative of 2 independent experiments.

Article Snippet: Endogenous peroxidase was blocked with H2O2 (in the Leica kit D59800) for 5 min followed by protein blocking with TCT buffer (0.05 M Tris, 0.15 M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 +/- 0.1) for 10 min. Primary rabbit anti-ROR1 antibody at 1:500 (Cell Signaling #16540) or rabbit anti-CD8 antibody at 1:200 (Cell Signaling #98941) was applied for 60 minutes followed by the Refine reagent Rbt-HRP polymer (in the Leica kit D59800) for 12 minutes.

Techniques: Retroviral, Construct, Control, Expressing, Ex Vivo, In Vivo, Immunohistochemistry

Journal: bioRxiv

Article Title: Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1 + Tcf1 + stem-like reservoir and overcomes resistance to PD-1 axis blockade

doi: 10.1101/2025.04.10.648245

Figure Lengend Snippet: A) Schematic of ROR1 CAR, cJun.ROR1 CAR, and BATF.ROR1 CAR retroviral constructs used to generate murine CAR-T cells. TM = transmembrane. tCD19 = truncated murine CD19. B) Tcf1 expression (left) and accumulation (right) of CD8 + GFP + CAR-T cells in infusion products ( IP ) and within tumors 8 days post-infusion into B6 mice transplanted with KP-ROR1 lung tumors. One-way ANOVA with Tukey’s post-test. N=5-6 mice per group. C) Survival of B6 mice transplanted with KP-ROR1 lung tumors and treated with Cy and indicated CAR-T cells. Log-rank Mantel-Cox test. N=4-5 mice per groups. D) Treatment scheme showing co-infusion of CAR-T cells. KP = Kras LSL-G12D/+ p53 f/f . I.T = intratracheal. Cy = cyclophosphamide. E) Frequency of CD45.1 + CD8 + GFP + ROR1 (pink) and CD45.2 + CD8 + GFP + cJun.ROR1 CAR-T cells (teal) of total live cells in spleens or lungs from non-tumor bearing WT or tumor-bearing KP ROR1 mice 8 and 22 days post-CAR-T cell infusion. Lines indicate paired comparisons between ROR1 CAR-T and cJun.ROR1 CAR-T cells in the same mice. N=3-6 mice per group. Paired Student’s two-way t-test. F) Treatment scheme showing separate CAR-T infusion. G) Frequency of CD8 + GFP + ROR1 (pink) and cJun.ROR1 CAR-T cells (teal) of total live cells in lungs of tumor-bearing KP ROR1 mice 8 and 35 days post-CAR-T cell infusion. N=5-9 mice per group. Unpaired Student’s two-way t-test. H) Representative IHC staining for CD8 on lungs of KP ROR1 mice 21 days post-infusion with ROR1 CAR-T or cJun.ROR1 CAR-T cells. Yellow line indicates tumor margin. I) IHC analysis showing quantification of CD8 + cells at various distances from tumor margin in KP ROR1 lungs 21 days post-infusion of ROR1 CAR-T or cJun.ROR1 CAR-T cells. Two-way ANOVA with Tukey’s post-test. N=3 mice per group. Data are representative of 2-5 independent experiments.

Article Snippet: Endogenous peroxidase was blocked with H2O2 (in the Leica kit D59800) for 5 min followed by protein blocking with TCT buffer (0.05 M Tris, 0.15 M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 +/- 0.1) for 10 min. Primary rabbit anti-ROR1 antibody at 1:500 (Cell Signaling #16540) or rabbit anti-CD8 antibody at 1:200 (Cell Signaling #98941) was applied for 60 minutes followed by the Refine reagent Rbt-HRP polymer (in the Leica kit D59800) for 12 minutes.

Techniques: Retroviral, Construct, Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1 + Tcf1 + stem-like reservoir and overcomes resistance to PD-1 axis blockade

doi: 10.1101/2025.04.10.648245

Figure Lengend Snippet: A, B): Representative flow cytometry plots (A) and summary (B) of inhibitory receptor expression on CD8 + GFP + ROR1 CAR-T (pink) and cJun.ROR1 CAR-T cells (teal) from KP ROR1 lungs 9 days and 30 days post-infusion. N=3-5 mice per group. Unpaired Student’s two-way t-test. C, D) Representative flow cytometry plots (C) and summary (D) of intracellular cytokine staining of CD8 + GFP + ROR1 CAR-T (pink) and cJun.ROR1 CAR-T cells (teal) in KP ROR1 lungs 8 and 35 days post-infusion after ex vivo restimulation with PMA and ionomycin. N=5-9 mice per group. Unpaired Student’s two-way t-test. E, F) Representative flow cytometry plots (E) and summary (F) of Tox expression in CD8 + GFP + ROR1 CAR-T (pink) and cJun.ROR1 CAR-T cells (teal) in KP ROR1 lungs 9 and 30 days post-infusion. N=3-5 mice per group. Two-way ANOVA with Tukey’s post-test. Data are representative of 2-5 independent experiments.

