cd8 ko mice  (Jackson Laboratory)

Journal: iScience

Article Title: Skin-resident T cells play an important role in controlling skin colonization of Candidozyma (Candida) auris

doi: 10.1016/j.isci.2026.115862

Figure Lengend Snippet: Skin-resident CD8 + T cells play an important role in controlling C. auris colonization in the skin (A) Percentages of skin γδT, CD4 + αβT cells, and ILCs in CD8-KO and WT mice 6 days after 1× C. auris application. (B and C) Percentages of skin γδT, CD4 + αβT cells, and ILCs that express IL-17A (B) or IFNγ (C) in CD8-KO and WT mice 6 days after 1× C. auris application. (D) Average numbers of colonies of C. auris in the skin of CD8-KO and WT mice 6 after 1× C. auris application. One dot is of one mouse. N = 6. Data were compiled from two separate experiments. The data are presented as mean ± SD. ns: not significantly different ( p > 0.05), ∗ p < 0.05 from ∗∗ p < 0.01. Statistical significance was determined by an unpaired t test.

Article Snippet: CD8-KO mice , The Jackson Lab , Strain # 002665.

Techniques:

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: Biomolecules

Article Title: Enhancing CD8 + T Cells Infiltration Through the Protein Arginine Methyltransferase 5 (PRMT5)/CXCL10 Axis Restricts Cervical Cancer Progression

doi: 10.3390/biom15121717

Figure Lengend Snippet: Increases in PRMT5 expression were found to be linked to adverse prognosis in cervical cancer. ( A ) PRMT5 expression was conducted in both healthy cervical tissues and tumor tissues obtained from the TCGA database (num (T) = 306; num (N) = 13). ( B ) Correlation between PRMT5 expression and immune cells from TCGA database (ssgsea test). ( C ) The analyses were performed to investigate the association among high/low PRMT5 expression levels and T cells/CD8 + T cells/Macrophages/NK/DC in cervical cancer patients derived from TCGA database. ( D ) The relationship between PRMT5 expression and T cells, CD8 + T cells, Macrophages, NK, DC using the TCGA database (ssgsea test). OS ( E ), DSS ( F ) and PFI ( G ) analyses were conducted on cervical cancer patients with high/low levels of PRMT5 using data from the TCGA database. The data represent the mean ± SEM. Blue represents low PRMT5 expression, red represents high PRMT5 expression. ns = no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: B6.129P2-Cd8atm1Mak/J (CD8 KO) mice and B6.129P2-Cxcr3 tm1Dgen /J (CXCR3 KO) mice were acquired from Jackson Laboratory.

Techniques: Expressing, Derivative Assay

Journal: Biomolecules

Article Title: Enhancing CD8 + T Cells Infiltration Through the Protein Arginine Methyltransferase 5 (PRMT5)/CXCL10 Axis Restricts Cervical Cancer Progression

doi: 10.3390/biom15121717

Figure Lengend Snippet: Disruption of PRMT5 suppressed less tumor growth in CD8 KO mice. 6-week-old female C57BL/6 mice and CD8 KO mice received subcutaneous injections of either control cells or U14 cells with PRMT5 knockdown ( n = 5 per group). ( A – C ) The mice were euthanized, and the tumors that were resected were subsequently analyzed on day 16 after inoculation. ( A ) The tumor growth curve observed on the mice is depicted in a line graph. On day 16, images ( B ) and weight measurements ( C ) of the excised tumor were recorded. ( D ) A survival curve was generated for tumor-bearing mice that received subcutaneous injections of control and U14 cells with PRMT5 knockdown. The data represent the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: B6.129P2-Cd8atm1Mak/J (CD8 KO) mice and B6.129P2-Cxcr3 tm1Dgen /J (CXCR3 KO) mice were acquired from Jackson Laboratory.

Techniques: Disruption, Control, Knockdown, Generated

Journal: Biomolecules

Article Title: Enhancing CD8 + T Cells Infiltration Through the Protein Arginine Methyltransferase 5 (PRMT5)/CXCL10 Axis Restricts Cervical Cancer Progression

doi: 10.3390/biom15121717

Figure Lengend Snippet: Disruption of PRMT5 enhanced CXCL10 secretion by tumor cells. ( A ) Venn analysis comparing control cells and U14 cells with PRMT5 knockdown using RNA-seq. ( B ) Comparison of chemokine expression profiles between control cells and U14 cells with PRMT5 knockdown analyzed through RNA-seq. ( C ) Real-time PCR experiments were conducted to examine CCL5, CCL11, CCL4, CXCL9, and CXCL10 expression in control cells and U14 cells with PRMT5 knockdown. ( D ) CXCL10 level was measured by ELISA. ( E ) Correlation between CXCL10 expression and immune cell populations was assessed using the Kruskal–Wallis test from TCGA database. ( F ) Analysis of high PRMT5 expression enrichment compared to low PRMT5 expression in T cells and CD8 + T cells of cervical cancer patients from TCGA database. Blue represents low CXCL10 expression, red represents high CXCL10 expression. Correlation between CXCL10 expression and T cells ( G )/CD8 + T cells ( H ) was evaluated using the Kruskal–Wallis test from TCGA database. The data represent the mean ± SEM. ns = no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: B6.129P2-Cd8atm1Mak/J (CD8 KO) mice and B6.129P2-Cxcr3 tm1Dgen /J (CXCR3 KO) mice were acquired from Jackson Laboratory.

