Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Protein Kinase CK2 Controls the Fate Between Th17 Cell and Regulatory T Cell Differentiation CK2 Regulates the Th17/Treg Axis

doi: 10.4049/jimmunol.1601912

Figure Lengend Snippet: (A, B) CD4+ T cells were activated with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies. (A) At 24 and 72 h, cells were stained for intracellular CK2α and CK2β; numbers represent corresponding mean fluorescence intensities (MFIs), and data are representative of 3 independent experiments. (B) RNA was extracted and qPCR performed using primers for Csnk2a1 and Csnk2b (n=3). (C) At 72 h, naïve and activated cells were assayed for CK2 kinase activity. Data represent mean of 2 technical replicates, and are representative of 3 independent experiments. (D–J) Naïve CD4+ T cells were activated in the absence or presence of CX-4945 (2 μM) or rapamycin (100 nM) for 24 h. (D–F) Cells were stained for phosphorylated S235/236 S6 kinase and phosphorylated S473 Akt. (D) Representative histograms and (E, F) MFIs normalized to the corresponding naïve control +/− SEM are shown (n=3). (G–I) Cells were stained for surface expression of the activation markers CD25 and CD69. (G) Representative histograms and (H) MFIs normalized to the corresponding naïve control +/− SEM are shown (n=3). (I) IL-2 was detected in the supernatant by ELISA. Concentrations +/− SEM are shown (n=3). (J) Cells were stained for phosphorylated Y694 STAT5. MFIs normalized to the corresponding naïve control +/− SEM are shown (n=3). (K, L) Naïve CD4+ T cells were incubated with CFSE dye and activated with anti-CD3 and anti-CD28 antibodies in the absence or presence of CX-4945 or rapamycin for 72 h, and proliferation assessed by CFSE dilution. (K) Representative histograms and (L) frequencies of cells undergoing 3 or more divisions +/− SEM (n=3). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Anti-mouse CD3, anti-mouse CD28 and mouse and human neutralizing antibodies to IL-4 and IFN-γ were purchased from BioXCell.

Techniques: Staining, Fluorescence, Activity Assay, Control, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Protein Kinase CK2 Controls the Fate Between Th17 Cell and Regulatory T Cell Differentiation CK2 Regulates the Th17/Treg Axis

doi: 10.4049/jimmunol.1601912

Figure Lengend Snippet: (A–C) Naïve CD4+ T cells from Il17fThy1.1.Foxp3GFP reporter mice were cultured in Th17 conditions. At 72 h Thy1.1+GFP− cells were sorted and reactivated with anti-CD3 and anti-CD28 antibodies and IL-12 (10 ng/ml) in the absence or presence of CX-4945 (2 μM). (A, B) After 48 h of restimulation, cells were stained for IFN-γ. (A) Representative FACS plots and (B) mean frequencies of Thy1.1+IFN-γ+ cells +/− SEM are shown (n=3). (C) At 72 h, mRNA was extracted and gene expression analyzed by qRT-PCR. Data represent the mean relative expression to DMSO control (n=3). (D) 2D2 T cells were activated and polarized under Th17 conditions in the absence or presence of CX-4945 (2 μM) [CX-4945 (day 0)]. On day 3, cells were reactivated for 24 h. Cells that were polarized in the absence of CX-4945 were reactivated in the absence (DMSO) or presence of CX-4945 [CX-4945 (day 3)]. On day 4, 2 x 106 cells from each condition were transferred into Rag1−/− recipient mice. (E) Mice were scored daily for symptoms of classical EAE. Data are pooled form 2 separate experiments; n=5/group. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Anti-mouse CD3, anti-mouse CD28 and mouse and human neutralizing antibodies to IL-4 and IFN-γ were purchased from BioXCell.

