Journal: Nature Communications

Article Title: Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

doi: 10.1038/s41467-018-07444-y

Figure Lengend Snippet: Starvation translocates TGN membrane proteins to endosomes. All cells are HeLa cells. a , b Furin loses its Golgi localization during starvation. Cells treated with indicated medium for 1 h and endogenous furin and Golgin-245 were stained. The fraction of Golgi-localized furin is quantified in b . c The recovery of Golgi localization of furin after supplying nutrient. After starvation in HBSS for 2 h, cells were treated with DMEM for indicated time and stained as in a . d Kinetics of Golgi-localized furin-GFP during HBSS and subsequent DMEM treatment. Cells expressing furin-GFP were first starved in HBSS for 2 h and subsequently stimulated by DMEM for 2 h. At indicated time, cells were stained for endogenous Giantin and the fraction of Golgi-localized furin-GFP is quantified. e , f Nutrient starvation significantly reduces the Golgi localization of CD8a-furin. Cells transiently expressing CD8a-furin were treated by indicated medium for 2 h and stained as in e . The fraction of Golgi-localized CD8a-furin is quantified in f . g The translocation of CD8a-furin to the endosome during nutrient starvation. Cells transiently expressing indicated constructs were treated with HBSS for 2 h and stained. Boxed regions are enlarged at the upper right corner. Arrows indicate colocalization. h , i The endosomal pool of CD8a-furin increases during nutrient starvation. Similar results have been observed in four independent experiments. Cells stably expressing CD8a-furin were treated with HBSS for 2 h or with additional 20 min treatment of DMEM. Lysates were subjected to sucrose gradient centrifugation to separate organelles. 20 fractions were collected and immunoblotted for CD8a-furin and markers. Percentages of CD8a-furin distributed in the endosomal (fractions 1–5) and TGN pool (fractions 10–16) are quantified in i . b , d , and f are representative results from three independent experiments. Complete, complete medium, n , the number of cells analyzed; error bar, mean ± s.e.m.; scale bar, 10 µm. P values were from t test (unpaired and two-tailed). *** P ≤ 0.0005

Article Snippet: Mouse anti-Lamp1 mAb (H4A3)(1:500 for IF) and mouse anti-CD8a mAb (OKT8)(1:500 for IF) were from Developmental Studies Hybridoma Bank.

Techniques: Membrane, Staining, Expressing, Translocation Assay, Construct, Stable Transfection, Gradient Centrifugation, Two Tailed Test

Journal: Nature Communications

Article Title: Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

doi: 10.1038/s41467-018-07444-y

Figure Lengend Snippet: AAs stimulate the endosome-to-Golgi trafficking. All cells are HeLa cells. a , b AAs but not growth factors and glucose stimulate the retrograde trafficking to the Golgi. Cells stably expressing CD8a-furin were treated with DMEM-base for 2 h. The surface-exposed CD8a-furin was labeled by anti-CD8a antibody and chased in respective medium for 20 min. The fraction of CD8a-furin at the Golgi is quantified after staining. d-serum, 10% dialyzed serum. c – e AAs stimulate the endosome-to-Golgi trafficking. Cells transiently expressing CD8a-furin or CD8a-CI-M6PR were surface labeled by anti-CD8a antibody and synchronized at endosomes at 18 °C in HBSS for 2 h before being chased at 37 °C in HBSS or DMEM for 12 min. The fraction of Golgi-localized CD8a-chimeras is quantified after staining. f – h Gln has one of the most acute stimulating effects on endocytic trafficking to the Golgi. Similar to a , b except that the nutrient starvation was conducted in HBSS before surface labeling. The labeled CD8a-furin was then chased in HBSS, DMEM, DMEM/-AAs supplemented with indicated AA at 0.8 mM in f or DMEM selectively leaving out indicated AA(s) in g . The fraction of Golgi-localized CD8a-furin is quantified in h . i AAs decrease the Golgi-to-PM trafficking of furin. Cells expressing SBP-GFP-CD8a-furin were treated with biotin for 2 h at 20 °C before the system was warmed up during the chase. The surface-labeling intensity was normalized by the total cellular GFP intensity. j AAs reduce cell surface CD8a-furin, -sortilin, and -CI-M6PR. Transfected cells were labeled for both surface and intracellular pools of CD8a-chimeras. The intensity of the surface pool was normalized by that of the intracellular pool. b , e , f , h – j are representative results from three independent experiments. Scale bar, 10 µm; n, the number of cells analyzed; error bar, mean ± s.e.m.; P values were from t test (unpaired and two-tailed). N.S. not significant ( P > 0.05); * P ≤ 0.05; ** P ≤ 0.005; *** P ≤ 0.0005; **** P ≤ 0.00005; ***** P ≤ 0.000005

Article Snippet: Mouse anti-Lamp1 mAb (H4A3)(1:500 for IF) and mouse anti-CD8a mAb (OKT8)(1:500 for IF) were from Developmental Studies Hybridoma Bank.

Techniques: Stable Transfection, Expressing, Labeling, Staining, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

doi: 10.1038/s41467-018-07444-y

Figure Lengend Snippet: Signaling components essential for the AA-stimulated retrograde trafficking. All cells are HeLa cells. a Cells stably expressing CD8a-furin were starved in HBSS for 2 h followed by surface-labeling and subsequent incubation with either HBSS or DMEM for 20 min. 1% DMSO or 2.5 µM conA was present throughout the incubation. Cells were stained and the AA-stimulated Golgi trafficking is quantified by imaging. b Endogenous SLC38A9 was depleted by lentivirus-transduced shRNAs as assessed by RT-qPCR from n = 3 independent experiments. c The knockdown of endogenous SLC38A9 attenuated the AA-stimulated mTORC1 activity. Knockdown cells were incubated with DMEM/-AAs for 50 min followed by incubation with DMEM for 20 min. Cell lysates were immuno-blotted for phospho-S6K1 (p-S6K1) and GAPDH. d SLC38A9 is required for the AA-simulated Golgi trafficking. Cells treated with indicated shRNAs were transfected to express CD8a-furin and subjected to treatment and analysis similar to a . e Immuno-blots showing that endogenous Lamtor1 and RagA/B were depleted by respective lentivirus-transduced shRNAs. f Lamtor1 and 3 but not RagA/B are required for the AA-stimulated Golgi trafficking. The experiment was similar to d . g , h Under Lamtor1 knockdown condition similar to e , an RNAi-resistant Lamtor1 was able to express and rescue the AA-stimulated Golgi trafficking. i Lamtor1 is required for the AA-stimulated reduction of surface CD8a-furin-mEos2. Knockdown and surface labeling were similar to e and Fig. , respectively. Surface intensity was normalized by mEos2 total intensity. Surface DMEM/HBSS-ratio is the normalized surface intensity under DMEM divided by that under HBSS treatment. j mTORC1 is not required for the AA-stimulated retrograde trafficking. The experiment was conducted similarly to a except that 1% DMSO, 100 nM rapamycin, or 250 nM Torin1 was present throughout the treatment. In a , b , d , f , h – j , the displayed value is the mean of n = 3 independent experiments and individual data points are shown as red dots. Error bar, mean ± s.d.; P values were from t test (unpaired and two-tailed); N.S., not significant ( P > 0.05); * P ≤ 0.05. GL2 is a non-targeting control siRNA or shRNA

Article Snippet: Mouse anti-Lamp1 mAb (H4A3)(1:500 for IF) and mouse anti-CD8a mAb (OKT8)(1:500 for IF) were from Developmental Studies Hybridoma Bank.