Techniques: Disruption, Control, Knockdown, RNA Sequencing, Comparison, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: Enhancing CD8 + T Cells Infiltration Through the Protein Arginine Methyltransferase 5 (PRMT5)/CXCL10 Axis Restricts Cervical Cancer Progression

doi: 10.3390/biom15121717

Figure Lengend Snippet: PRMT5 regulated CD8 + T cells recruitment through CXCL10/CXCR3 axis. Control cells and PRMT5 knockdown U14 cells were subcutaneously injected into 6-week-old female C57BL/6 mice and CXCR3 KO mice. Mice were euthanized at day 18 after inoculation ( n = 4 per group). The tumor single cell suspension was prepared and analyzed by flow cytometry. ( A ) A line graph shows the tumor growth curve of mice. Images ( B ) and weight ( C ) of the resected tumor at day 18 after inoculation. ( D ) The percentage of CD4 + and CD8 + T cells in CD45 + cells. The data represent the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: B6.129P2-Cd8atm1Mak/J (CD8 KO) mice and B6.129P2-Cxcr3 tm1Dgen /J (CXCR3 KO) mice were acquired from Jackson Laboratory.

Techniques: Control, Knockdown, Injection, Suspension, Flow Cytometry

Journal: Biomolecules

Article Title: Enhancing CD8 + T Cells Infiltration Through the Protein Arginine Methyltransferase 5 (PRMT5)/CXCL10 Axis Restricts Cervical Cancer Progression

doi: 10.3390/biom15121717

Figure Lengend Snippet: EPZ015666 effectively suppressed cervical cancer growth by enhancing intracellular cytokine expression in T cells. ( A – I ) 6-week-old female C57BL/6 mice received daily intraperitoneal injections of EPZ015666 (150 mg kg −1 ) 3 days after inoculation of U14 cells ( n = 5 per group). ( A ) The growth curve of tumors in the mice is illustrated by a line graph. On day 12 post-inoculation, images ( B ) and weight measurements ( C ) of the excised tumors were collected. ( D ) Analysis was conducted to determine PRMT5 expression and SDMA levels within the tumor. ( E ) The proportion of CD4 + and CD8 + T cells among CD45 + cells was determined. ( F ) IFN-γ, TNF-α and granzyme B expression levels were assessed in CD8 + T cells. ( G ) PD-1, TIM-3 and LAG-3 expression levels were revealed through surface examination on CD8 + T cells. ( H ) IFN-γ, TNF-α and Foxp3 expression levels were examined in CD4 + T cells. ( I ) PD-1, TIM-3 and LAG-3 expression levels was assessed on CD4 + T cells. The data represent the mean ± SEM. * p < 0.05 and *** p < 0.001.

Article Snippet: B6.129P2-Cd8atm1Mak/J (CD8 KO) mice and B6.129P2-Cxcr3 tm1Dgen /J (CXCR3 KO) mice were acquired from Jackson Laboratory.

Techniques: Expressing

Journal: iScience

Article Title: Smoking-induced gut microbial dysbiosis mediates cancer progression through modulation of anti-tumor immune response

doi: 10.1016/j.isci.2025.112002

Figure Lengend Snippet: Modulation of adaptive immune response drives the smoking-induced dysbiosis-mediated cancer progression (Also refer to Figures S2 and ) (A–C) Graphical representation of tumor flow-cytometric analysis (left) and bar graphs (right) of control, CSE, antibiotics alone, and CSE + antibiotics group showing percentage infiltration of CD3CD4+ T cells (A), CD3CD8+ T cells (B), and CD11b+Ly6G + MDSC (C) cells of single cells in the tumor microenvironment, respectively, showing CSE-induced immunosuppressive changes reversed with gut microbiome depletion ( n = 7–10 per group). (D–F) Tumor flow-cytometric analysis showing immunosuppressive changes characterized by decreased CD3CD4+ T cells (D), CD3CD8+ T cells (E), and increased CD11b+Ly6G + MDSC cells (F), respectively, induced in mice that received FMT from NNK-exposed mice vs. controls ( n = 7–8 per group). (G) Tumor volumes in WT and Rag1-KO mice exposed to CSE with or without gut microbiome depletion with broad-spectrum antibiotics ( n = 7–10 per group). (H) Tumor volumes in WT and Rag1-KO mice receiving FMT from smoke donors (smoke FMT) vs. control donors (control FMT) ( n = 9–12 for WT mice, n = 8–9 for Rag1-KO mice). (I) Tumor volumes in WT ( n = 14–15) and CD8-KO mice ( n = 7–8) exposed to CSE. (J) Tumor kinetics in WT CSE mice with or without Ly6G-depleting monoclonal antibody with appropriate controls ( n = 10 per group). ∗ p value <0.05; ∗∗ p value <0.01; ∗∗∗ p value <0.001. Unpaired t test or ANOVA used as appropriate for statistical comparison. Data presented as mean ± SEM.

