Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: a Gating strategy in screening experiment using the panel of TCR Vβ antibodies. First, CD3 lymphocyte were separated into CD4 + and CD8 + populations using monoclonal antibodies (CD8 + : Pe-Cy7, CD4 + : PerCP). Next, the samples were stained with Vβ specific antibody cocktails included in IO Test Beta Mark TCR beta Repertoire Kit (Beckman-Coulter Immunotech, USA). The β variable chain family was determined based on FITC and PE positivity from CD4 + and CD8 + populations according to the manufacturer’s instruction. Vβ20 clone was detected from total CD4 + T cells (52.9%, middle panel) and total CD8 + T cells (1.74%, right). b Flow cytometry Vβ screening results from the index patient’s peripheral blood sample. T cell clonality with antibodies which target Vβ region of TCR was analysed of CD4 + T cells. The increased distribution suggests that the cells have large T cell clone. c Increased Vβ20 bearing clonotype over time in the index patient’s CD4 + T cells. Source data are provided as a Source data file. d T cell repertoire of FACS-sorted CD4 + Vβ20+ and CD8 + T cells analysed with TCRβ deep sequencing (Adaptive Biotechnologies). The TCRBV30-01 clone was detected in the CD4 + Vβ20+ fraction, but not in the CD8 + fraction. e Multicolor flow cytometry was applied to identify the immune phenotype of HSCT donor and index patient’s memory T cell subtypes. Central memory (CM), naïve, effector memory (EM), and terminal effector memory (TEMRA) cells. f The relative proportion of granzyme B positive (GrB + ) CD4 + T cells and GrB + CD8 + T cells in index patient. Index patient’s PBMCs were stained with anti-CD45, −CD3, −CD4, and −CD8 (surface markers), and then GrB stained after fixation and permeabilization. Stained cells were analyzed using FACSVerse.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Staining, Flow Cytometry, Sequencing

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: Somatic mutations discovered in CD4 + T cells in the index patient, detected from 2013 sample.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Mutagenesis

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: a Locations of mTOR, TLR2 , and NFκB2 somatic mutations. Linearized structure of MTOR, NFκB2 , and TLR2 presenting the location of somatic mutations. mTOR P2229R mutation is located in the kinase domain, NFκB2 P882Q in the C-terminus, and TLR2 W558L between LRR (Leucine-rich repeats) domain and transmembrane (TM) domain. b A heterozygous mTOR mutation (G to C, P2229R ) was detected in CD4 + T cells by Sanger sequencing. c Variant allele frequencies (VAFs) of mTOR P2229R , NFκB2 P882Q , and TLR2 W558L mutations in the index patient’s CD4 + T cells over time as measured with amplicon sequencing. Source data are provided as a Source data file. d VAFs (%) of mTOR P2229R mutation from the index patient’s skin, liver, and eyes biopsy. e Immunofluorescence staining indicated CD3 + CD4 + and CD3 + CD8 + T cell infiltration in the skin. Paraffin embedded skin biopsy from the index patient was sectioned and stained with antibody specific human CD3 (cyan), CD4 (green), and CD8 (red). White arrows indicate infiltrated CD3 + CD4 + or CD3 + CD8 + T cells. Original imaging magnification: 20×, the figure has been further zoomed to 25× for visualization. Scale bar: 50 μm.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Mutagenesis, Sequencing, Variant Assay, Amplification, Immunofluorescence, Staining, Imaging

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: Somatic MTOR and NFκB2 mutations validated by amplicon sequencing in the index patient.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Amplification, Sequencing