Article Snippet: Endogenous peroxidase was blocked with H2O2 (in the Leica kit D59800) for 5 min followed by protein blocking with TCT buffer (0.05 M Tris, 0.15 M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 +/- 0.1) for 10 min. Primary rabbit anti-ROR1 antibody at 1:500 (Cell Signaling #16540) or rabbit anti-CD8 antibody at 1:200 (Cell Signaling #98941) was applied for 60 minutes followed by the Refine reagent Rbt-HRP polymer (in the Leica kit D59800) for 12 minutes.

Techniques: Flow Cytometry, Expressing, Staining, Ex Vivo

Journal: bioRxiv

Article Title: Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1 + Tcf1 + stem-like reservoir and overcomes resistance to PD-1 axis blockade

doi: 10.1101/2025.04.10.648245

Figure Lengend Snippet: A) Unsupervised clustering of ROR1 and cJun.ROR1 CAR-T cells from the Infusion Product or from KP ROR1 tumors 9 and 30 days post-infusion and analyzed by scRNA-seq. B) Clusters in (A) colored by time point. C) Dot plot showing expression of genes that define each CAR-T cell subcluster. D) Geneset scores of enriched pathways from Jansen et al. in CAR-T cell subclusters. E) Clusters in (A) colored by CAR-T cell product. F) K-nearest neighbor graph using Milo showing neighborhoods enriched in cJun.ROR1 CAR-Ts (teal) or ROR1 CAR-Ts (pink). G) Differential abundance analysis using Milo showing fold change enrichment of neighborhoods within specific CAR-T subclusters in either cJun.ROR1 CAR-Ts (teal) or ROR1 CAR-Ts (pink). H) Heatmap showing genes differentially expressed between neighborhoods enriched in ROR1 CAR-T or cJun.ROR1 CAR-T cells. I, J) Representative flow cytometry plots (I) and summary (J) showing frequency of Tcf1 + TIM-3 - stem-like subset among CD8 + GFP + PD-1 + CAR-T cells in KP ROR1 lungs 9 and 30 days post-infusion. N=3-4 mice per group. Two-way ANOVA with Tukey’s post-test. K) Frequency of Tcf1 + TIM-3 - stem-like cells among CD45.1 + CD8 + GFP + PD-1 + ROR1 CAR-T (pink) and CD45.2 + CD8 + GFP + PD-1 + cJun.ROR1 CAR-T cells (teal) in KP ROR1 lungs 9 days post-co-infusion. Lines indicated matched ROR1 and cJun.ROR1 CAR-T cells within the same mice. N=6 mice per group. Paired Student’s two-way t-test. Data are representative of 2 independent experiments.

Article Snippet: Endogenous peroxidase was blocked with H2O2 (in the Leica kit D59800) for 5 min followed by protein blocking with TCT buffer (0.05 M Tris, 0.15 M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 +/- 0.1) for 10 min. Primary rabbit anti-ROR1 antibody at 1:500 (Cell Signaling #16540) or rabbit anti-CD8 antibody at 1:200 (Cell Signaling #98941) was applied for 60 minutes followed by the Refine reagent Rbt-HRP polymer (in the Leica kit D59800) for 12 minutes.

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1 + Tcf1 + stem-like reservoir and overcomes resistance to PD-1 axis blockade

doi: 10.1101/2025.04.10.648245

Figure Lengend Snippet: A) Treatment schematic. I.T. = intratracheal. Cy = cyclophosphamide. B) Representative flow cytometry plots showing expression of the CAR transduction marker (GFP) and PD-1 in CD8 + T cells from KP ROR1 lungs 5 days post-infusion. C) Absolute number and PD-1 expression in CD8 + GFP + CAR-T cells (top) and CD8 + GFP - endogenous T cells (bottom) in lungs of KP ROR1 mice treated as indicated. N=4-5 mice per group. One-way ANOVA with Tukey’s post-test. D) Representative flow cytometry plots showing Tcf1 and TIM-3 expression in CD8 + GFP + PD-1 + CAR-T cells in KP ROR1 lungs 5 days post-infusion. E) Frequency of Tcf1 + TIM-3 - stem-like precursor exhausted ( Tpex ) and Tcf1 - TIM-3 + terminally exhausted ( Tex ) subsets of CD8 + GFP + PD-1 + CAR-T cells in KP ROR1 lungs 5 days post-infusion. N=4-5 mice per group. One-way ANOVA with Tukey’s post-test. Data are representative of 2 independent experiments