Article Snippet: C57BL/6J WT mice, A/J mice (stock no. 000646), Rag1-KO mice (B6.129S7- Rag1 tm1Mom /J), as well as CD8-KO mice (B6.129S2- Cd8a tm1Mak /J), were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Control, Comparison

Journal: iScience

Article Title: Smoking-induced gut microbial dysbiosis mediates cancer progression through modulation of anti-tumor immune response

doi: 10.1016/j.isci.2025.112002

Figure Lengend Snippet: Smoke-induced dysbiosis is characterized by unique microbial and metabolomic signatures amenable to selective targeting (Also refer to Figures S4 and ) (A) Fecal samples were obtained from subcutaneous KPC tumor-bearing control and CSE mice and analyzed using 16s rRNA amplicon sequencing for metagenomic characterization. (Left panel) The significantly enriched microbes based on linear model analysis in the CSE (green bars) and control (red bars) groups are shown. (Right panel) PCoA plot comparing beta diversity between control (red) and CSE (green) groups. Distance was calculated using Bray-Curtis analysis, and PERMANOVA was used for statistical significance. (B) Fecal samples obtained from C57BL/6J mice receiving FMT from control or CSE donors and implanted with KPC pancreatic tumors were similarly interrogated through 16 s rRNA sequencing. Linear model (left panel) and Bray-Curtis analysis (right panel) are shown. (C) Heatmap representation of all differential fecal metabolites (BH FDR <0.25) between tumor-bearing control ( n = 10) and CSE ( n = 11) mice. A total of 194 differential metabolites were obtained upon untargeted LC-MS analysis. (D) Significantly altered metabolic pathways were obtained using pathway enrichment analysis of significantly altered fecal metabolites upon CSE using MetaboAnalyst 5.0. Only the metabolites annotated in HMDB were used for the analysis (88/194 differential metabolites). (E) Subcutaneous tumor volumes at endpoint after CSE with selective targeting of gut microbiome using oral neomycin gavage (200 mg/kg) or complete gut microbiome depletion using broad-spectrum antibiotics cocktail ( n = 8–12 per group). (F) Flow cytometric analysis of tumors showing % of CD3 + CD8 + of single cells after selective targeting of gut microbiome using oral neomycin gavage (200 mg/kg) or broad-spectrum antibiotic cocktail ( n = 7–8 per group). ∗ p value <0.05, ∗∗ p value <0.01. CSE, cigarette smoke exposure; PCoA, principle coordinate analysis; PERMANOVA, permutational analysis of variance; FMT, fecal microbiome transplant; LC-MS, liquid chromatography-mass spectrometry; BH, Benjamini-Hochberg; FDR, false discovery rate; HMDB, human metabolome database. Unpaired t test or ANOVA used, as appropriate, for statistical comparison. Data presented as mean ± SEM.

Article Snippet: C57BL/6J WT mice, A/J mice (stock no. 000646), Rag1-KO mice (B6.129S7- Rag1 tm1Mom /J), as well as CD8-KO mice (B6.129S2- Cd8a tm1Mak /J), were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Control, Amplification, Sequencing, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Comparison

Journal: iScience

Article Title: Smoking-induced gut microbial dysbiosis mediates cancer progression through modulation of anti-tumor immune response

doi: 10.1016/j.isci.2025.112002

Figure Lengend Snippet:

Article Snippet: C57BL/6J WT mice, A/J mice (stock no. 000646), Rag1-KO mice (B6.129S7- Rag1 tm1Mom /J), as well as CD8-KO mice (B6.129S2- Cd8a tm1Mak /J), were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Recombinant, Sequencing, Software

Journal: Frontiers in Immunology

Article Title: Coxiella burnetii Nine Mile phase I primary infection derived protective immunity against C. burnetii reinfection in mice depends on both B and T cells, but T cells play a critical role

doi: 10.3389/fimmu.2024.1427822

Figure Lengend Snippet: Compare NMI reinfection induced innate and adaptive cellular responses between PBS and different doses of primary NMI-infected mice. BALB/c mice were IP infected with PBS, 1 × 10 2 , 1 × 10 4 , or 1 × 10 5 GE of NMI bacteria. All dose of NMI-infected and PBS control mice were IP challenged with 1 × 10 7 GE of NMI bacteria at 35 days post primary NMI infection. In addition, naive BALB/c mice were used as uninfected normal mouse controls. The absolute cell numbers of macrophages plus monocytes, neutrophils, dendritic cells, B cells, CD8 + T cells, and CD4 + T cells in the spleen were determined by flow cytometry and compared between PBS control and primary NMI-infected mice at 14 days after NMI reinfection. (A) , macrophages plus monocytes (CD11b + CD11c − Ly6G − ). (B) , neutrophils (CD11b + Ly6G + ). (C) , Dendritic cells (CD11c + ). (D) , B cells (CD19 + ). (E) , CD8 + T cells (CD3 + CD8 + ). (F) , CD4 + T cells (CD3 + CD4 + ). Data presented in each group are the averages with SD for four mice, with error bars representing the SD from the means. *P < 0.05; **P < 0.01 and ****P < 0.0001 were determined by one-way ANOVA with Tukey’s post-test.

Article Snippet: BALB/c (Strain # 000651), C57BL/6 (Strain # 000664), B-cell KO ( Ighm tm1Cgn/J , strain # 002288) mice, T-cell KO ( Foxn1 nu , strain # 000819) mice, CD4 + T-cell KO ( Cd4 tm1Mak , strain # 002663) and CD8 + T-cell KO ( Cd8a tm1Mak , strain # 002665) mice were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Infection, Bacteria, Control, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Coxiella burnetii Nine Mile phase I primary infection derived protective immunity against C. burnetii reinfection in mice depends on both B and T cells, but T cells play a critical role

doi: 10.3389/fimmu.2024.1427822

Figure Lengend Snippet: The protective efficacy of primary NMI-infection induced protection against NMI reinfection in B cell, T cell, CD4 + T cell or CD8 + T cell deficient mice. C57BL/6J WT and B cell, T cell, CD4 + T cell or CD8 + T cell deficient mice were IP infected with 1 × 10 4 GE of NMI bacteria and challenged with 1 × 10 7 GE of NMI bacteria at 35 days post-primary NMI-infection. Additionally, naive WT mice were mock infected with PBS and served as controls. Splenomegaly and bacterial burden in the spleen were measured at 14 days post-challenge. (A) , Relative body weights (current body weight/day 0 body weight) were measured throughout the challenge period. (B) , Splenomegaly (% of spleen weight/body weight). (C) , bacterial burden in the spleen was determined by real-time qPCR and is expressed as log10 C . burnetii com1 gene copy numbers. Data presented in each group are the averages with SD for five mice, with error bars representing the SD from the means. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 were determined by one-way ANOVA with Tukey’s post-test.