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: Real-time cell analysing (RTCA) systems, xCELLigence TM , was applied to monitor real-time killing effect of primary fibroblasts obtained from the index patient and healthy donors. a Index patient’s primary fibroblasts were cultured as monolayers for 24 h to reach full confluence. Once confluent, the effector cells; NK-92 cell (positive control, 8:1), primary CD4 + T cells (4, 8, 16:1) and primary CD8 + T cells (16:1) with different ratios (effector cells:fibroblasts) were added to each well followed by co-culture (arrow indicates the point of adding the effector cells, 0 h). The control (black line, media) shows the impedance of the fibroblasts without any added effectors. The cell impedance was measured every 30 min for 21 h. The measured impedance was expressed as Cell Index with the normalization performed at time of addition of effector cells. b Visualization of monolayers of the primary fibroblast before and 21 h after addition of effector cells (8:1 for all effector cells). c CD4 + T cells and CD8 + T cells from healthy donors were added to healthy donors’ own fibroblasts ( n = 3). The cell impedance was measured for 21 h. Data is representative of three independent individuals. d Index patient’s fibroblasts were seeded with HLA1 and HLA2 antibodies (10 and 20 μg/mL). After CD4 + T cells (CD4 + T cells:fibroblasts = 8:1) were added the cell impedance was measured for 21 h. Dots represent mean values and error bars indicate range ( n = 2 for all conditions, technical duplicates). Source data are provided as a Source data file.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Cell Culture, Positive Control, Co-Culture Assay

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A Double staining of CD8 and αSMA in mouse various subcutaneous tumors. Scale bar, 200 μm. The quantitative evaluation of sections from each tumor stained for B αSMA ( n = 12 fields from four mice per group) and C CD8 ( n = 12 fields from four mice per group). D The correlation between the number of CD8 + T cells and αSMA + area in each mouse tumor tissue. E Double staining of collagen 1 and CD45 to detect fibrocytes in mouse various subcutaneous tumors. Scale bar, 100 μm. F The quantitative evaluation of sections from each tumor stained for fibrocytes ( n = 12 fields from four mice per group). G The correlation between the number of fibrocytes and αSMA + area in each mouse tumor tissue. The correlation was estimated by Spearman’s correlation and a linear regression analysis (the best-fit line is indicated). Representative images of tumor sections resected from non-small cell lung carcinoma (NSCLC) patients stained to detect αSMA + cancer-associated fibroblasts ( H , αSMA + FAP + ) and fibrocytes ( I , CD45 + FSP-1 + ). Scale bar, 100 μm. J Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in NSCLC tumor tissue stained in Fig. 1H and I ( n = 50 patients). K Representative images of resected NSCLC tissue stained to detect CD8 + T cells and EpCAM + tumor cells. Scale bar, 100 μm. L Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in each NSCLC group divided by the immune phenotypes of tumor-infiltrating CD8 + T cells (inflamed, desert, and exclusion). * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Double Staining, Staining

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume of AB1-HA-bearing balb/c nude mice treated with KL001 from 5 days after tumor cell injection ( n = 6 per group). B Double staining of CD8 and αSMA in AB1-HA tumors from balb/c mice treated with or without KL001 (studied in Fig. ). Scale bar, 200 μm. C Representative images and D quantitative evaluation of sections from KL001-treated AB1-HA tumors stained for CD8, CD4, Foxp3, and F4/80 ( n = 15 fields from five mice per group). Scale bar, 200 μm. E Quantitative evaluation of sections from CLK8-treated AB1-HA tumors stained for CD8, CD4, Foxp3 ( n = 15 fields from five mice per group). * P < 0.05 by the Mann-Whitney U test. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Injection, Double Staining, Staining, MANN-WHITNEY