Techniques: Cell Culture, Staining, Gene Expression, Quantitative RT-PCR, Expressing, Control

Journal: bioRxiv

Article Title: Humanizing a CD28 signaling domain affects CD8 activation, exhaustion and stem-like precursors

doi: 10.1101/2025.03.10.642460

Figure Lengend Snippet: A) Phylogenetic tree of CD28 C-terminal proline-rich domain amino acid sequence in tetrapod mammals, generated from uniprot sequence database. B) CD28 A210P mice were generated by CRISPR-mediated substitution of proline for alanine at amino acid position 210 of mouse CD28. Gene and amino acid sequence of WT and CD28 A210P CD28. C-E : Thymus of adult CD28 A210P and WT mice assessed by flow cytometry for T cell development stage subsets (C), thymic FoxP3 + Tregs (D) and surface CD5 expression (E). F-H : Peripheral LN and spleen of adult WT and CD28 A210P mice were assessed by flow cytometry for proportions of T cell subsets (F), Foxp3 + Tregs (G) and surface expression of CD28 (H). I-M : Mice were injected with CD28 superagonist (CD28SA) and monitored for weight loss (J), on day 4 post-injection cytokines were assessed in serum (K), and spleen analyzed by flow cytometry for frequencies and absolute counts of Foxp3 + Tregs (L) and activated CD4 + and CD8 + T cells (M). Data are pooled from at least two experiments with 3-6 per group, except E&H are one experiment representative of two, dots show individual mice, significance assessed by Student’s t-test or one-way ANOVA, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Anti-CD28 (D665) monoclonal antibody was purchased from BioXcell.

Techniques: Sequencing, Generated, CRISPR, Flow Cytometry, Expressing, Injection

Journal: bioRxiv

Article Title: Humanizing a CD28 signaling domain affects CD8 activation, exhaustion and stem-like precursors

doi: 10.1101/2025.03.10.642460

Figure Lengend Snippet: A) Experimental timeline for LCMV Armstrong infection and splenocyte analysis 7 days post infection. B) UMAP clustering and marker heatmap overlay of CD4 + and CD8 + cells by flow cytometry. Data pooled from WT and CD28 A210P splenocytes to construct representative clustering. C) Absolute numbers of CD44 + CD8 + splenocytes. D) Representative gating and quantification of H-2D b GP33 + CD8 + T cells. E) Absolute numbers of effector CX3CR1 + and KLRG1 + CD8 + T cells. F) Representative gating and absolute numbers of cytokine producing CD8 + T cells following 4-hour ex vivo stimulation with LCMV peptide GP33. G) Weight loss following LCMV Armstrong infection shown as percentage of starting weight on day 0. H) Experimental timeline of LCMV clone 13 infection experiments. I) Absolute numbers of activated (CD44 + PD-1 + ) CD8 + T cells 8 days post infection with LCMV clone 13. J) Absolute numbers of IFNγ and IFNγ TNF producing CD8 + T cells following 4-hour ex vivo stimulation with LCMV peptide GP33. Data are pooled from at least two experiments with 4-8 per group. Significance assessed by Student’s t-test or one-way ANOVA, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Anti-CD28 (D665) monoclonal antibody was purchased from BioXcell.

Techniques: Infection, Marker, Flow Cytometry, Construct, Ex Vivo

Journal: bioRxiv

Article Title: Humanizing a CD28 signaling domain affects CD8 activation, exhaustion and stem-like precursors