Techniques: Stable Transfection, Expressing, Labeling, Incubation, Staining, Imaging, Quantitative RT-PCR, Knockdown, Activity Assay, Transfection, Western Blot, Two Tailed Test, Control, shRNA

Journal: Nature Communications

Article Title: Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

doi: 10.1038/s41467-018-07444-y

Figure Lengend Snippet: Arl5’s localization and its essential role in the AA-stimulated Golgi trafficking. HeLa cells were used. a The Golgi localization of different mutant forms of Arl5b. Cells transiently expressing Arl5b-GFP in QL, TN, or wt form were fixed and endogenous Golgin-245 was stained. b Endogenous Arl5b localizes to the Golgi. Cells were fixed and endogenous Arl5b and GS28 were co-stained. c , d Arl5b colocalizes with Lamtor1 at the endosome and lysosome. Cells transiently co-expressing indicated GFP or mCherry-tagged proteins were imaged under live-cell condition. e Lamtor1 localizes to the EE, LE and lysosome. Endogenous Lamtor1 was co-stained with exogenously expressed GFP-Rab7, endogenous EEA1, or Lamp1, respectively. In c – e , the boxed region was enlarged in the upper right corner to show the colocalization at puncta (denoted by arrows). Scale bar, 10 µm. f The immuno-blot showing that endogenous Arl5b was knocked down by a mixture of siRNAs targeting Arl5a, b, and c. g Arl5 is required for the AA-stimulated Golgi trafficking of CD8a-furin. The experiment was conducted as in Fig. . h – j When endogenous Arl5b was depleted by lentivirus-transduced shRNA, the expression of an RNAi-resistant Arl5b significantly increased the cellular level of Arl5b and rescued the AA-stimulated Golgi trafficking. k The knockdown of endogenous Vps51 and Vps54 by respective lentivirus-transduced shRNAs as assessed by RT-qPCR. The data were from n = 3 independent experiments. l GARP is required for the AA-stimulated Golgi trafficking. The experiment was conducted as in Fig. . In g , j , k , and l , the displayed value is the mean of n = 3 independent experiments and individual data points are shown as red dots. Error bar, mean ± s.d.; P values were from t test (unpaired and two-tailed); N.S, . not significant ( P > 0.05); * P ≤ 0.05. GL2 is a non-targeting control siRNA or shRNA

Article Snippet: Mouse anti-Lamp1 mAb (H4A3)(1:500 for IF) and mouse anti-CD8a mAb (OKT8)(1:500 for IF) were from Developmental Studies Hybridoma Bank.

Techniques: Mutagenesis, Expressing, Staining, shRNA, Knockdown, Quantitative RT-PCR, Two Tailed Test, Control

Journal: Arthritis Research & Therapy

Article Title: Differences in serum and synovial CD4+ T cells and cytokine profiles to stratify patients with inflammatory osteoarthritis and rheumatoid arthritis

doi: 10.1186/s13075-017-1305-1

Figure Lengend Snippet: Patients with osteoarthritis ( OA ) manifest qualitative and quantitative differences in the immune cell infiltrates in the synovial compartment as compared to patients with rheumatoid arthritis ( RA ). Frequencies ( a ) and cumulative statistical analysis ( b ) of immune cell populations (monocytes, CD14 + , B cells CD19 + , natural killer cells, CD56 + , CD3 + CD4 + T cells and CD3 + CD8 + T cells) in peripheral blood ( a , b ) from healthy donors ( HD , n = 25) patients with OA ( n = 11) and patients with RA ( n = 24). Frequencies ( c ) and cumulative statistical analysis ( d ) of immune cell populations in synovial fluid ( SF ) from patients with OA ( n = 6) and patients with RA ( n = 8): * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, Mann-Whitney unpaired two-tailed t test. Mean value ± SEM are reported. ns not significant, PBMC peripheral blood mononuclear cells, MC mononuclear cells

Article Snippet: Single-cell suspension of cells was stained with the following antibodies: CD3 (UCHT1), CD4 (RPA-T4; VIT4), CD8 (RPA-T8; OKT8), CD19 (HIB19), CD56 (B159,), CD127 (eBioRDR5), CD25 (M-A251 BD; BC96), ICOS (ISA-3), CXCR5 (51505), CCR6 (11A9) and CCR5 (27D/CCR5), obtained from BD Bioscience (Becton, Dickinson and Company, NJ, USA), eBioscience (eBioscience Inc, CA, USA), Miltenyi (Miltenyi Biotec GmbH, DE) or R&D (R&D Systems, MN, USA).

Techniques: MANN-WHITNEY, Two Tailed Test

Journal: European journal of immunology

Article Title: A human immune system (HIS) mouse model that dissociates roles for mouse and human FcR + cells during antibody-mediated immune responses.

doi: 10.1002/eji.202350454

Figure Lengend Snippet: Figure 2. Human immune cell reconstitution in BRGSA2DR2 Fcer1g−/−mice. Quantification of human immune cell populations in human immune system (HIS) Fcer1g wt mice (black) versus HIS Fcer1g−/−mice (red). (A–C) In blood at 12 weeks postgrafting (wpg), (A) percentage of hCD45 calculated as %hCD45 cells = 100*hCD45/(hCD45 + mCD45), (B) frequency of CD19+ B cells in hCD45 cells, and (C) frequency of CD3+ T cells in hCD45 cells. (D–I) For the spleen, BM and liver at 16 wpg. (D) Absolute numbers of hCD45 per organ (for BM for one femur and one tibia). Frequency of (E) CD19+ B cells, (F) CD3+ T cells, (G) NKp46+ and CD94+ NK cells in hCD45 and (H) CD4+ and (I) CD8+ in total CD3+ T cells. Each dot represents one mouse, and data from the blood are representative for 21 mice (>6 independent experiments) and from the organs for 2 independent experiments. Pairwise comparison in the blood was done by Wilcoxon test, and Kruskal–Wallis test was used for multiple comparisons of means in the organs.