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume and B the representative image of tumor tissue of LLC-bearing mice treated with KL001 and/or αPD-L1 Ab (n = 7 per group). * P < 0.05 by a one-way ANOVA. C Representative images and D the quantitative evaluation of sections from LLC tumors stained for αSMA ( n = 15 fields from five mice per group). The tumors were harvested at day 21 from each group studied in Fig. 6A. Scale bar, 200 μm. Representative images and the quantitative evaluation of sections from LLC tumors stained for collagen 1 + CD45 + fibrocytes ( E, F ), CD8 + T cells ( G, H ) and CD4 + T cells ( I, J ). Scale bar, 100 μm. * P < 0.05 by a one-way ANOVA. K The evaluation of the tumor volume of AB1-HA-bearing mice treated with KL001 and/or αCTLA-4 Ab ( n = 6 per group). * P < 0.05 by a one-way ANOVA. The quantitative evaluation of sections from AB1-HA tumors ( n = 15 fields from five mice per group) stained for CD8 ( L ), CD4 ( M ), and Foxp3 ( N ). The tumors were harvested at day 18 from each group studied in Fig. 6K. * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Staining

Journal: Nature

Article Title: Sleep modulates hematopoiesis and protects against atherosclerosis

doi: 10.1038/s41586-019-0948-2

Figure Lengend Snippet: a, Image of a sleep fragmentation cage. b, Body weight (n=10 per group). c, Plasma cholesterol at ZT3 (n=5 per group). d, Plasma glucose at ZT3 (n=5 per group). e , Glucose tolerance test (GTT) beginning at ZT3 and ZT12 (n=4 per group). f-h, Apoe −/− mice were placed in sleep fragmentation chambers where the sweep bar operated during the dark period (ZT12–0) when mice are normally awake. Control mice were maintained in SF chambers with a stationary sweep bar. f , Assessment of atherosclerosis and lesion area (n=5 per group). g, Assessment of blood Ly-6C hi monocytes and neutrophils (n=5 per group). h, Assessment of bone marrow LSKs and proliferation (n=5 per group). i, Aortic macrophage proliferation in Apoe −/− and Apoe −/− SF mice after 16 weeks of SF at ZT3 and ZT14 (n=5 Apoe −/− ; n=4 Apoe −/− SF). j , Quantification at ZT3 in Apoe −/− and Apoe −/− SF mice of B cells, CD4 + T cells and CD8 + T cells in blood (n=10 Apoe −/− ; for B and CD4 T cells n=6 Apoe −/− SF for CD8 T cells n=7 Apoe −/− SF), k, spleen (n=10 Apoe −/− ; n=7 Apoe −/− SF) and l, B cells in bone marrow (n=10 Apoe −/− ; n=7 Apoe −/− SF). Data presented as mean ± s.e.m.

Article Snippet: The following monoclonal antibodies were used for flow cytometric analysis: anti-CD45 (BioLegend, clone30-F11, Cat#103147, Lot#B243834), anti-CD45.1 (BioLegend, clone A20, Cat#110708), anti-CD45.2 (BioLegend, clone 104, Cat#109802), anti-CD3 (BioLegend, clone 17A2, Cat#100206), anti-CD90.2 (BioLegend, clone 53–2.1, Cat#105308, Lot#B260050), anti-CD19 (BioLegend, clone 6D5, Cat#115508, Lot#B226581), anti-B220 (BD Biosciences, clone RA3–6B2, Cat#553089, Lot#6012954), anti-NK1.1 BioLegend, clone PK136, Cat#108708), anti-Ly6G (BioLegend, clone 1A8, Cat127614#, Lot#B259670), anti-Ly6C (BioLegend, AL-21, Cat#128006, Lot#B247728), anti-MHCII (BioLegend, clone M5/114.152, Cat#107602, Lot#B217859), anti-F4/80 (Biolegend, clone BM8, Cat#123114, Lot#B237342), anti-CD11b (BioLegend, clone M1/70, Cat#101226, Lot#B238268), anti-CD115 (BioLegend, clone AFS98, Cat#135517, Lot#B265220), anti-Ter119 (BioLegend, clone TER-119, Cat#116208, Lot#B220899), anti-CD34 (eBioscience, clone RAM34, Cat#11–0341-85, Lot#E00265–1634), anti-CD49b (BioLegend, clone DX5, Cat#1089008, Lot#B258302), ant-CD11c (BioLegend, clone N418, Cat#117310, Lot#B206713), anti-IL7Rα (BioLegend, clone SB/199, Cat#121112, Lot#B189668), anti-CD16/32 (BioLegend, clone 93, Cat#101324, Lot#B250025), anti-CD150 (BioLegend, clone TC15–12F12.2, Cat#115922, Lot#B220585), anti-cKit (BioLegend, clone 2B8, Cat#105814, Lot#B252918), anti-CD135 (BioLegend, clone A2F10, Cat#135310, Lot#B234045), anti-CD48 (BioLegend, clone HM48–1, Cat#103426, Lot#B236445), anti-Sca1 (BioLegend, clone D7, Cat#108126, Lot#B234288), anti-CD8 (BD Bioscience, clone 53–6.7, Cat#553035, Lot#2296946), anti-CD4 (BioLegend, clone GK1.5, Cat#100428, Lot#B237336), anti-SiglecF (BD Pharmingen, clone E50–2440, Cat#562680, Lot#7054789), anti-CXCR4 (Invitrogen, clone 2B11, Cat#12–9991-81, Lot#B251481), anti-CXCR2 (BioLegend, clone SA044G4, Cat#149307, Lot#B251481), anti-BrdU (eBioscience, clone BU20A, Cat#17–5071-42, Lot#4319920).