Article Snippet: BALB/c (Strain # 000651), C57BL/6 (Strain # 000664), B-cell KO ( Ighm tm1Cgn/J , strain # 002288) mice, T-cell KO ( Foxn1 nu , strain # 000819) mice, CD4 + T-cell KO ( Cd4 tm1Mak , strain # 002663) and CD8 + T-cell KO ( Cd8a tm1Mak , strain # 002665) mice were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Infection, Bacteria

Journal: Nature Communications

Article Title: Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/β/δ treatment

doi: 10.1038/s41467-021-27833-0

Figure Lengend Snippet: A PTEN null CAP2, CAP8, PC3 and LNCaP prostate cancer cell lines and Pten -null prostate cancer in vivo model were treated with vehicle or 5μM BAY1082439 for 48 h (for cell lines), or 180 mg/kg bullet dose (for in vivo model). RNA-seq and GSEA analyses were performed and commonly down- or up-regulated pathways were presented with p < 0.05 as significant. Statistical test was performed by GSEA. B RNA-seq analysis identifies the core for PI3K-dependent immune modulating effect. RNAseq data from ( A ) and 72 h after BAY1082439 withdrawal were analyzed. The relative gene expression levels of indicated genes in each sample and cell line were determined and presented as heatmaps with p < 0.05 by two-sided T test (except in CAP8 cell line, p = 0.264 for STAT1 and p = 0.067 for Fas ). C RT-PCR analyses showed the relative expression levels of CCL5/CXCL10 chemokines. D ELISA measurement for the relative CCL5/CXCL10 secretion levels. PTEN null CAP2 and CAP8 cells were treated with BAY1082439 or vehicle for 48 h. Culture supernatant was collected and analyzed as suggested by manufactural recommendation. E Western blot analyses for P-AKT, Pan-AKT and B2M levels. PTEN null CAP2 and CAP8 cells were treated with BAY1082439 or vehicle for indicated time periods, and cell lysates were analyzed by Western blot using indicated antibodies. The experiment was repeated 3 independent times with similar results. F The positive correlations between IFNα/γ activity scores and CCL5/CXCL10/B2M/CD8A gene expression levels in human prostate cancer samples (499 patients). Linear regression was used, error bands represent 95% confidence intervals. Statistical test done by two-sided T test. C - D : each experiment was repeated 3 times and mean ± SEM were presented in C - D with * p < 0.05, ** p < 0.01, *** p < 0.001 by two-sided T test. Source data and exact p values are provided in the Source Data file.

Article Snippet: Cd8 atm1Mak mice ( Cd8 -KO mice) was purchased from the Jackson lab (002665) then crossed with the Pten -null mice to generate Pb-Cre + ;Pten LoxP/LoxP ;Cd8 -KO ( Pten -null; Cd8 -KO) mice.

Techniques: In Vivo, RNA Sequencing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay

Journal: Nature Communications

Article Title: Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/β/δ treatment

doi: 10.1038/s41467-021-27833-0

Figure Lengend Snippet: A A schematic illustration of treatment schedules. B FACS analyses for tumor-associated immune cells. Castrated Pten -null mice were treated with vehicle ( n = 18), BAY-D ( n = 7) or BAY-I ( n = 11) for 4 weeks. Tumor tissues were dissociated and weighted, the numbers of tumor-associated CD45 + , CD4 + and CD8 + T cells were measured by FACS analysis. Prostates were fixed and stained with HE, and cancer cell areas in anterior lobes were measured and fold of differences between vehicle and treatment groups were presented. Data were presented as dot plots with mean as the central lines; * p < 0.05; ** p < 0.01, *** p < 0.001 by two-sided T-test. C H&E and Immunohistochemistry analyses for immune cell infiltration. Consecutive sections were stained with H&E or antibodies against CD45, CD8 and GZMb. Dashed red lines: the boundaries between cancer acini and stroma areas. The same staining was performed with 6 mice in vehicle and BAY-I cohort and 7 mice in BAY-D cohort and similar results were observed. D RNA-seq and GSEA for BAY-I responses. RNAs were extracted from BAY-I treated tumor tissues for RNA-seq analysis. GSEA analysis showed enriched IFN-γ, T cell reporter and cytokine-cytokine receptor interaction signaling pathways in BAY-I treated cohort. Statistical test was performed by GSEA. Source data and exact p values are provided in the Source Data file.

Article Snippet: Cd8 atm1Mak mice ( Cd8 -KO mice) was purchased from the Jackson lab (002665) then crossed with the Pten -null mice to generate Pb-Cre + ;Pten LoxP/LoxP ;Cd8 -KO ( Pten -null; Cd8 -KO) mice.

