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

Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: Fig. 4 Activation of primary CD4+ T cells selectively alters SR kinase and SR protein levels. Primary CD4+ T cells were isolated from healthy (HIV uninfected) human donors and untreated (control) or treated with activators (anti-CD3/CD28 and IL-2). Cells were harvested at different times (24 h, 48 h, 4 d, and 6 d) with or without activation for analyses by western blots or RT-qPCR to look for changes in the expression of SR kinases and SR proteins. a Top and bottom panels on the left are the representative western blots probed for CLK1, CLK2, CLK3, and SRPK1. Top and bottom panels on the right are the quantitation of blots for at least 3 donors (except for 4 d and 6 d post-activation for CLK2, CLK3, and SRPK1 expression levels where only one donor was used). b Quantification of CLK1 and SRPK1 mRNA levels in CD4+ T cells of 3 donors by RT-qPCR assay. mRNA levels were normalized to ß2-microglobulin and mean mRNA levels were expressed relative to untreated control. c Quantitation of western blots for SR protein expression levels in untreated versus treated/activated CD4+ T cell lysates (see Additional file 1: Fig. S4 for representative western blots) across at least 3 donors. For western blots, band intensity was quantified relative to untreated control and normalized to total protein load using Bio-Rad ImageLab software. Data are indicated as mean ± SD, n = 3 or 4 independent experiments, *p ≤ 0.05 and **p ≤ 0.01

Article Snippet: Following isolation of CD4+ T cells, 5 million cells were plated onto each well of a 12-well non-treated TC dish that was coated overnight with 2 μg/mL of anti-CD3 antibody (Ultra-LeafTM Purified anti-human CD3 Clone: OKT3, BioLegend, Cat#317326) and washed with 1XPBS, in the presence of RPMI-1640 complete medium containing 2 μg/mL of anti-CD28 antibody (Fitzgerald, Batch#0979) and 10 ng/mL IL-2 (Sigma-Aldrich, Cat#I7908-10KU).

Techniques: Activation Assay, Isolation, Control, Western Blot, Quantitative RT-PCR, Expressing, Quantitation Assay, Software