cd8 knockout ko mice  (Jackson Laboratory)

Journal: Cancer Research

Article Title: GFER Represents a Target for Dual Disruption of Redox Homeostasis and Reactivation of the Immune Response in Pancreatic Adenocarcinoma

doi: 10.1158/0008-5472.CAN-24-4211

Figure Lengend Snippet: Immunogenicity increases upon GFER depletion in PDAC cells. A–E, Gene set enrichment analysis of RNA-seq data reveals the upregulation of the IFNα response ( A ), IFNγ response ( B ), TNFα signaling via NFκB ( C ), inflammatory response ( D ), and IL2–STAT5 signaling ( E ) pathways in GFER-depleted PATC 124 cells infected with sgGFER. A total of 100,000 PATC cells were cultured in six-well plates for 7 days and then measured. F, RNA-seq analysis demonstrates significant changes in gene expression in orthotopic tumor samples derived from 10,000 KPC (p53 mut) cells infected with nontargeting shRNA ( n = 2) as well as shGFER (shGFER#4, n = 2; shGFER#5, n = 3). Tumors were harvested when all tumors were of comparable size. Immune system–related genes are highlighted in red boxes. G, Protein levels of chemokines ( CXCL9, CXCL10 , and CXCL11 ) related to CD8 + T-cell infiltration in orthotopic shCTRL and shGFER KPC (p53 mut) tumors. Statistical analysis was performed using a two-tailed Student unpaired t test. Data represent the mean ± SD; n = 4 mice. H and I, Immunophenotyping of orthotopic shCTRL and shGFER KPC tumors. A total of 10,000 KPC cells were injected into the pancreas, and tumors were harvested for multicolor flow cytometry analysis. The percentage of CD8 + T cells, CD4 + T cells, NK cells (NK1.1+ cells), B cells (B220+ cells), macrophages (F4/80+ cells), and dendritic cells (DC; CD11c + cells) were identified in CD45 + cells ( H ). The percentage of PD-1–positive and granzyme B–positive cells were identified in CD8 + and CD8 + PD-1 + cells, respectively ( I ). Fibroblasts were identified as CD45 − EpCAM − αSMA + cells ( J ). Statistical analysis was performed using a two-tailed Student unpaired t test. Data represent the mean ± SD; n = 5 mice. K, Kaplan–Meier survival analysis of C57BL/6 mice orthotopically implanted with 10,000 KPC (p53 mut) cells infected with shCTRL or shGFER. Mice received either anti-CD8 antibody (10 mg/kg) or rat IgG2a starting 1 day prior to surgery and then intraperitoneally every 7 days for four cycles. Animals were euthanized when tumor volume reached ∼2000 mm 3 . Survival curves were analyzed by one-way ANOVA ( n = 10 per group). L, Growth curves of 10,000 KPC (p53 mut) cells subcutaneously implanted into C57BL/6 mice. Prior to implantation, KPC cells were infected with shCTRL or shGFER. Tumor-bearing mice were treated with IgG control or anti–PD-1 antibodies. Tumor volume was measured every 4 days for 36 days. Statistical analysis was performed using one-way ANOVA. Data represent the mean ± SD; n = 4–7 per group. M, Pancreatic cancer patient with anti–PD-1/anti-CD40 treatment prior to chemotherapy were selected from the PRINCE trial and divided in to GFER-high and -low groups. The overall survival analysis was done with log-rank test ( N = 17).

Article Snippet: We used 8-week-old male or female nude mice (Strain #002019, The Jackson Laboratory) and immunocompetent C57BL/6J mice (Strain #000664, The Jackson Laboratory) and CD8 knockout (KO) mice (Strain #002665, The Jackson Laboratory) to establish subcutaneous and orthotopic models, with n = 3–10 mice per treatment group.

Techniques: Immunopeptidomics, RNA Sequencing, Infection, Cell Culture, Gene Expression, Derivative Assay, shRNA, Two Tailed Test, Injection, Flow Cytometry, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synthetic high-density lipoprotein nanodiscs for personalized immunotherapy against gliomas

doi: 10.1158/1078-0432.CCR-20-0341

Figure Lengend Snippet: A-E) C57BL/6 mice inoculated s.c. with 1.2 × 106 GL261 tumor cells were vaccinated on day 20 and administered with anti-PD-L1 on days 21 and 24. On day 26, tumor tissues were stained with antibodies and analyzed by flow cytometry for CD8a+ T-cells, Tregs, and DCs (n = 4 per group).

Article Snippet: Immunocompetent female C57BL/6 or immunocompromised CD4−/− and CD8−/− knockout (KO) mice (Jackson Laboratory) were stereotactically injected with 20,000 GL261 cells into the right striatum using a 22-gauge Hamilton syringe (1 μL over 1 minute) with the following coordinates: +1.00 mm anterior, 2.5 mm lateral, and 3.00 mm deep to establish brain tumors ( 36 – 39 ).

Techniques: Staining, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synthetic high-density lipoprotein nanodiscs for personalized immunotherapy against gliomas

doi: 10.1158/1078-0432.CCR-20-0341

Figure Lengend Snippet: A) Treatment regimen and study timeline. B) IFN-γ ELISPOT assays were performed using PBMCs on day 7 after prime (left) and boost (right) vaccinations (n = 3 per group). C) Kaplan-Meier overall survival curves for all treatment groups (n = 10 per group). D) Kaplan-Meier survival analysis of long-term survivors from (C) re-challenged with GL261 cells in the contralateral hemisphere. E) CD4−/− KO mice or F) CD8−/− KO mice carrying orthotopic GL261 tumors were treated with nanodisc vaccine + anti-PD-L1 therapy as in A) and monitored for survival (n = 5 per group).

Article Snippet: Immunocompetent female C57BL/6 or immunocompromised CD4−/− and CD8−/− knockout (KO) mice (Jackson Laboratory) were stereotactically injected with 20,000 GL261 cells into the right striatum using a 22-gauge Hamilton syringe (1 μL over 1 minute) with the following coordinates: +1.00 mm anterior, 2.5 mm lateral, and 3.00 mm deep to establish brain tumors ( 36 – 39 ).

Techniques: Enzyme-linked Immunospot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synthetic high-density lipoprotein nanodiscs for personalized immunotherapy against gliomas

doi: 10.1158/1078-0432.CCR-20-0341

Figure Lengend Snippet: A-I) C57BL/6 mice were inoculated with GL261 cells and treated as in Figure 5, and tumors were isolated on day 23 and analyzed by flow cytometry (n = 5 per group). Shown are A) CD8α+ T cells among all T cells, B) PD-1 receptor expression on CD8α+ T cells, C) CD25+Foxp3+ regulatory T cells among CD4+ T cells, D) ratio of CD8α+ T cells to regulatory T cells, and E) the ratio of M1 (CD206- F4/80+) to M2 (CD206+ F4/80+) macrophages.

Article Snippet: Immunocompetent female C57BL/6 or immunocompromised CD4−/− and CD8−/− knockout (KO) mice (Jackson Laboratory) were stereotactically injected with 20,000 GL261 cells into the right striatum using a 22-gauge Hamilton syringe (1 μL over 1 minute) with the following coordinates: +1.00 mm anterior, 2.5 mm lateral, and 3.00 mm deep to establish brain tumors ( 36 – 39 ).

Techniques: Isolation, Flow Cytometry, Expressing