Techniques: Staining, Immunohistochemistry, RNA Sequencing, Protein-Protein interactions

Journal: Nature Communications

Article Title: Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/β/δ treatment

doi: 10.1038/s41467-021-27833-0

Figure Lengend Snippet: A - B BAY-I treatment induced prolonged T cell-inflamed phenotype after drug withdrawal. Castrated Pten -null mice were treated with vehicle ( n = 24), BAY-I ( n = 32), or BAY-I then drug withdrawal for 4 ( n = 15) weeks, BAY-I then drug withdrawal then S1PR modulator Fingolimod for 4 weeks ( n = 6) or BAY-I then drug withdrawal for 10 weeks ( n = 5) before the analyses. Prostate tumor sizes and weight were presented in ( A ), and prostate tumor-associated CD8 + T cell number and the percentages of CD8 + in CD45 + cells were determined by FACS analysis ( B ). C IHC analysis showed that CD8 + T cells remained in the cancer acini 4 weeks after drug withdrawal. Red dash line marks the boundary between cancer acini and stroma areas. The same staining was performed with 6 mice in vehicle and BAY-I cohort and 8 mice in BAY-I withdraw cohort and similar results were observed. D RNA-seq analysis shows T cell inflamed phenotype after drug withdrawal. RNAs were extracted from tumor tissues in ( A , B ), and RNA-seq analyses were performed. The relative expression levels of indicated genes in each sample were determined and the statistical analysis was performed based on the average of expression levels of each cohort. n = 6, 7 and 7 for vehicle, BAY-I and BAY-I withdraw 4 weeks group. p was calculated by two-sided T-test. E The relative expression levels of the Ifn-γ gene were determined by RT-PCR analysis. n = 6 at each treatment cohort. F The enrichment of T cell receptor pathway was determined by GSEA based on RNA-seq data in E . Statistical test was performed by GSEA. A and B , data were presented as dot plots with mean as the central lines; E Data were presented as mean ± SEM; N.S p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 by two-sided T-test. Source data and exact p values are provided in the Source Data file.

Article Snippet: Cd8 atm1Mak mice ( Cd8 -KO mice) was purchased from the Jackson lab (002665) then crossed with the Pten -null mice to generate Pb-Cre + ;Pten LoxP/LoxP ;Cd8 -KO ( Pten -null; Cd8 -KO) mice.

Techniques: Staining, RNA Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/β/δ treatment

doi: 10.1038/s41467-021-27833-0

Figure Lengend Snippet: A The differential inhibitory effects of BAY1082439 on T cells. Primary CD8 + , helper T and Treg cells from spleen of WT mice were FACS sorted, and cultured with different concentrations of BAY1082439. This experiment was repeated 3 times the percentages of cell growth as compared to vehicle controls were presented. Data were presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 by two-sided T-test. IC50s were calculated by GraphPad Prism 6 software. B , C . Prostate- and spleen-associated Treg cell numbers and the percentages of Treg in CD4 + T cell in vehicle ( n = 17), BAY-D ( n = 7) and BAY-I ( n = 13) treated cohorts were analyzed by FACS. Data were presented as dot plots with mean as the central lines; ** p < 0.01, *** p < 0.001 by two-sided T-test. D The negative correlation between the percentages of CD8 + in CD45 + and Treg in CD4 + cells by Pearson correlation coefficient. E The CD8 + /Treg ratios in vehicle, BAY-D and BAY-I treated cohorts analyzed by FACS. Data were presented as dot plots with mean as the central lines; * p < 0.05 by two-sided T-test. Source data and exact p values are provided in the Source Data file.

Article Snippet: Cd8 atm1Mak mice ( Cd8 -KO mice) was purchased from the Jackson lab (002665) then crossed with the Pten -null mice to generate Pb-Cre + ;Pten LoxP/LoxP ;Cd8 -KO ( Pten -null; Cd8 -KO) mice.

Techniques: Cell Culture, Software

Journal: Nature Communications

Article Title: Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/β/δ treatment

doi: 10.1038/s41467-021-27833-0

Figure Lengend Snippet: A – C . A schematic illustration of treatment procedures and the percentages of CD8 + in CD45 + in peripheral blood and the prostates. Pten -null mice were treated with vehicle ( n = 6), S1PR modulator Fingolimod alone ( n = 5) or in combination with BAY-I ( n = 6) for 4 weeks. CD45 + and CD8 + T cells from peripheral blood or the prostates were sorted and presented as the percentage of CD8 + in CD45 + cells. D BAY-I treatment induced CD8 + T cell proliferation. Pten -null mice was treated with vehicle ( n = 7) or BAY-I ( n = 7) for 2 weeks, then BrdU labeled (10 mg single dose) for 24 h before analyzing the percentages of BrdU + cells in CD8 + T cell by FACS analysis. E BAY-I treatment induced CD8 + T cell activation. Castrated Pten -null mice were treated with vehicle ( n = 6), BAY-D ( n = 7) or BAY-I ( n = 9) for 4 weeks, and tumor-associated CD8 + T cells were sorted then analyzed by RNA-seq. The relative expression levels of Il2 , Cd25 and Cd40l were presented. F GSEA analysis of IL2 and TCR single pathway between Vehicle and BAY-I/BAY-D group. Statistical test was performed by GSEA. Data were presented as dot plots with mean as the central lines (for A , B , C and D ) or as mean ± SEM (for E ); * p < 0.05, ** p < 0.01, *** p < 0.001 by two-sided T-test. Source data and exact p values are provided in the Source Data file.

Article Snippet: Cd8 atm1Mak mice ( Cd8 -KO mice) was purchased from the Jackson lab (002665) then crossed with the Pten -null mice to generate Pb-Cre + ;Pten LoxP/LoxP ;Cd8 -KO ( Pten -null; Cd8 -KO) mice.