Article Snippet: Antigen Fluorochrome Clone Reactivity Supplier Catalog number CD3 BV510 SK7 Human BioLegend 344828 CD3 APC-Vio770 REA613 Human Miltenyi Biotec 130-113-136 CD4 APC RPA-T4 Human BD 555349 CD4 BUV496 SK3 Human BD 612936 CD11b AF700 M1/70 Mouse/human eBioscience 56-0112-82 CD16 BUV737 3G8 Human BD 564434 CD16 BUV805 3G8 Human BD 748850 CD16/CD32 PE 2.4G2 Mouse BD 561727 CD19 PE HIB19 Human BioLegend 302208 CD19 PE-CF594 HIB19 Human BD 562294 CD20 BV786 2H7 Human BioLegend 302356 CD45 APC-Cy7 30-F11 Mouse BD 557659 CD45 PerCP-Cy5.5 HI30 Human BD 564105 CD45 FITC 30-F11 Mouse BioLegend 103108 CD45 BUV395 HI30 Human BD 563792 CD45 BV650 30-F11 Mouse BD 563410 CD45 BUV805 HI30 Human BD 612891 CD64 PE X54-5/7.1 Mouse BD 558455 CD8a PE-CF594 RPA-T8 Human BD 562282 CD8a BV786 RPA-T8 Human BioLegend 301046 CD8b PE-Cy7 SIDI8BEE Human eBioscience 25-5273-42 CD94 FITC DX22 Human BioLegend 305504 CD94 PerCP-Vio700 REA113 Human Miltenyi Biotec 130-119-763 FCER1A PE MAR-1 Mouse eBioscience 12-5898-82 FCER1G FITC Human/mouse Merck- Millipore FCABS400F Gr-1 FITC RB6-8C5 Mouse BD 553127 NKp46 BV421 9E2/NKp46 Human BD 564065 Antibody-mediated depletion of human cell subsets in vivo For CD8a+ cell depletion, HIS mice were prebled 3 days before i.p. (intraperitoneal) injection with 100 μg/mouse of antihuman CD8a antibody OKT8 (BioXcell) in PBS, and control mice were injected with only PBS and bled at day 4 (d4) postinjection.

Techniques: Comparison

Journal: Arthritis Research & Therapy

Article Title: Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

doi: 10.1186/ar2406

Figure Lengend Snippet: IL-17 expression on T cells within rheumatoid synovium and in synovial fluid CD4 + CD3 + T cells . (a) Rheumatoid synovial tissue was examined by immunohistochemistry. IL-17 (red) was found to colocalise with CD3 + T cells (blue) in perivascular cuffs (purple). Blood vessels were localised with von Willebrand factor (vWF) (green). Nuclear staining is shown in grey. (b) IL-17 (red) expression is associated with CD4 + T cells (blue) but not CD8 + (green) T cells. Nuclear staining is shown in grey. (c) flow cytometric analysis of peripheral blood (PB) and synovial fluid (SF) CD3 + T cells demonstrates that IL-17 is expressed in SF CD4 + T cells. PE, phycoerythrin; FITC, fluorescein isothyocyanate.

Article Snippet: The sections were rehydrated with PBS pH 7.4 with 5% foetal calf serum (Biosera Ltd, Ringmer, UK), and indirect immunofluorescence was performed using the following primary antibody combinations: IgG 2b mouse anti-human CD3 (UCHT-1, 17 μg/ml; gift from Peter Beverley, University College Hospital, London, UK) with rabbit anti-human von Willibrand factor (A0082, 6.2 μg/ml; Dako, Glostrup, Denmark) and IgG 1 mouse anti-human IL-17 (12-7179, 1.25 μg/ml; eBiosciences); or IgG 2b mouse anti-human CD4 (OKT4) with IgG 2a mouse anti-human CD8 (OKT8, both OKT clones used as ascitic fluid 1/100; American Type Culture Collection, Middlesex, UK) and rabbit anti-human IL-17 (AHP455G, 20 μg/ml; AbD Serotec, Oxfordshire, UK).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Advanced Science

Article Title: Tumor‐Associated Monocytes Reprogram CD8 + T Cells into Central Memory‐Like Cells with Potent Antitumor Effects

doi: 10.1002/advs.202304501

Figure Lengend Snippet: Tumor‐associated monocytes are related to T M cell abundance. a) The correlation and p value between monocyte scores and SELL , IL7R , CCR7 mRNA levels in different types of tumor tissues. b) PBMCs from lung cancer patients were stimulated with anti‐CD3 and anti‐CD28 antibodies plus rhIL‐2 for 96 h. The percentage of monocytes in untreated PBMCs and expression levels of the memory markers CD62L and CCR7 in CD8 + T cells from stimulated PBMCs were determined by flow cytometry, followed by Pearson's correlation test ( n = 13). c) Overall survival curves of TCGA LIHC and SKCM patients grouped by mean expression values of monocyte signature genes ( n (LIHC) = 364 patient samples, n (SKCM) = 458 patient samples). d) , CD14 expression levels in tumor tissue samples from melanoma patients with PD, PR, or CR using RNA‐seq data from GSE100797_ACT_Melanoma or GSE91061_αPD‐1_Melanoma cohorts. Data are shown as means ± SEM d). Statistical significance was assessed using Pearson's correlation test a,b) a log‐rank (Mantel‐Cox) test c) or a two‐tailed unpaired Student's t test d). * p < 0.05, ** p < 0.01.

Article Snippet: The following fluorescence tagged antibodies were used for flow cytometry: anti‐human CD45 (clone HI30, PE), anti‐mouse/human CD11b (clone M1/70, FITC), anti‐human CD3 (clone OKT3, PerCP‐Cy5.5), anti‐human CD8 (clone HIT8a, FITC), anti‐mouse CD45 (clone 30‐F11, PE‐Cy7), anti‐mouse Ly6C (clone HK1.4, PE), anti‐phospho‐ZAP70/Syk (Tyr319, Tyr352) (clone n3kobu5, PE), anti‐human/mouse TOX (clone TXRX10, EF660), and anti‐mouse CD8 (clone 53–6.7, FITC) (all eBioscience); anti‐human CD14 (clone M5E2, APC), anti‐human CD4 (clone RPA‐T4, APC), anti‐human CD197 (CCR7) (clone G043H7, PerCP‐Cy5.5), anti‐human CD62L (clone DREG‐56, PE), anti‐mouse Ly6G (clone 1A8, PerCP‐Cy5.5), anti‐mouse CD8 (clone 53–6.7, PE/Cyanine7), anti‐mouse CD44 (clone IM7, PerCP‐Cy5.5), anti‐mouse CD44 (clone IM7, APC), anti‐mouse CD62L (clone MEL14, APC), anti‐mouse CD62L (clone MEL14, PE), anti‐mouse TCR Vα2 (clone B20.1, PE), anti‐mouse TCR Vα2 (clone B20.1, FITC), anti‐mouse TNF (clone MP6‐XT22, PE), anti‐mouse IFN‐γ (clone XMG1.2, APC), anti‐mouse CD69 (clone H1.2F3, APC), anti‐mouse CD300LG (clone ZAQ5, PE), and anti‐mouse CD279 (PD‐1) (clone 29F.1A12, PerCP‐Cy5.5) (all BioLegend); anti‐human CD8 (clone OKT8, PerCP‐Cy5.5), and anti‐human CD45RO (clone UCHL1, APC) (Tonbo Biosciences); anti‐mouse ACE (CD143) (clone 230214, Alexa Fluor 647) (R&D); anti‐human/mouse TCF‐1 (clone C63D9, PE) (CST); and anti‐mouse EOMES (clone Dan11mag, AF488) (Thermo Fisher).

