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: PLOS ONE

Article Title: B cell phenotypes and maturation states in cows naturally infected with Mycobacterium avium subsp. Paratuberculosis

doi: 10.1371/journal.pone.0278313

Figure Lengend Snippet: Primary and secondary antibodies used to discriminate B cell subpopulations.

Article Snippet: CD28 , CC219-PE , , Bio-Rad , MCA5779PE , .

Techniques: Marker

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