Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) Wild type (WT) and MMTV-PyMT (PyMT) mice on the FVB background were allowed to exercise voluntarily (in running wheels, runners) or left non-exercised (locked running wheels, non-runners) between 4 and 12 weeks of age. ( B ) Running distance (km/day) in WT and PyMT mice. *p<0.05, two-tailed unpaired t test. ( C ) Tumor volume measured twice weekly for PyMT running and non-running mice. Mean and SEM (n = 10–11). ( D ) Survival of PyMT mice, runners, and non-runners. Survival curve ns = not significant, Log-rank (Mantel-Cox) test, (n = 10–11). ( E ) Tumor initiation as age (day) of first indication of a palpable tumor in running (Runners) and non-running (Non-runners) PyMT mice. n = 11, ns = not significant, two-tailed t test. ( F ) Tumor stage from histological scoring (1-4) in mammary glands of running and non-running PyMT mice at 12 (n = 6–10) and 8 (n = 4) weeks of age, ns = not significant, two-tailed t test. ( G ) Individual body weight for WT and PyMT running and non-running mice. n = 7–10, ns = not significant, one-way ANOVA with Tukey’s multiple comparison test. ( H ) Immunohistological characterization of PyMT tumors from non-running and running mice using CD3, F4/80, PCNA, Podocalyxin (PODXL), and Granzyme B (GZMB) antibodies, respectively. n = 8–15, ns = not significant, *p<0.05, Two-tailed unpaired t test. ( I ) Flow-cytometry-based frequency of macrophages within I3TC tumor. Ns = not significant, two-tailed t-test. ( J ) Flow-cytometry-based frequency of neutrofiles within I3TC tumor. Ns = not significant, two-tailed t-test.

Article Snippet: The cells were characterized using Flow cytometry described below or plated at 1 × 10 6 (24-well plate) or 5 × 10 5 (48-well plate) and activated with Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher, 11456D ) in a 1:1 beat to T-cell ratio and cultured in 2 ml (24WP) or 1 ml (48WP) RPMI 1640 (Thermo Fisher, 21875 ) supplemented with 10% Fetal Bovine Serum, (Thermo Fisher, 10270 – 106 ), 50 µM 2-mercaptoethanol (Thermo Fisher, 21985023 ), 10 U/ml penicillin-streptomycin (Thermo Fisher, 15140122 ), and 10 U/ml recombinant human IL-2 (Sigma, 11011456001 ), and incubated at 37 °C for 3 days in a humidified CO 2 incubator.

Techniques: Two Tailed Test, Flow Cytometry

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet:

Article Snippet: The cells were characterized using Flow cytometry described below or plated at 1 × 10 6 (24-well plate) or 5 × 10 5 (48-well plate) and activated with Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher, 11456D ) in a 1:1 beat to T-cell ratio and cultured in 2 ml (24WP) or 1 ml (48WP) RPMI 1640 (Thermo Fisher, 21875 ) supplemented with 10% Fetal Bovine Serum, (Thermo Fisher, 10270 – 106 ), 50 µM 2-mercaptoethanol (Thermo Fisher, 21985023 ), 10 U/ml penicillin-streptomycin (Thermo Fisher, 15140122 ), and 10 U/ml recombinant human IL-2 (Sigma, 11011456001 ), and incubated at 37 °C for 3 days in a humidified CO 2 incubator.

Techniques: Transgenic Assay, Marker, Derivative Assay, Staining, Software, Plasmid Preparation, Avidin-Biotin Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Immune isolation-enabled nanoencapsulation of donor T cells: a promising strategy for mitigating GVHD and treating AML in preclinical models

doi: 10.1136/jitc-2023-008663

Figure Lengend Snippet: Key resource table

Article Snippet: APC-conjugated anti-mouse CD28 (REA806) , Miltenyi , Cat#130-111-973; RRID: AB_2656964.

Techniques: Purification, Recombinant, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Proliferation Assay, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Immune isolation-enabled nanoencapsulation of donor T cells: a promising strategy for mitigating GVHD and treating AML in preclinical models

doi: 10.1136/jitc-2023-008663

Figure Lengend Snippet: Successful conformal nanoencapsulation of T cells and preservation of original cell functions. ( A ) Illustration of T-cell encapsulation progression. ( B ) Depiction of zeta potential changes in T cells throughout the layer-by-layer (LbL) encapsulation process. ( C ) Absorption peak plots of both alginate and FITC-alginate at 480 nm are presented, accompanied by FITC fluorescence images of the encapsulated T cell’s outer layer. ( D ) Representative flow scatter plots, demonstrating the encapsulation efficiency achieved when employing a combination of 0.2% gelatin and 0.25% alginate. ( E ) Comparative scanning electron microscopy images of non-encapsulated and encapsulated T cells are displayed, supplemented by differential interference contrast images. Quantification of cell diameters was executed using ImageJ software. ( F–G ) Following 48 hours of purified CD3/CD28 antibody-stimulated proliferation, representative flow peak plots of CFSE for both encapsulated and non-encapsulated T cells are exhibited. The attenuation of cell proliferation fluorescence was observed relative to the fluorescence at 0 hours. ( H–J ) The secretion levels of TNF-α, IL-2, and IFN-γ by T cells at 48 hours and 96 hours post-activation by CD3/CD28 antibody were detected by ELISA. ( K–L ) Comparison of the Anti-CD3 binding capacity between non-encapsulated and encapsulated T cells. All data are represented as mean values±SE, results of at least three ( G–J ) or five ( B, L ) repeat experiments each with three samples. * p<0.05, ** p<0.01. APC, antigen-presenting cell; CFSE, carboxyfluorescein succinimidyl ester; DPBS, Dulbecco's phosphate-buffered saline; FITC, fluorescein isothiocyanate; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

Article Snippet: APC-conjugated anti-mouse CD28 (REA806) , Miltenyi , Cat#130-111-973; RRID: AB_2656964.