Techniques: Expressing, Flow Cytometry, RNA Sequencing, Two Tailed Test

Journal: Advanced Science

Article Title: Tumor‐Associated Monocytes Reprogram CD8 + T Cells into Central Memory‐Like Cells with Potent Antitumor Effects

doi: 10.1002/advs.202304501

Figure Lengend Snippet: TAMos derived from the spleens of tumor‐bearing mice promote T CM cell generation. a) CD8 + T cells were cocultured with TAMos or TANs from the spleens of LLC tumor‐bearing mice (four pooled mice) and stimulated with anti‐CD3 and anti‐CD28 antibodies (α‐CD3 + α‐CD28), followed by flow cytometry to determine the proportion of CD44 + CD62L + cells in the activated CD8 + T cells ( n = 3 cell cultures). FMO, fluorescence minus one. b–d) Expression levels of CD62L b), CCR7 c), and IL‐7R d) in CD8 + T cells cocultured with TAMos or TANs in the presence of anti‐CD3 and anti‐CD28 antibodies ( n = 3 cell cultures). FMO, fluorescence minus one. e) OT‐I cells were cocultured with TAMos or TANs sorted from the spleens of LLC tumor‐bearing mice (six pooled mice) under OVA 257‐264 stimulation, and the percentages of CD44 + CD62L + cells were determined by flow cytometry ( n = 3 cell cultures). FMO, fluorescence minus one. f) CD62L, CCR7, and IL‐7R expression levels in OT‐I CD8 + T cells cocultured with TAMos or TANs in the presence of OVA 257‐264 peptides ( n = 3 cell cultures). g,h) TANs, TAMos, or TAMs were sorted from B16‐OVA tumor‐bearing mice (ten pooled mice) and cocultured with OT‐I cells under OVA 257‐264 stimulation. CD44 + CD62L + cell proportions (g, n = 3 cell cultures) and CD62L levels (h, n = 3 cell cultures) in CD8 + T cells were determined by flow cytometry. Data are representative of three independent experiments and shown as means ± SEM. Statistical significance was assessed using a two‐tailed unpaired Student's t test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS, not significant.

Article Snippet: The following fluorescence tagged antibodies were used for flow cytometry: anti‐human CD45 (clone HI30, PE), anti‐mouse/human CD11b (clone M1/70, FITC), anti‐human CD3 (clone OKT3, PerCP‐Cy5.5), anti‐human CD8 (clone HIT8a, FITC), anti‐mouse CD45 (clone 30‐F11, PE‐Cy7), anti‐mouse Ly6C (clone HK1.4, PE), anti‐phospho‐ZAP70/Syk (Tyr319, Tyr352) (clone n3kobu5, PE), anti‐human/mouse TOX (clone TXRX10, EF660), and anti‐mouse CD8 (clone 53–6.7, FITC) (all eBioscience); anti‐human CD14 (clone M5E2, APC), anti‐human CD4 (clone RPA‐T4, APC), anti‐human CD197 (CCR7) (clone G043H7, PerCP‐Cy5.5), anti‐human CD62L (clone DREG‐56, PE), anti‐mouse Ly6G (clone 1A8, PerCP‐Cy5.5), anti‐mouse CD8 (clone 53–6.7, PE/Cyanine7), anti‐mouse CD44 (clone IM7, PerCP‐Cy5.5), anti‐mouse CD44 (clone IM7, APC), anti‐mouse CD62L (clone MEL14, APC), anti‐mouse CD62L (clone MEL14, PE), anti‐mouse TCR Vα2 (clone B20.1, PE), anti‐mouse TCR Vα2 (clone B20.1, FITC), anti‐mouse TNF (clone MP6‐XT22, PE), anti‐mouse IFN‐γ (clone XMG1.2, APC), anti‐mouse CD69 (clone H1.2F3, APC), anti‐mouse CD300LG (clone ZAQ5, PE), and anti‐mouse CD279 (PD‐1) (clone 29F.1A12, PerCP‐Cy5.5) (all BioLegend); anti‐human CD8 (clone OKT8, PerCP‐Cy5.5), and anti‐human CD45RO (clone UCHL1, APC) (Tonbo Biosciences); anti‐mouse ACE (CD143) (clone 230214, Alexa Fluor 647) (R&D); anti‐human/mouse TCF‐1 (clone C63D9, PE) (CST); and anti‐mouse EOMES (clone Dan11mag, AF488) (Thermo Fisher).

Techniques: Derivative Assay, Flow Cytometry, Fluorescence, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Tumor‐Associated Monocytes Reprogram CD8 + T Cells into Central Memory‐Like Cells with Potent Antitumor Effects

doi: 10.1002/advs.202304501

Figure Lengend Snippet: TAMos reprogram the T cell landscape during T cell activation. RNA‐seq analysis a–d) of CD8 + T cells purified from coculture systems in which murine OT‐I cells were exposed to vehicle, TAMos, or TANs sorted from the spleens of LLC tumor‐bearing mice and stimulated with OVA 257‐264 peptides ( n = 2 cell cultures). a) Principal components analysis of transcriptomes of CD8 + T cells under different conditions. b) Enrichment analysis of upregulated genes in T cells cocultured with TAMos compared with T cells cultured alone using GO datasets. Pathways related to T cell biological processes are highlighted in red. c) Bubble map displaying the similarities among vehicle, TAN, or TAMo‐treated murine T cells and different T cell subsets isolated from the PBMCs and tumor tissues of human NSCLC patients. T Naïve , naïve T cells; T CM , central memory T cells; T EM , effector memory T cells; T EFF , effector T cells; T EX , exhausted T cells. d) GSEA plots showing representative pathways enriched in murine CD8 + T cells cocultured with TAMos or cultured alone. NES, normalized enrichment score. e) CD8 + T cells were cultured alone or cocultured with TAMos derived from the spleens of LLC tumor‐bearing mice (five pooled mice) and stimulated with anti‐CD3 and anti‐CD28 antibodies for 48 h, followed by flow cytometry to determine TCF‐1 and EOMES protein levels. Unstained activated CD8 + T cells were used as a negative control ( n = 3 cell cultures). f) IGV plots of ATAC‐seq peaks at Ccr7 , Sell , Tcf7 , Eomes , Ifng , Prf1 , Ctla4 , or Il10 loci in OT‐I CD8 + T cells cultured alone or cocultured with TAMos derived from the spleens of LLC tumor‐bearing mice. Data are representative of three independent experiments e) and shown as means ± SEM. Statistical significance was assessed using a two‐tailed unpaired Student's t test e). ** p < 0.01.