Article Snippet: Endogenous peroxidase was blocked with H2O2 (in the Leica kit D59800) for 5 min followed by protein blocking with TCT buffer (0.05 M Tris, 0.15 M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 +/- 0.1) for 10 min. Primary rabbit anti-ROR1 antibody at 1:500 (Cell Signaling #16540) or rabbit anti-CD8 antibody at 1:200 (Cell Signaling #98941) was applied for 60 minutes followed by the Refine reagent Rbt-HRP polymer (in the Leica kit D59800) for 12 minutes.

Techniques: Flow Cytometry, Expressing, Transduction, Marker

Journal: bioRxiv

Article Title: Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1 + Tcf1 + stem-like reservoir and overcomes resistance to PD-1 axis blockade

doi: 10.1101/2025.04.10.648245

Figure Lengend Snippet: A) Flow cytometry plots showing murine c-Jun expression in ROR1 CAR-T (pink) and cJun.ROR1 CAR-T cells (teal) pre-infusion and within KP ROR1 lungs 9 days post-infusion. B) Frequency of c-Jun (left) and ratio of c-Jun to GFP median fluorescence intensity ( MFI ) (right) in CD8 + GFP + CAR-T cells in the infusion product ( IP ) and in KP ROR1 lungs 9 days post-infusion. N=3-5 mice per group. Two-way ANOVA with Tukey’s post-test. C) Schematic of retroviral CAR and c-Jun constructs. D) Total c-Jun expression in CD8 + GFP + CAR-T cells in the IP or in KP ROR1 lungs 8 days post-infusion. N=5 mice per group. Two-way ANOVA with Tukey’s post-test. E) Total c-Jun expression in CD8 + GFP + Thy1.1 + CAR-T cells co-transduced with ROR1 CAR-P2A-tCD19/GFP and cJun-P2A-Thy1.1 vectors in the IP and in KP ROR1 lungs 8 days post-infusion. N=5 mice per group. Unpaired Student’s two-way t-test. F) Summary (left) and representative flow cytometry plot (right) of c-Jun expression in CD8 + GFP + ROR1 CAR-T (pink) and cJun.ROR1 CAR-T cells (teal) in KP ROR1 lungs 5 days post-infusion and treatment with vehicle (filled) or αPD-L1 antibody (unfilled). N=4-5 mice per group. One-way ANOVA with Tukey’s post-test. G) PD-1 expression on CRISPR/Cas9 mock-edited and PD-1 KO cJun.ROR1 CAR-T cell infusion products 24 hr post-stimulation with PMA/ionomycin. N=3 technical replicates. Unpaired Student’s two-way t-test. H) c-Jun levels in tumor-infiltrating cJun.ROR1 CAR-T cells treated as indicated 9 days post-infusion into B6 mice bearing transplanted KP ROR1 tumors. N=5-6 mice per group. One-way ANOVA with Tukey’s post-test. I) c-Jun levels in mock-edited and PD-1 KO cJun.ROR1 CAR-T cells 9 days post-co-infusion into tumor-bearing KP ROR1 mice. N=4 mice per group. Paired Student’s two-way t-test. Data are representative of 2 independent experiments.

Article Snippet: Endogenous peroxidase was blocked with H2O2 (in the Leica kit D59800) for 5 min followed by protein blocking with TCT buffer (0.05 M Tris, 0.15 M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 +/- 0.1) for 10 min. Primary rabbit anti-ROR1 antibody at 1:500 (Cell Signaling #16540) or rabbit anti-CD8 antibody at 1:200 (Cell Signaling #98941) was applied for 60 minutes followed by the Refine reagent Rbt-HRP polymer (in the Leica kit D59800) for 12 minutes.