Techniques: Preserving, Encapsulation, Zeta Potential Analyzer, Fluorescence, Electron Microscopy, Software, Purification, Activation Assay, Enzyme-linked Immunosorbent Assay, Comparison, Binding Assay, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Immune isolation-enabled nanoencapsulation of donor T cells: a promising strategy for mitigating GVHD and treating AML in preclinical models

doi: 10.1136/jitc-2023-008663

Figure Lengend Snippet: Single-cell nanoencapsulation reduced the expression of co-stimulatory molecules between donor T cells and recipient antigen-presenting cells and affected the formation of immune synapses. Mature DCs were co-cultured with encapsulated or non-encapsulated donor T cells to activate unidirectional mixed lymphocyte responses. ( A–B ) Proliferation of CFSE-labeled T cells was monitored at 48 hours and 96 hours. The attenuation of cell proliferation fluorescence was observed relative to the fluorescence at 0 hours. ( C–F ) Representative flow cytometry histograms and associated statistical analysis of the co-stimulatory molecules CD28, ICOS, and CD40L on T cells and CD80, ICOSL, and CD40 on DCs. ( G–K ) DCs were activated, sensitized with OVA antigen, and co-cultured with either encapsulated or non-encapsulated donor T cells for 6 hours. Cells within this co-culture system were then collected for further analysis. ( G–H ) Imaging flow cytometry results comparing the encapsulated group to the non-encapsulated group. ( I ) Representative immunofluorescence images of T cells co-cultured with DCs in both the encapsulated and non-encapsulated groups. ( J–K ) Scanning electron microscopy and TEM images of T cells co-cultured with DCs in both the encapsulated and non-encapsulated groups. Mean value±SEM, results of at least five repeat experiments each with three samples. *p<0.05, **p<0.01, ***p<0.01. CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; DC, dendritic cell; MHC, major histocompatibility complex; TEM, transmission electron microscopy.

Article Snippet: APC-conjugated anti-mouse CD28 (REA806) , Miltenyi , Cat#130-111-973; RRID: AB_2656964.

Techniques: Expressing, Cell Culture, Labeling, Fluorescence, Flow Cytometry, Co-Culture Assay, Imaging, Immunofluorescence, Electron Microscopy, Immunopeptidomics, Transmission Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Immune isolation-enabled nanoencapsulation of donor T cells: a promising strategy for mitigating GVHD and treating AML in preclinical models

doi: 10.1136/jitc-2023-008663

Figure Lengend Snippet: Transplantation of encapsulated T cells in combination with BMCs inhibits the development of GVHD in recipient mice while preserving the GVL effect. ( A ) Experimental design diagram, demonstrating the use of T cells encapsulated with BMCs for the inhibition of GVHD progression in female BALB/c recipient mice. Mice (20 g each) were administered intraperitoneal (IP) injections of 0.4 mg busulfan and 2 mg cyclophosphamide 7 days prior to BMT. WEHI-3B cells were subsequently infused via tail vein 1-day pre-transplantation. The transplantation involved injecting BMCs (CD45.1) and splenic T cells (CD45.2) from H2-b C57BL/6 mice into the recipient's tail veins. ( B–D ) Graphical representation of alterations in body weight, clinical scores, and survival rates of mice across all groups, monitored over a period of 60 days. ( E–H ) Flow cytometry scatter plots and associated statistical results, indicating the proportions of CD3+, CD4+, and CD8+T cell subsets in mice peripheral blood. A comparison is made between groups receiving encapsulated and non-encapsulated T cells. ( I ) Flow cytometry quantification of cells derived from CD45. One donor mice in recipient mice peripheral blood reflects donor bone marrow-derived cell engraftment. Splenic lymphocytes from recipient mice were analyzed. ( J–K ) Representative scatter plots depicting Treg and T helper cell 17 cell subsets. ( L ) Representative flow cytometry peak plots and statistical analysis of CD28, CD40L, and ICOS expression within the H2kb+CD3+ subset. ( M ) Representative flow cytometry peak plots and statistical analysis of CD80, CD40, and ICOSL expression within the H2kd+LIN-CD11c+MHC-II+ subpopulation was presented. ( N–Q ) Bar graphs representing the secretion levels of IL-6, IL-10, IFN-γ, and C-X-C motif chemokine ligand 10 in plasma and peritoneal macrophages across both encapsulated and non-encapsulated groups. ( S–V ) Statistical plot of pathological scores of H&E-stained images of each target organs from mice. Pooled data from three independent experiments each with seven recipients. Survival ( D ) Kaplan-Meier curve, clinical score ( C ) weight ( B ) from two or three independent experiments, each with seven mice per group, are shown. Mean value±SEM; *p<0.05, ***p<0.001. BMC, bone marrow cell; GVHD, graft-versus-host disease; IFN, interferon; IL, interleukin; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; Treg, regulatory T cell.

Article Snippet: APC-conjugated anti-mouse CD28 (REA806) , Miltenyi , Cat#130-111-973; RRID: AB_2656964.

Techniques: Transplantation Assay, Preserving, Inhibition, Flow Cytometry, Comparison, Derivative Assay, Expressing, Clinical Proteomics, Staining, Fluorescence, Immunopeptidomics