Techniques: Labeling, Activation Assay, RNA Sequencing, Expressing

Journal: Nature Communications

Article Title: Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/β/δ treatment

doi: 10.1038/s41467-021-27833-0

Figure Lengend Snippet: A H&E and IHC analyses showing a representative tertiary lymphoid structure in BAY-I treated prostates. Castrated Pten -null mice were treated with 4 cycles of BAY-I then drug was withdrawal for 4–10 weeks. Tumor tissues were fixed, and tertiary lymphoid structures were determined by staining consecutive sections with H&E, and CD8 \B220\CD11C\Ki67 antibodies. The same staining were performed with 3 mice and similar results were observed. B CD8 + cells within the tertiary lymphoid structures are highly proliferative. BAY-I treated Animals were pulse-labeled by a bullet dose of BrdU before the analysis. Tumor tissues were fixed and CD8 + BrdU + double positive cells were visualized by co- Immunofluorescence staining with anti-BrdU and CD8 antibodies. The same staining were performed with 3 mice and similar results were observed. C TLS score is increased in BAY-I treated cohort and positively correlates with Cd8a expression. Castrated Pten -null mice were treated with 4 cycles of vehicle, BAY-I then withdrawal for 4 weeks, or treated with anti-PD-1 antibody as indicated in Fig. . TLS score was calculated from tumor RNAseq dataset. Correlation between TLS score and Cd8a expression was calculated. Statistic was performed by Pearson correlation coefficient. D BAY-I treated and BAY-I treated then drug withdrew prostate-associated CD8 + T cells remained activated. Castrated Pten -null mice were treated with vehicle ( n = 6), BAY-D ( n = 7) and BAY-I for 4 weeks ( n = 9), and BAY-I for 4 weeks then drug withdrew for 4 weeks ( n = 6). Tumor-associated CD8 + T cell were sorted by FACS. The relative expression levels of genes associated with T cell activation, effector cytokines, co-inhibitory/stimulatory molecules and T memory were presented based on RNA-seq data. p was calculated by two-sided T-test. E Cell surface expression levels of CD44 and CD62L molecules on tumor-associated CD8 + T cell in vehicle ( n = 8) or BAY-I withdraw cohort ( n = 6) mice were determined by FACS. * p = 0.0386 by two-sided T-test. F Vehicle ( n = 6), BAY-I ( n = 9) treated and BAY-I treated then drug withdrew ( n = 6) prostate-associated CD8 + T cells remained clonal selected. TCR clonotype diversities were calculated and presented as dot plots with mean as the central lines; *** p < 0.001 by two-sided T-test. Source data and exact p values are provided in the Source Data file.

Article Snippet: Cd8 atm1Mak mice ( Cd8 -KO mice) was purchased from the Jackson lab (002665) then crossed with the Pten -null mice to generate Pb-Cre + ;Pten LoxP/LoxP ;Cd8 -KO ( Pten -null; Cd8 -KO) mice.

Techniques: Staining, Labeling, Immunofluorescence, Expressing, Activation Assay, RNA Sequencing

Journal: Nature Communications

Article Title: Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/β/δ treatment

doi: 10.1038/s41467-021-27833-0

Figure Lengend Snippet: A , B . BAY-I induced anti-tumor immunity was diminished in Pten -null; Cd8 -KO mice. 10 weeks old Pten-null and Pten -null; Cd8 -KO mice were treated with vehicle ( n = 25 for Pten -null mice, n = 13 for Pten -null; Cd8 -KO mice) or BAY-I ( n = 13 for Pten -null mice, n = 12 for Pten -null; Cd8 -KO mice) for 4 weeks. Tumor tissues were harvested and fixed, and consecutive sections were stained for H&E and antibodies against CD8 and GZMb. C , D . BAY-I treatment efficacies in prostate from Pten -null ( n = 25 in vehicle cohort, n = 13 in BAY-I cohort), Pten -null; Cd8 -KO ( n = 13 in vehicle cohort, n = 12 in BAY-I cohort), castrated Pten -null with ( n = 6) or without anti-CD8 antibody ( n = 6 in vehicle cohort, n = 8 in BAY-I cohort) were determined by counting GZMb + cells density in cancer areas ( C ), and calculating anterior lobe’s cancer cell area in H&E stained slides ( D ) for each treatment cohorts. Data were presented as dot plots with mean as the central lines; N.S p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 by two-sided T-test. Source data and exact p values are provided in the Source Data file.

Article Snippet: Cd8 atm1Mak mice ( Cd8 -KO mice) was purchased from the Jackson lab (002665) then crossed with the Pten -null mice to generate Pb-Cre + ;Pten LoxP/LoxP ;Cd8 -KO ( Pten -null; Cd8 -KO) mice.

Techniques: Staining

Journal: Nature Communications

Article Title: Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/β/δ treatment