doi: 10.1101/2025.03.10.642460

Figure Lengend Snippet: A) Experimental timeline of LCMV clone 13 infection. B) Absolute numbers of splenic PD-1 + Tim-3 + CD8 + T cells 8 days post LCMV clone 13 infection. C) PD-1 GMFI of PD-1 + Tim-3 + CD8 + cells 8 days post infection. D) Serum IFNγ 8 days post infection. E) Ratio of CD8 + T eff (CX3CR1 + ) to T ex (CX3CR1 - PD-1 + Tim-3 + ) splenocytes. F) Weight loss following LCMV infection, shown as a percentage of starting weight on day 0, data pooled from multiple experiments, (WT n= 9-28/time point) (CD28 A210P n= 8-25/time point) student’s t test used for each day. G) Representative gating and quantification of absolute numbers of CD8 + effectors (CX3CR1 + ) and exhausted cells (CX3CR1 - PD-1 + Tim-3 + ) on day 18 post infection. H) Ratio of absolute numbers of CD8 + CX3CR1 + effectors/ CD8 + CX3CR1 - PD-1 + Tim-3 + exhausted cells. I) Representative gating and quantification of absolute numbers of IFNγ + CD8 + T cells of indicated subsets based on previously described surface marker gating. Splenocytes were stimulated ex vivo for 4 hours with LCMV GP33 peptide in the presence of GolgiPlug followed by FACS staining. J) RT-qPCR quantification of ifng from LCMV clone 13 infected mouse kidney 18 days post infection. K) RT-qPCR quantification of viral copies in Kidney tissue from LCMV clone 13 infected mice 18 days post infection. Data are pooled from at least two experiments with 3-6 per group, except C and D one experiment representative of two and J is one experiment. Dots show individual mice. Significance assessed by Student’s t-test or one-way ANOVA, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Anti-CD28 (D665) monoclonal antibody was purchased from BioXcell.

Techniques: Infection, Marker, Ex Vivo, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Humanizing a CD28 signaling domain affects CD8 activation, exhaustion and stem-like precursors

doi: 10.1101/2025.03.10.642460

Figure Lengend Snippet: A-J) CD8 + T cells cells were stimulated with plate-bound anti-CD3 and anti-CD28 agonistic antibodies (1μg/mL in A,B E-J) (5μg/mL in C,D). A) Supernatant IL-2 was quantified by ELISA following 6 hours of stimulation. Each point represents the mean of replicate wells from an independent experiment. Data normalized to WT control in each independent experiment. B) Representative gating and quantification of PD-1 and Tim-3 on CD8 + cells stimulated for 2-3 days. Day 2: points indicate replicate wells pooled from 3 independent experiments. Day 3: points indicate replicate wells pooled from 2 independent experiments. C) Representative immunoblot of JunB from WT or CD28 A210P CD8 + T cell nuclear extracts following stimulation for indicated times. Representative of 5 independent experiments. D) Representative immunoblot of JunB from nuclear extracts from WT or CD28 A210P CD8 + T cells stimulated 2 hours in the presence of DMSO vehicle control or indicated concentrations of the MEK1/2 inhibitor, trametinib. Representative of 3 independent experiments. Quantification of vehicle treated extracts is pooled from 9 independent experiments and inhibitor treated extracts are pooled from 4 independent experiments. E) CD8 + cells were stimulated for 6 hours in the presence of vehicle or indicated concentrations of the MEK1/2 inhibitor. Supernatant IL-2 was quantified by ELISA. Data normalized to WT control in each independent experiment. Points indicate mean of pooled experimental replicates from each experiment. F) CD8 + cells were stimulated for 24 hours. Vehicle or MEK1/2 inhibitor were added to wells prior to cell seeding. Supernatant IFNγ was quantified by ELISA. Data normalized to WT control in each independent experiment. Points indicate mean of pooled experimental replicates from each experiment. G) Representative gating and quantification of PD-1 and Tim-3 on CD8 + cells stimulated for 24 hours with vehicle or MEK1/2 inhibitor added to wells prior to cell seeding. Data normalized to WT control in each independent experiment. Points indicate mean of pooled replicates from 2 independent experiments. H) CD8 + T cells were stimulated for 6 hours. Vehicle or indicated concentrations of the JNK1/2 inhibitor, JNK-IN-8, were added to wells prior to cell seeding. Supernatant IL-2 was quantified by ELISA. Data normalized to WT control in each independent experiment. Points indicate mean of pooled experimental replicates from 3 independent experiments. I) CD8 + cells were stimulated for 24 hours. Vehicle or JNK1/2 inhibitor were added to wells prior to cell seeding. Supernatant IFNγ quantified by ELISA. Data normalized to WT control in each independent experiment. Points indicate mean of pooled experimental replicates from 3 independent experiments. J) Representative gating and quantification of PD-1 and Tim-3 on CD8 + T cells stimulated 24 hours with vehicle or JNK1/2 inhibitor added prior to cell seeding. Data normalized to WT control in each independent experiment. Points indicate mean of pooled replicates from 2 independent experiments. Data are pooled or representative of at least two experiments with significance assessed by Student’s t-test or one-way ANOVA, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Anti-CD28 (D665) monoclonal antibody was purchased from BioXcell.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: bioRxiv