Techniques: Flow Cytometry, Expressing, Fluorescence, Retroviral, Construct, Transduction, CRISPR

Journal: bioRxiv

Article Title: Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1 + Tcf1 + stem-like reservoir and overcomes resistance to PD-1 axis blockade

doi: 10.1101/2025.04.10.648245

Figure Lengend Snippet: A) Unsupervised clustering of spatial transcriptomic profiles of cells from lungs of KP ROR1 mice 7 days post-treatment with ROR1 CAR-T or cJun.ROR1 CAR-T cells +/- anti-PD-L1. N=1 lung per treatment group. B) Spatial plot showing localization of ROR1_CART + cells (black) in tumor (red), airway/blood vessels (blue), and normal lung parenchyma (grey). C) Proportion of cells in indicated spatial regions of each sample expressing the ROR1_CART transgene. CAR-T frequency was calculated within each individual tumor nodule. N=8-35 tumor nodules from 1 lung per group. One-way ANOVA with Tukey’s post-test. D) Pie chart showing proportion of each cluster in (A) within tumor region of each sample. E) Left: Proportion of various myeloid clusters among total cells in each tumor nodule. N=8-35 tumor nodules from 1 lung per group. One-way ANOVA with Tukey’s post-test. F) Left: Representative image illustrating proximity analysis calculation. Red = cells co-expressing ROR1_CART and Pdcd1 (PD-1 + CAR-T cells). Blue = cells expressing Cd274 (PD-L1 + cells). Right: mean number of PD-L1 + neighbors within a 20um radius of a PD-1 + CAR-T cell in each tumor nodule. N=17-35 tumor nodules from 1 lung per group. G) Sub-clustering of CAR-T cells. H) Dot plot showing expression of genes defining each cluster. I) Proportion of indicated CAR-T subcluster among total cell within each tumor nodule. N=8-35 tumor nodules from 1 lung per group. One-way ANOVA with Tukey’s post-test. J) Distance of indicated CAR-T subset from tumor border normalized by tumor radius. Connecting lines indicate Tpex and Teff CAR-T subsets within the same tumor nodule. Paired Student’s two-way t-test. N=8-35 tumor nodules from 1 lung per group.

Article Snippet: Endogenous peroxidase was blocked with H2O2 (in the Leica kit D59800) for 5 min followed by protein blocking with TCT buffer (0.05 M Tris, 0.15 M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 +/- 0.1) for 10 min. Primary rabbit anti-ROR1 antibody at 1:500 (Cell Signaling #16540) or rabbit anti-CD8 antibody at 1:200 (Cell Signaling #98941) was applied for 60 minutes followed by the Refine reagent Rbt-HRP polymer (in the Leica kit D59800) for 12 minutes.

Techniques: Expressing

Journal: bioRxiv

Article Title: Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1 + Tcf1 + stem-like reservoir and overcomes resistance to PD-1 axis blockade

doi: 10.1101/2025.04.10.648245

Figure Lengend Snippet: A) Treatment schematic. I.T. = intratracheal. Cy = cyclophosphamide. B) Representative IHC staining for ROR1 on lungs of KP ROR1 mice 42 days post-infusion with ROR1 CAR-T or cJun.ROR1 CAR-T cells. +/- anti-PD-L1. C) Quantification of ROR1 + tumor (top) and total tumor (bottom) from IHC images of KP ROR1 lungs treated as indicated 42 days post-infusion. N=3-7 mice per group. One-way with ANOVA with Tukey’s post-test. D) Treatment schematic. E) Left: ROR1 expression on KP-ROR1 tumor cell lines derived from GEM model and sorted to be >95% ROR1 + . Right: representative IHC showing ROR1 expression on lungs of B6 mice transplanted with KP-ROR1 lung tumors 21 days post-tumor injection and pre-CAR-T cell infusion. F) Representative flow plots showing GFP and PD-1 expression on CD8 + T cells within lung tumors 14 days post-infusion. G) Absolute number of CD8 + GFP + CAR-T cells and CD8 + PD-1 + endogenous T cells within lung tumors 14 days post-infusion. N=6-7 mice per group. One-way ANOVA with Tukey’s post-test. H) Survival. Log-rank Mantel-Cox test. N=7-8 mice per group. Data are representative of 2 independent experiments.

Article Snippet: Endogenous peroxidase was blocked with H2O2 (in the Leica kit D59800) for 5 min followed by protein blocking with TCT buffer (0.05 M Tris, 0.15 M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 +/- 0.1) for 10 min. Primary rabbit anti-ROR1 antibody at 1:500 (Cell Signaling #16540) or rabbit anti-CD8 antibody at 1:200 (Cell Signaling #98941) was applied for 60 minutes followed by the Refine reagent Rbt-HRP polymer (in the Leica kit D59800) for 12 minutes.

Techniques: Immunohistochemistry, Expressing, Derivative Assay, Injection