Techniques:

Journal: Translational Oncology

Article Title: Vaccine-induced ErbB (EGFR/HER2)-specific immunity in spontaneous canine cancer

doi: 10.1016/j.tranon.2021.101205

Figure Lengend Snippet: cEGFR p527 immunization mediates CD8 infiltration and antibody deposition into canine tumors. Postmortem bladder cancer tissue from a cEGFR p527 immune dog and tissue from healthy, non-cancerous bladder was examined for infiltration of CD8 T cells and endogenous antibody. A. CD8 isotype control. B. CD8 T cell staining (green). Arrows illustrate presence of CD8+ cells within the tumor tissue. C. & D. Antibody deposition in canine bladder tumor tissue from a cEGFR p527 immunized dog (two different fields). E. & F. Canine IgG deposition in normal canine bladder tissue (two different fields). Magnification is 40x (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Article Snippet: CD8 T cells were stained in tissues using mouse anti-dog CD8 (MAB6709; R&D Systems, Minneapolis, MN), followed by goat anti-mouse IgG ( H + L ) Alexa Fluor 488 (A11017; Life Technologies, Eugene, OR) and counterstaining with DAPI (Molecular Probes; Eugene, OR).

Techniques: Control, Staining

Journal: Journal of Nanobiotechnology

Article Title: Orchestrated Cu 2+ -coordinated tetracycline-porphyrin self-assembly remodels tumor microenvironment for photo-enhanced immuno-chemodynamic therapy

doi: 10.1186/s12951-025-03486-9

Figure Lengend Snippet: In vivo immunomodulatory effects of LP/CuTT. Representative flow cytometric analysis and quantification of tumor-infiltrating M1 TAMs gated on CD11b + and CD80 + cells ( A , B ), CD4 + T cells gated on CD3 + and CD4 + cells ( C , D ), and CD8 + T cells gated on CD3 + and CD8a + cells ( E , F ) from the upper right quadrant of the scatter diagrams in tumors after 17 days treatments. G - L ) Representative flow cytometric analysis and quantification of M1 TAMs, CD4 + T cells and CD8 + T cells in spleen after different treatments. Serum levels of cytokines M ) TNF-𝛼, N ) IL-1β, and O ) IL-10 in mice after different treatments detected by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared to control. # p < 0.05, ns indicates no significant difference

Article Snippet: PE anti-mouse CD206/MMR antibody, FITC anti-mouse CD86 antibody, APC anti-mouse CD80 antibody, FITC anti-mouse CD3 antibody, PerCP anti-mouse CD8 antibody, and APC anti-mouse CD4 antibody were purchased from Elabscience (Wuhan, China).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Control