Article Snippet: The following fluorescence tagged antibodies were used for flow cytometry: anti‐human CD45 (clone HI30, PE), anti‐mouse/human CD11b (clone M1/70, FITC), anti‐human CD3 (clone OKT3, PerCP‐Cy5.5), anti‐human CD8 (clone HIT8a, FITC), anti‐mouse CD45 (clone 30‐F11, PE‐Cy7), anti‐mouse Ly6C (clone HK1.4, PE), anti‐phospho‐ZAP70/Syk (Tyr319, Tyr352) (clone n3kobu5, PE), anti‐human/mouse TOX (clone TXRX10, EF660), and anti‐mouse CD8 (clone 53–6.7, FITC) (all eBioscience); anti‐human CD14 (clone M5E2, APC), anti‐human CD4 (clone RPA‐T4, APC), anti‐human CD197 (CCR7) (clone G043H7, PerCP‐Cy5.5), anti‐human CD62L (clone DREG‐56, PE), anti‐mouse Ly6G (clone 1A8, PerCP‐Cy5.5), anti‐mouse CD8 (clone 53–6.7, PE/Cyanine7), anti‐mouse CD44 (clone IM7, PerCP‐Cy5.5), anti‐mouse CD44 (clone IM7, APC), anti‐mouse CD62L (clone MEL14, APC), anti‐mouse CD62L (clone MEL14, PE), anti‐mouse TCR Vα2 (clone B20.1, PE), anti‐mouse TCR Vα2 (clone B20.1, FITC), anti‐mouse TNF (clone MP6‐XT22, PE), anti‐mouse IFN‐γ (clone XMG1.2, APC), anti‐mouse CD69 (clone H1.2F3, APC), anti‐mouse CD300LG (clone ZAQ5, PE), and anti‐mouse CD279 (PD‐1) (clone 29F.1A12, PerCP‐Cy5.5) (all BioLegend); anti‐human CD8 (clone OKT8, PerCP‐Cy5.5), and anti‐human CD45RO (clone UCHL1, APC) (Tonbo Biosciences); anti‐mouse ACE (CD143) (clone 230214, Alexa Fluor 647) (R&D); anti‐human/mouse TCF‐1 (clone C63D9, PE) (CST); and anti‐mouse EOMES (clone Dan11mag, AF488) (Thermo Fisher).

Techniques: Activation Assay, RNA Sequencing, Purification, Cell Culture, Isolation, Derivative Assay, Flow Cytometry, Negative Control, Two Tailed Test

Journal: Advanced Science

Article Title: Tumor‐Associated Monocytes Reprogram CD8 + T Cells into Central Memory‐Like Cells with Potent Antitumor Effects

doi: 10.1002/advs.202304501

Figure Lengend Snippet: TAMos potentiate the T cell memory phenotype and attenuate the T cell exhaustion state after antigen rechallenge. a) Flow diagram illustrating the protocol used for antigen rechallenge experiments. b) Expanded CD8 + T cells were cocultured with TAMos derived from the spleens of LLC tumor‐bearing mice at different ratios and reactivated with anti‐CD3 and anti‐CD28 antibodies for 48 h, followed by flow cytometry to determine the percentages of CD44 + CD62L + cells and CD62L levels (four pooled mice, n = 3 cell cultures). c) CD8 + T cells were sorted from the TME of LLC tumor‐bearing mice and cocultured with autogenous TAMos in the presence of anti‐CD3 and anti‐CD28 antibodies plus IL‐2 for 48 h, followed by flow cytometry to determine the percentages of CD44 + CD62L + cells in CD8 + T cells (eight pooled mice, n = 3 cell cultures). d) Expression levels of PD‐1 in CD8 + T cells sorted from the TME of LLC or B16‐OVA tumor‐bearing mice and cocultured with TAMos after anti‐CD3 and anti‐CD28 plus IL‐2 stimulation for 48 h (eight pooled LLC tumor‐bearing mice or six pooled B16‐OVA tumor‐bearing mice, n = 3 cell cultures). e,f) Human CD8 + T cells and TAMos were sorted from PBMCs of lung cancer patients ( n = 7 pooled samples). CD8 + T cells were cultured alone or cocultured with the sorted TAMos and activated with anti‐CD3 and anti‐CD28 antibodies plus rhIL‐2 for 96 h, followed by flow cytometry to determine the proportions of different T cell subsets (e, T Naïve , CCR7 + CD45RO − ; T CM , CCR7 + CD45RO + ; T EM , CCR7 − CD45RO + ; T EFF , CCR7 − CD45RO − ; n = 3 cell cultures) and CD62L expression levels in CD8 + T cells (f, n = 3 cell cultures). FMO, fluorescence minus one. Data are representative of two independent experiments and shown as means ± SEM b–f). Statistical significance was assessed using a two‐tailed unpaired Student's t test b–f). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS, not significant.

Article Snippet: The following fluorescence tagged antibodies were used for flow cytometry: anti‐human CD45 (clone HI30, PE), anti‐mouse/human CD11b (clone M1/70, FITC), anti‐human CD3 (clone OKT3, PerCP‐Cy5.5), anti‐human CD8 (clone HIT8a, FITC), anti‐mouse CD45 (clone 30‐F11, PE‐Cy7), anti‐mouse Ly6C (clone HK1.4, PE), anti‐phospho‐ZAP70/Syk (Tyr319, Tyr352) (clone n3kobu5, PE), anti‐human/mouse TOX (clone TXRX10, EF660), and anti‐mouse CD8 (clone 53–6.7, FITC) (all eBioscience); anti‐human CD14 (clone M5E2, APC), anti‐human CD4 (clone RPA‐T4, APC), anti‐human CD197 (CCR7) (clone G043H7, PerCP‐Cy5.5), anti‐human CD62L (clone DREG‐56, PE), anti‐mouse Ly6G (clone 1A8, PerCP‐Cy5.5), anti‐mouse CD8 (clone 53–6.7, PE/Cyanine7), anti‐mouse CD44 (clone IM7, PerCP‐Cy5.5), anti‐mouse CD44 (clone IM7, APC), anti‐mouse CD62L (clone MEL14, APC), anti‐mouse CD62L (clone MEL14, PE), anti‐mouse TCR Vα2 (clone B20.1, PE), anti‐mouse TCR Vα2 (clone B20.1, FITC), anti‐mouse TNF (clone MP6‐XT22, PE), anti‐mouse IFN‐γ (clone XMG1.2, APC), anti‐mouse CD69 (clone H1.2F3, APC), anti‐mouse CD300LG (clone ZAQ5, PE), and anti‐mouse CD279 (PD‐1) (clone 29F.1A12, PerCP‐Cy5.5) (all BioLegend); anti‐human CD8 (clone OKT8, PerCP‐Cy5.5), and anti‐human CD45RO (clone UCHL1, APC) (Tonbo Biosciences); anti‐mouse ACE (CD143) (clone 230214, Alexa Fluor 647) (R&D); anti‐human/mouse TCF‐1 (clone C63D9, PE) (CST); and anti‐mouse EOMES (clone Dan11mag, AF488) (Thermo Fisher).