doi: 10.1038/s41467-021-27833-0

Figure Lengend Snippet: A Castrated Pten -null mice were treated with 4 weeks of vehicle ( n = 4) or BAY-I ( n = 4). Cell surface expression levels of co-inhibitory/stimulatory molecules on tumor-associated CD8 + T cell were determined by FACS. B . Increased PD-L1 expressions induced by BAY-I treatment. Lin - EPCAM + cancer cells from indicated cohorts ( n = 5 in vehicle cohort, n = 9 in BAY-I cohort, n = 6 in BAY-I withdraw cohort) were sorted by FACS, and the relative Pd-l1 expression levels were determined by RNA-seq analysis. Data were presented as mean with dot plots; ** p < 0.01 by two-sided T-test. A schematic illustration of treatment strategy. Castrated Pten -null mice were treated with vehicle ( n = 8), or BAY-I for 4 weeks followed by isotype antibody ( n = 11) or anti-PD-1 antibody ( n = 15), or anti-PD-1 antibody plus S1PR modulator Fingolimod ( n = 5) treatment for 4 weeks, or anti-PD-1 antibody then withdrawal for 4 weeks ( n = 6) ( C ). Low power HE stained images of the anterior prostate lobes from Pten -null mice treated with BAY-I follow isotype or anti-PD-1 antibody, or anti-PD-1 antibody then withdrawal for 4 weeks ( D ). The anti-tumor immunity induced by sequential BAY-I and anti-PD-1 combination treatment is not influenced by S1PR modulator Fingolimod and is CD8 + T cell dependent. The Pten -null and Pten -null; Cd8 -KO mice were treated as indicated in C . Tumor tissues were fixed, weighted, and stained with H&E and anti-GZMb antibody. The same staining were performed with 6 mice in all cohort and similar results were observed. ( E ), GZMb + cells density in cancer cell acini ( n = 8, 5, 7, 5, 7 and 8 in each cohort) and cancer cell areas in the anterior lobes ( n = 6, 11, 7, 7, 6, 7, 7 and 7 in each cohort) ( F ) were calculated. The percentages of tumor associated CD8 + cell in CD45 + cell ( n = 8, 15 and 6 in each cohort) ( G ) and CD44 + in CD8 + cells ( n = 8 and 6 in each cohort) ( H ) was determined by FACS. Data were presented as mean with dot plots; N.S p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 by two-sided T-test. Source data and exact p values are provided in the Source Data file.

Article Snippet: Cd8 atm1Mak mice ( Cd8 -KO mice) was purchased from the Jackson lab (002665) then crossed with the Pten -null mice to generate Pb-Cre + ;Pten LoxP/LoxP ;Cd8 -KO ( Pten -null; Cd8 -KO) mice.

Techniques: Expressing, RNA Sequencing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Critical role of the CD44 low CD62L low CD8 + T cell subset in restoring antitumor immunity in aged mice

doi: 10.1073/pnas.2103730118

Figure Lengend Snippet: Loss of antitumor activity and reduced P4 cell induction in aged PD-1 KO mice. (A and B) MC38 cells were i.d. inoculated into young and aged PD-1 KO mice. (A) MC38-tumor sizes in young (3–4 mo old) and aged (15 mo old) PD-1 KO mice. (B) Kaplan–Meier plot of percent survival of MC38-tumor–bearing PD-1 KO mice. (C and D) Analysis of CD8+ T cell subsets in young (C) (2–3 mo old) or aged (D) (15–21 mo old) PD-1 KO mice with or without injection of MC38 cells. Stained PLN and DLN cells on day 9. Representative plots showing CD44 and CD62L expression gated on CD3+CD8+ T cells and percentage of CD8+ T cell subsets; CD44low/CD62Lhigh (naive; P1), CD44high/CD62Lhigh (central memory; P2), CD44high/CD62Llow (effector/memory; P3), and CD44low/CD62Llow (P4). P values were calculated by log-rank test or two-tailed unpaired Student’s t test. **P < 0.01; ***P < 0.001; n.s., not significant. Data are presented as the mean ± SEM (n = 8–10 for A and B; n = 5–6 for C and D).

Article Snippet: OT-1 TCR-transgenic and CD8 −/− (CD8 KO) mice were purchased from The Jackson Laboratory (originally from M. B. Bevan at University of Washington [Seattle, WA], or T. Mak at University of Toronto [Toronto, ON, Canada]).

Techniques: Activity Assay, Injection, Staining, Expressing, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Critical role of the CD44 low CD62L low CD8 + T cell subset in restoring antitumor immunity in aged mice

doi: 10.1073/pnas.2103730118

Figure Lengend Snippet: Antitumor activity of P4 cells through differentiation to P3 cells. (A and B) To obtain the OT-I P3 and P4 subsets, MC38-OVA cells were i.v. injected into young OT-1 mice and each subset was isolated from splenocytes. The P3 or P4 subset cells were adoptively transferred into CD8 KO mice 5 d after MC38-OVA i.d. injection. (A) Tumor volume in MC38-OVA–bearing CD8 KO mice with or without (Ctrl) the transfer of P3 or P4 cells. (B) FACS analysis of the transferred CD8+ T cell subsets in peripheral blood on day 11. P1 to P4 subsets were defined by CD44 and CD62L positivity after gating on CD8+ T cells. (C) Scheme of CD8+ T cell subset isolation from splenocytes of young PD-1 KO mice. (D–G) FACS analysis of cultured P4 (D), P1 (E and G), or P2 (F) cells isolated from young (2–3 mo old) or aged (16–18 mo old) PD-1 KO mice 2 or 3 d after the stimulation using anti-CD3/CD28 mAbs and IL-2. Representative plots showing CD44 and CD62L expression in the indicated culture cells and percentage of CD8+ T cell subsets. Data are presented as the mean ± SEM (n = 3–5); **P < 0.01 (one-way ANOVA followed by Tukey’s test).

Article Snippet: OT-1 TCR-transgenic and CD8 −/− (CD8 KO) mice were purchased from The Jackson Laboratory (originally from M. B. Bevan at University of Washington [Seattle, WA], or T. Mak at University of Toronto [Toronto, ON, Canada]).