Article Title: Humanizing a CD28 signaling domain affects CD8 activation, exhaustion and stem-like precursors

doi: 10.1101/2025.03.10.642460

Figure Lengend Snippet: A) Experimental timeline of LCMV Armstrong or clone 13 infection with CD4-depletion. Anti-CD4 (GK1.5) was given to indicated mice I.P. (250 μg/mouse) on days –1 and +1. B) Representative gating and quantification of absolute numbers of CD8 + Tpex (PD-1 + SLAMF6 + Tim-3 - ) 18 days post infection. For assessment of IFNγ potential, splenocytes were stimulated with LCMV GP33 peptide ex vivo for 4 hours in the presence of GolgiPlug. C) Experimental timeline of LCMV clone 13 infection with CD4-depletion. Anti-CD4 (GK1.5) was given I.P. (250 μg/mouse) on days –1 and +1. For mice treated with anti-PD-L1 (10F.9G2), 5 injections of anti-PD-L1 were administered I.P. (200 μg/injection) every 3 days for 2 weeks prior to analysis. D) Representative histograms and GMFIs of CD28 on CD8 + Tpex (PD-1 + SLAMF6 + Tim-3 - ), effectors (CX3CR1 + ), and exhausted (CX3CR1 - PD-1 + Tim-3 + ) cells 35+ days post LCMV infection. E) Representative gating and quantification of absolute counts of splenic CD8 + Tpex (PD-1 + SLAMF6 + Tim-3 - ), effectors (CX3CR1 + ), and exhausted (CX3CR1 - PD-1 + Tim-3 + ) cells 35+ days post LCMV infection. To determine absolute counts of IFNγ-producing CD8 + cells, 4-hour ex vivo LCMV GP33 peptide restimulation in the presence of GolgiPlug was performed. F) Quantification of absolute counts of splenic CD8 + Tpex (PD-1 + SLAMF6 + Tim-3 - ) 8-, 18-, or 42-days post infection with LCMV clone 13 with or without anti-PD-L1 treatment for two weeks. G) Experimental timeline of LCMV Armstrong or clone 13 infection. Anti-CD4 (GK1.5) was given to indicated mice I.P. (250 μg/mouse) on days –1 and +1. H) Representative gating and absolute counts of splenic CD8 + Tpex (PD-1 + SLAMF6 + Tim-3 - ) 7-8 days post infection. I) Absolute counts of IFNγ-producing CD8 + Tpex (PD-1 + SLAMF6 + Tim-3 - ) following 4-hour ex vivo LCMV GP33 peptide restimulation in the presence of GolgiPlug. J) Representative histograms and GMFIs of PD-1 on Tpex following LCMV infection. K) Representative gating and quantification of absolute numbers of CD8 + SLECs (CD44 + KLRG1 + CD127 - ) and MPECs (CD44 + KLRG1 - CD127 + ). Data are pooled or representative of at least two experiments with 3-6 per group, dots show individual mice, significance assessed by Student’s t-test or one-way ANOVA, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Anti-CD28 (D665) monoclonal antibody was purchased from BioXcell.

Techniques: Infection, Ex Vivo, Injection

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: Figure 1 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 (130- 111- 973) and Anti- PE MicroBeads (130- 048- 801) were purchased from Miltenyi (Germany).

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: Figure 3 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 (130- 111- 973) and Anti- PE MicroBeads (130- 048- 801) were purchased from Miltenyi (Germany).

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: Figure 4 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 (130- 111- 973) and Anti- PE MicroBeads (130- 048- 801) were purchased from Miltenyi (Germany).

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