Techniques: Derivative Assay, Flow Cytometry, Expressing, Cell Culture, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Tumor‐Associated Monocytes Reprogram CD8 + T Cells into Central Memory‐Like Cells with Potent Antitumor Effects

doi: 10.1002/advs.202304501

Figure Lengend Snippet: TAMos promote T CM cell differentiation independent of NOS2‐mediated inhibition of T cell proliferation. a,b) CFSE‐labeled CD8 + T cells were cultured alone or cocultured with TAMos derived from the spleens of LLC tumor‐bearing mice (ten pooled mice) in the presence or absence of L‐NMMA under stimulation of anti‐CD3 and anti‐CD28 antibodies, and T cell proliferation was determined by mean fluorescence intensity of CFSE at 12, 24, or 48 h post T cell activation ( n = 3 cell cultures). c) NO concentrations in supernatants of CD8 + T cell activated with anti‐CD3 and anti‐CD28 antibodies alone (Vehicle), in the presence of L‐NMMA, in the presence of TAMos or in the presence of TAMos and L‐NMMA ( n = 3 cell cultures). ND, not detected. d,e) Flow cytometry analysis of the proportions of different T cell subsets (d, n = 3 cell cultures) and the expression levels of CD62L, CCR7, IL‐7R, and TCF‐1 (e, n = 3 cell cultures) in activated CD8 + T cells cultured alone or cocultured with TAMos sorted from LLC tumor‐bearing mice (ten pooled mice) for 12, 24, or 48 h with or without L‐NMMA. Data are shown as means ± SEM. Statistical significance was assessed using a two‐tailed unpaired Student's t test. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS, not significant.

Article Snippet: The following fluorescence tagged antibodies were used for flow cytometry: anti‐human CD45 (clone HI30, PE), anti‐mouse/human CD11b (clone M1/70, FITC), anti‐human CD3 (clone OKT3, PerCP‐Cy5.5), anti‐human CD8 (clone HIT8a, FITC), anti‐mouse CD45 (clone 30‐F11, PE‐Cy7), anti‐mouse Ly6C (clone HK1.4, PE), anti‐phospho‐ZAP70/Syk (Tyr319, Tyr352) (clone n3kobu5, PE), anti‐human/mouse TOX (clone TXRX10, EF660), and anti‐mouse CD8 (clone 53–6.7, FITC) (all eBioscience); anti‐human CD14 (clone M5E2, APC), anti‐human CD4 (clone RPA‐T4, APC), anti‐human CD197 (CCR7) (clone G043H7, PerCP‐Cy5.5), anti‐human CD62L (clone DREG‐56, PE), anti‐mouse Ly6G (clone 1A8, PerCP‐Cy5.5), anti‐mouse CD8 (clone 53–6.7, PE/Cyanine7), anti‐mouse CD44 (clone IM7, PerCP‐Cy5.5), anti‐mouse CD44 (clone IM7, APC), anti‐mouse CD62L (clone MEL14, APC), anti‐mouse CD62L (clone MEL14, PE), anti‐mouse TCR Vα2 (clone B20.1, PE), anti‐mouse TCR Vα2 (clone B20.1, FITC), anti‐mouse TNF (clone MP6‐XT22, PE), anti‐mouse IFN‐γ (clone XMG1.2, APC), anti‐mouse CD69 (clone H1.2F3, APC), anti‐mouse CD300LG (clone ZAQ5, PE), and anti‐mouse CD279 (PD‐1) (clone 29F.1A12, PerCP‐Cy5.5) (all BioLegend); anti‐human CD8 (clone OKT8, PerCP‐Cy5.5), and anti‐human CD45RO (clone UCHL1, APC) (Tonbo Biosciences); anti‐mouse ACE (CD143) (clone 230214, Alexa Fluor 647) (R&D); anti‐human/mouse TCF‐1 (clone C63D9, PE) (CST); and anti‐mouse EOMES (clone Dan11mag, AF488) (Thermo Fisher).

Techniques: Cell Differentiation, Inhibition, Labeling, Cell Culture, Derivative Assay, Fluorescence, Activation Assay, Flow Cytometry, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Tumor‐Associated Monocytes Reprogram CD8 + T Cells into Central Memory‐Like Cells with Potent Antitumor Effects

doi: 10.1002/advs.202304501

Figure Lengend Snippet: T cells cocultured with TAMos exhibited enhanced antitumor activity in vitro and in vivo independent of NO signaling. a) Diagram illustrating protocol for T cell killing assay. b) OT‐I cells were cultured alone or cocultured with TAMos derived from the spleens of LLC tumor‐bearing mice (ten pooled mice) and activated with OVA 257‐264 peptides for 48 h in the presence or absence of L‐NMMA, followed by incubation with LLC‐OVA tumor cells. The percentages of specific lysis were determined by flow cytometry ( n = 3 cell cultures). E refers to effector cells (OT‐I cells); T refers to target cells (LLC‐OVA tumor cells). c) Flow diagram illustrating the adoptive T cell transfer model. d–h) OT‐I cells were cultured alone or cocultured with TAMos derived from the spleens of LLC tumor‐bearing mice (twenty pooled mice) in the presence or absence of L‐NMMA upon OVA 257‐264 stimulation for 48 h. After expansion for 4 days using IL‐2, those T cells were transferred into NOD‐SCID mice bearing LLC‐OVA tumor. d) Fold changes in the number of T cells in different groups after IL‐2 expansion normalized by the vehicle‐treated T cell group ( n = 3 replicates). e) Tumor volumes in the different groups of mice were monitored every two days ( n = 5 mice per group). f,g) On day 4 after T cell transfer, the proportions of CD8 + T cells in TDLNs (f, n = 6 or 7 mice per group) and tumors (g, n = 7 or 8 mice per group) were determined by flow cytometry. h) Flow cytometry analysis showing the expression levels of TCF‐1 and TOX in transferred CD8 + T cells infiltrating tumors ( n = 7 or 8 mice per group). FMO, fluorescence minus one. Data are shown as means ± SEM. Statistical significance was assessed using a two‐tailed unpaired Student's t test b,d,f–h) or two‐way ANOVA e). ** p < 0.01, **** p < 0.0001. NS, not significant.