Techniques: Activity Assay, Injection, Isolation, Cell Culture, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Critical role of the CD44 low CD62L low CD8 + T cell subset in restoring antitumor immunity in aged mice

doi: 10.1073/pnas.2103730118

Figure Lengend Snippet: Increased expression of 1C-metabolism–related genes in P4 cells. (A–E) Microarray analysis of P1, P2, P3, and P4 cells sorted from young PD-1 KO mice (1–3 mo old; nine mice pooled). (A) Hierarchical clustering heat map of all detected genes. (B) Scatter plots represent normalized log intensities of individual probes. The dashed lines indicate log 2-fold change. Genes previously linked to CD8+ T cell activation and differentiation are listed. (C) Top 10 enriched gene ontology (GO) terms from the up-regulated genes in P4 cells. GO terms related to 1C metabolism are highlighted in red. (D) Schematic of 1C-metabolic pathways. THF, tetrahydrofolate. (E) Heat map showing the expression of 1C-metabolism–related genes in CD8+ T cell subsets. (F) Relative mRNA expression of 1C-metabolism–related genes in CD8+ T cell subsets from splenocytes of young PD-1 KO mice. (G) Microarray analysis of WT or PD-1 KO CD8+ T cells in PLNs or DLNs of young (2 mo old, three mice pooled) or aged (17 mo old, six mice pooled) mice with or without MC38 tumors (day 9). Heat map showing the expression of 1C-metabolism–related genes in the indicated cells. (H and I) Oxygen consumption rate (OCR) of WT or PD-1 KO CD8+ T cells from DLNs of young (2–3 mo old) or aged (15–19 mo old) mice was measured using a Seahorse XFe96 analyzer. OCR trace (H) and basal respiration and spare respiratory capacity were calculated from OCR values (I). *P < 0.05; **P < 0.01 (two-tailed unpaired Student’s t test). Data are presented as the mean ± SEM (n = 4).

Article Snippet: OT-1 TCR-transgenic and CD8 −/− (CD8 KO) mice were purchased from The Jackson Laboratory (originally from M. B. Bevan at University of Washington [Seattle, WA], or T. Mak at University of Toronto [Toronto, ON, Canada]).

Techniques: Expressing, Microarray, Activation Assay, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Critical role of the CD44 low CD62L low CD8 + T cell subset in restoring antitumor immunity in aged mice

doi: 10.1073/pnas.2103730118

Figure Lengend Snippet: P4 cell induction in aged CD8+ T cells is attenuated by TCR signal suppression through high CD45RB expression. (A and B) Analysis of CD8+ T cell subsets in PLNs of young (2–3 mo old) (A) or aged (12–15 mo old) (B) OT-1 mice with or without i.v. injection of MC38-OVA cells. Representative plots showing CD44 and CD62L expression on CD8+ T cells and the percentage of each CD8+ T cell subset in the indicated mice 5 d after MC38-OVA injection. (C) The percentages of pZAP70+ CD8+ T cells in PLNs from young or aged OT-1 mice with or without MC38-OVA injection are shown in representative plots. (D) CD45RB expression levels in total CD8+ T cells or each subset from PLNs of young (2–3 mo old) or aged (14–17 mo old) OT-1 mice are shown in representative plots. (E) CD8+ T cells were isolated from PLNs of young or aged OT-1 mice and cultured with or without MMC-treated MC38-OVA cells for 1 d. In the last 1 h, vehicle or CD45 inhibitor (0.05 μM) was added. The graphs show the percentages of p-ZAP-70+ cells relative to all CD8+ T cells. Data are represented as the mean ± SEM (n = 3–6). *P < 0.05; **P < 0.01; n.s., not significant (one-way ANOVA followed by Tukey’s test or two-tailed unpaired Student’s t test).

Article Snippet: OT-1 TCR-transgenic and CD8 −/− (CD8 KO) mice were purchased from The Jackson Laboratory (originally from M. B. Bevan at University of Washington [Seattle, WA], or T. Mak at University of Toronto [Toronto, ON, Canada]).

Techniques: Expressing, Injection, Isolation, Cell Culture, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Critical role of the CD44 low CD62L low CD8 + T cell subset in restoring antitumor immunity in aged mice

doi: 10.1073/pnas.2103730118

Figure Lengend Snippet: Recovery of P4 cell induction and antitumor activity in aged mice by xenogeneic cell injection. (A) Schematic diagram of the experimental schedule. (B–D) Young (3–4 mo old) and aged (16–18 mo old) PD-1 KO mice were i.v. injected with Daudi cells (day −10). Ten days after injection (day 0), PLN cells of the mice were analyzed by FACS and real-time PCR. (B) Percentages of P1–P4 subsets relative to all CD8+ T cells in PLNs. (C) Expression levels of 1C-metabolism–related genes in CD8+ T cells. (D) CD45RB expression levels in P1 cells from PLNs. (E–I) Young (3–4 mo old) and aged (17–18 mo old) PD-1 KO mice were i.v. injected with or without (Ctrl) Daudi cells (day −10). Ten days after the transfer (day 0), the mice were i.d. inoculated with MC38 (E–G) or MC38-OVA (H and I) cells and used for the following experiments. MC38-tumor sizes (E) and percent survival (F). (G) The percentage of p-ZAP-70+ cells relative to all CD8+ T cells from DLNs on day 6. (H and I) Frequency of OVA tetramer+ CD8+ T cells in DLNs (H) or tumor tissues (I) on day 6. Data are presented as the mean ± SEM (n = 9–10 for B; n = 4–6 for C–I). *P < 0.05; **P < 0.01 (one-way ANOVA followed by Tukey’s test, log-rank test, or two-tailed unpaired Student’s t test).

Article Snippet: OT-1 TCR-transgenic and CD8 −/− (CD8 KO) mice were purchased from The Jackson Laboratory (originally from M. B. Bevan at University of Washington [Seattle, WA], or T. Mak at University of Toronto [Toronto, ON, Canada]).

Techniques: Activity Assay, Injection, Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test