Article Snippet: The following fluorescence tagged antibodies were used for flow cytometry: anti‐human CD45 (clone HI30, PE), anti‐mouse/human CD11b (clone M1/70, FITC), anti‐human CD3 (clone OKT3, PerCP‐Cy5.5), anti‐human CD8 (clone HIT8a, FITC), anti‐mouse CD45 (clone 30‐F11, PE‐Cy7), anti‐mouse Ly6C (clone HK1.4, PE), anti‐phospho‐ZAP70/Syk (Tyr319, Tyr352) (clone n3kobu5, PE), anti‐human/mouse TOX (clone TXRX10, EF660), and anti‐mouse CD8 (clone 53–6.7, FITC) (all eBioscience); anti‐human CD14 (clone M5E2, APC), anti‐human CD4 (clone RPA‐T4, APC), anti‐human CD197 (CCR7) (clone G043H7, PerCP‐Cy5.5), anti‐human CD62L (clone DREG‐56, PE), anti‐mouse Ly6G (clone 1A8, PerCP‐Cy5.5), anti‐mouse CD8 (clone 53–6.7, PE/Cyanine7), anti‐mouse CD44 (clone IM7, PerCP‐Cy5.5), anti‐mouse CD44 (clone IM7, APC), anti‐mouse CD62L (clone MEL14, APC), anti‐mouse CD62L (clone MEL14, PE), anti‐mouse TCR Vα2 (clone B20.1, PE), anti‐mouse TCR Vα2 (clone B20.1, FITC), anti‐mouse TNF (clone MP6‐XT22, PE), anti‐mouse IFN‐γ (clone XMG1.2, APC), anti‐mouse CD69 (clone H1.2F3, APC), anti‐mouse CD300LG (clone ZAQ5, PE), and anti‐mouse CD279 (PD‐1) (clone 29F.1A12, PerCP‐Cy5.5) (all BioLegend); anti‐human CD8 (clone OKT8, PerCP‐Cy5.5), and anti‐human CD45RO (clone UCHL1, APC) (Tonbo Biosciences); anti‐mouse ACE (CD143) (clone 230214, Alexa Fluor 647) (R&D); anti‐human/mouse TCF‐1 (clone C63D9, PE) (CST); and anti‐mouse EOMES (clone Dan11mag, AF488) (Thermo Fisher).

Techniques: Activity Assay, In Vitro, In Vivo, Cell Culture, Derivative Assay, Incubation, Lysis, Flow Cytometry, Expressing, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Tumor‐Associated Monocytes Reprogram CD8 + T Cells into Central Memory‐Like Cells with Potent Antitumor Effects

doi: 10.1002/advs.202304501

Figure Lengend Snippet: CD300LG upregulated in TAMos is associated with T CM cell differentiation. a) Schematic representation of the direct coculture and Transwell indirect coculture protocols with OT‐I T cells and TAMos. b) OT‐I T cells were cocultured with TAMos sorted from the spleens of LLC tumor‐bearing mice (nine pooled mice) together or in a Transwell system for 48 h, followed by flow cytometry to determine percentages of CD44 + CD62L + cells in CD8 + T cells ( n = 3 cell cultures). c) Venn diagram denoting the overlap of genes encoding the top upregulated surface transmembrane proteins in TAMos (fold change > 10, adjusted p value < 0.05 and FPKM > 0.5) from the spleens and the TME of tumor‐bearing mice. d) Schematic plot of the MCTOC screening assay. e) Flow cytometry analysis of fold changes of T CM cell percentages in CD8 + T cells from coculture of OT‐I T cells and mitomycin C‐treated HEK293T cells expressing mCherry‐tagged mock or surface transmembrane proteins ( n = 2 cell cultures). f) The proportion of T CM cells in CD8 + T cells from coculture of OT‐I T cells and mitomycin C‐treated HEK293T cells expressing mCherry‐tagged mock or CD300LG protein. g) Flow cytometry analysis showing CD300LG levels in TAMos expressing shRNA targeting Cd300lg or scramble shRNA. h) OT‐I T cells were cocultured with TAMos infected with lentivirus encoding shRNA targeting Cd300lg or scramble shRNA during T cell activation, followed by flow cytometry to determine proportion of T CM cells. i) Coomassie‐stained SDS‐polyacrylamide gel of recombinant CD300LG‐EC. j,k) OT‐I cells were cultured in the presence of recombinant CD300LG‐EC for 48 h during T cell activation, followed by flow cytometry to determine percentages of CD44 + CD62L + cells (j, n = 3 cell cultures) and the expression levels of TCF‐1, EOMES, IL‐7R, and CD62L (k, n = 3 cell cultures). Data are representative of two independent experiments b,e–h) and shown as means ± SEM b,e,h,j,k). Statistical significance was assessed using a two‐tailed unpaired Student's t test b,e,h,j,k). NS, not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The following fluorescence tagged antibodies were used for flow cytometry: anti‐human CD45 (clone HI30, PE), anti‐mouse/human CD11b (clone M1/70, FITC), anti‐human CD3 (clone OKT3, PerCP‐Cy5.5), anti‐human CD8 (clone HIT8a, FITC), anti‐mouse CD45 (clone 30‐F11, PE‐Cy7), anti‐mouse Ly6C (clone HK1.4, PE), anti‐phospho‐ZAP70/Syk (Tyr319, Tyr352) (clone n3kobu5, PE), anti‐human/mouse TOX (clone TXRX10, EF660), and anti‐mouse CD8 (clone 53–6.7, FITC) (all eBioscience); anti‐human CD14 (clone M5E2, APC), anti‐human CD4 (clone RPA‐T4, APC), anti‐human CD197 (CCR7) (clone G043H7, PerCP‐Cy5.5), anti‐human CD62L (clone DREG‐56, PE), anti‐mouse Ly6G (clone 1A8, PerCP‐Cy5.5), anti‐mouse CD8 (clone 53–6.7, PE/Cyanine7), anti‐mouse CD44 (clone IM7, PerCP‐Cy5.5), anti‐mouse CD44 (clone IM7, APC), anti‐mouse CD62L (clone MEL14, APC), anti‐mouse CD62L (clone MEL14, PE), anti‐mouse TCR Vα2 (clone B20.1, PE), anti‐mouse TCR Vα2 (clone B20.1, FITC), anti‐mouse TNF (clone MP6‐XT22, PE), anti‐mouse IFN‐γ (clone XMG1.2, APC), anti‐mouse CD69 (clone H1.2F3, APC), anti‐mouse CD300LG (clone ZAQ5, PE), and anti‐mouse CD279 (PD‐1) (clone 29F.1A12, PerCP‐Cy5.5) (all BioLegend); anti‐human CD8 (clone OKT8, PerCP‐Cy5.5), and anti‐human CD45RO (clone UCHL1, APC) (Tonbo Biosciences); anti‐mouse ACE (CD143) (clone 230214, Alexa Fluor 647) (R&D); anti‐human/mouse TCF‐1 (clone C63D9, PE) (CST); and anti‐mouse EOMES (clone Dan11mag, AF488) (Thermo Fisher).

Techniques: Cell Differentiation, Flow Cytometry, Screening Assay, Expressing, shRNA, Infection, Activation Assay, Staining, Recombinant, Cell Culture, Two Tailed Test

Journal: Advanced Science

Article Title: Tumor‐Associated Monocytes Reprogram CD8 + T Cells into Central Memory‐Like Cells with Potent Antitumor Effects

doi: 10.1002/advs.202304501

Figure Lengend Snippet: CD300LG high ACE low subset in TAMos plays a major role in T CM cell differentiation. ScRNA‐seq analysis of TAMos purified from the spleens of B16‐OVA tumor‐bearing mice (twelve pooled mice, a–e). a) UMAP plot showing ten subclusters of TAMos utilizing graph‐based clustering ( n = 7113 single cells). b) Heatmap showing scaled expression patterns of the top marker genes in each cell cluster. Clusters 8 and 9 represent B and T cells, respectively. Cluster 6 represents adherent cells. c) Trajectory of cells from the TAMo clusters 0–5 and 7 using the Monocle 2 algorithm. Each dot represents a single cell ( n = 6788 cells). d) UMAP plots showing expression levels of Cd300lg and Ace in the different clusters of TAMos. e) Cd300lg and Ace expression levels in clusters 0, 3, 4 and clusters 1, 2, 5, 7 of TAMos. f–h) CD8 + T cells were cocultured with purified TAMos, CD300LG low ACE high TAMos, or CD300LG high ACE low TAMos derived from B16‐OVA tumor‐bearing mice (five pooled mice) or cultured alone in the presence of anti‐CD3 and anti‐CD28 antibodies, followed by flow cytometry to determine proportions of CD44 + CD62L + cells f,g) and CD62L levels h) in CD8 + T cells ( n = 2 or 3 cell cultures). Data are representative of two independent experiments f–h) and shown as means ± SEM e,g,h). Statistical significance was assessed using a two‐tailed unpaired Student's t test e,g,h). * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The following fluorescence tagged antibodies were used for flow cytometry: anti‐human CD45 (clone HI30, PE), anti‐mouse/human CD11b (clone M1/70, FITC), anti‐human CD3 (clone OKT3, PerCP‐Cy5.5), anti‐human CD8 (clone HIT8a, FITC), anti‐mouse CD45 (clone 30‐F11, PE‐Cy7), anti‐mouse Ly6C (clone HK1.4, PE), anti‐phospho‐ZAP70/Syk (Tyr319, Tyr352) (clone n3kobu5, PE), anti‐human/mouse TOX (clone TXRX10, EF660), and anti‐mouse CD8 (clone 53–6.7, FITC) (all eBioscience); anti‐human CD14 (clone M5E2, APC), anti‐human CD4 (clone RPA‐T4, APC), anti‐human CD197 (CCR7) (clone G043H7, PerCP‐Cy5.5), anti‐human CD62L (clone DREG‐56, PE), anti‐mouse Ly6G (clone 1A8, PerCP‐Cy5.5), anti‐mouse CD8 (clone 53–6.7, PE/Cyanine7), anti‐mouse CD44 (clone IM7, PerCP‐Cy5.5), anti‐mouse CD44 (clone IM7, APC), anti‐mouse CD62L (clone MEL14, APC), anti‐mouse CD62L (clone MEL14, PE), anti‐mouse TCR Vα2 (clone B20.1, PE), anti‐mouse TCR Vα2 (clone B20.1, FITC), anti‐mouse TNF (clone MP6‐XT22, PE), anti‐mouse IFN‐γ (clone XMG1.2, APC), anti‐mouse CD69 (clone H1.2F3, APC), anti‐mouse CD300LG (clone ZAQ5, PE), and anti‐mouse CD279 (PD‐1) (clone 29F.1A12, PerCP‐Cy5.5) (all BioLegend); anti‐human CD8 (clone OKT8, PerCP‐Cy5.5), and anti‐human CD45RO (clone UCHL1, APC) (Tonbo Biosciences); anti‐mouse ACE (CD143) (clone 230214, Alexa Fluor 647) (R&D); anti‐human/mouse TCF‐1 (clone C63D9, PE) (CST); and anti‐mouse EOMES (clone Dan11mag, AF488) (Thermo Fisher).

Techniques: Cell Differentiation, Purification, Expressing, Marker, Derivative Assay, Cell Culture, Flow Cytometry, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Immune signatures of SARS-CoV-2 infection resolution in human lung tissues

doi: 10.1371/journal.ppat.1013469

Figure Lengend Snippet: (A) BLT-L mice were administered 200 μg of anti-CD3e (OKT3), anti-CD4 (OKT4), anti-CD8 (OKT8) or IgG2a isotype. Created with BioRender.com. (B) Viral titer (log10(PFU/mg)) of fLX collected from BLT-L mice treated with isotype, OKT3, OKT4, or OKT8 antibody at 12 dpi. (C) Immunohistochemistry for SARS-CoV-2 N protein of fLX collected from antibody-treated BLT-L mice at 12 dpi. Images are representative of two independent cohorts and donors. (D) Multiplex immunohistochemistry of fLX collected from antibody-treated BLT-L mice at 12 dpi. Dapi = gray, CD3 = teal, CD4 = red. Scale bar = 50 μm. Images are representative of two independent cohorts and donors. (E-F) Multiplex fluorescent immunohistochemistry (E) and CD163 + area quantification (AQ) analysis (F) of fLX following infection resolution (12 dpi, isotype-treated), or during acute (2 dpi) and persistent infection (12 dpi, OKT4-treated). Images are representative of two independent cohorts and donors. CD163: teal, CD4: yellow, CD3: orange, PPARγ: green, Dapi: gray. Scale bar = 50 μM.

Article Snippet: BLT-L mice were administered 200 mg of anti-CD3e (OKT3) (BioxCell; cat. # BE0001–2), anti-CD4 (OKT4) (BioxCell; cat. # BE0003–2), anti-CD8 (OKT8) (BioxCell; cat. # BE0004–2), or isotype IgG2a (Thermofisher; cat # 02–6200) antibody 3-, 2-, and 1-day prior to inoculation and 4- and 8-days post inoculation with 1x10 6 PFU SARS-CoV-2 WA-1.

Techniques: Immunohistochemistry, Multiplex Assay, Infection