cd28  (R&D Systems)

Journal: bioRxiv

Article Title: Identification of the cellular transcription factor KLF16 as a novel repressive epigenetic repressor of HIV-1 transcription

doi: 10.64898/2026.05.02.722432

Figure Lengend Snippet: (a) Experimental flowchart. Memory CD4 + T cells were isolated from PBMCs of PWOH were stimulated with α-CD3+α-CD28 antibodies for 3 days and then were nucleofected with non-targeting control (siNT) or KLF16-targeting (siKLF16) small interfering RNA (siRNA). Nucleofected cells were cultured in the presence of rhIL-2 (5 ng/ml) for 24 hours, then infected with VSV-G-pseudotyped HIV-1 encoding GFP (50 ng HIV-p24/10e6 cells) and cultured in the presence of rhIL-2 for an additional 3 days. (b) KLF16 mRNA expression was measured by RT-qPCR 24 hours post-nucleofection. (c) At day 3 post-infection, cells were harvested to quantify the levels of integrated HIV DNA by nested real-time PCR. (d) Levels of HIV-p24 capsid protein were measured by ELISA in cell culture supernatants collected at day 3 post-infection. Results were generated with cells from n=7 PWOH participants. (e)- (g) Memory CD4 + T cells isolated from PBMCs of PWOH (n = 9) or of ART-treated PWH (n = 9) were stimulated with α-CD3+α-CD28 antibodies or left unstimulated for 18 hours and used for the measurement of KLF16 mRNA expression. (e) KLF16 mRNA levels quantified by RT-qPCR in samples from PWOH and PWH ex vivo . (f) KLF16 mRNA levels quantified by RT-qPCR in samples from PWH upon TCR triggering in vitro . (g) KLF16 mRNA levels quantified by RT-qPCR in samples from PWOH upon TCR triggering in vitro . Statistical analysis methods and corresponding p values are indicated above each graph.

Article Snippet: Following isolation, CD4+ T cells were stimulated with anti-CD3 and anti-CD28 antibodies (T Cell TransActTM, Miltenyi Biotec, #130-111-160) for three days and then infected by spinoculation (2h, 800xg, 32°C) with VSV-G-pseudotyped HIVGKO particles at a multiplicity of infection (MOI) of 3000, where GFP and mKO2 expression were used to distinguish between productively-infected and latently-infected cells.

Techniques: Isolation, Control, Small Interfering RNA, Cell Culture, Infection, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Generated, Ex Vivo, In Vitro

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A ) Circos plots showing the percentage of cells from HC (Healthy control, n = 6), ICI ( n = 2), RAC (RA control, n = 5), or irAE ( n = 5). ( B ) Expression of CD45RA and CCR7 on CD8 + T cells. Right: Summaries of the percentage of cells from HC ( n = 53), irAE ( n = 29), RAC ( n = 41), and ICI ( n = 26). ( C ) Expression of CXCR3 and CCR6 on CD8 + T cells. Right: Summaries of T cell subsets. HC ( n = 45), irAE ( n = 27), RAC ( n = 31), and ICI ( n = 26). ( D ) The cytotoxic score was evaluated using the gene list identified previously . ( E ) Specific genes were evaluated on CD8 + T cells. ( F ) Pathways that were significantly enriched in the CD8 + T cells between irAE and ICI. NF-κB, nuclear factor κB; STAT5, signal transducer and activator of transcription 5; DN, down. Gene set enrichment analysis (GSEA) plots of the allograft rejection ( G ), oxidative phosphorylation ( H ), IFN-α response ( I ), and IFN-γ response ( J ) between irAE and ICI. NES, normalized enrichment score ( K to M ) PBMCs were stimulated with plate-coated anti-CD3 and anti-CD28 (10 μg/ml) for 5 days. Mean fluorescence intensities (MFIs) of MitoTracker Green (MTG) (K), MitoTracker deep red (MTDR) (L) [HC ( n = 31), irAE ( n = 18), RAC ( n = 32), and ICI ( n = 16)] or Cy5-linked-1-amino-glucose (GluCy5) (M) [HC ( n = 35), irAE ( n = 20), RAC ( n = 36), and ICI ( n = 18)] in CD8 + T cells were presented. Expression was normalized to the HC in each experiment. ( N ) UMAP shows the presence or absence of T cell receptor (TCR) in the major immune cells across all the samples. ( O ) Pie charts showing the distribution of the top 100 TCR clones across different T cell subsets. Data in graphs represent mean ± SEM. Significance was tested by one-way analysis of variance (ANOVA). [(A) to (E) and (G) to (O)] ICI, ICI control.

Article Snippet: A total of 0.5 million isolated CD4 + T cells or CD8 + T cells was stimulated with plate-coated anti-CD3 (10 μg/ml; BioXCell, catalog no. BE0001-2) and anti-CD28 (BioXCell, catalog no. BE0291), rhIL-6 (100 ng/ml), rhIFN-α 2 (100 ng/ml), rhIL-12 (100 ng/ml), or the combination of rhIL-6, rhIFN-α 2 , and rhIL-12 for 5 days.

Techniques: Control, Expressing, Phospho-proteomics, Fluorescence, Clone Assay

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A to G ) PBMCs from the patients with irAE were cultured in the plate coated with anti-CD3 and anti-CD28 (10 μg/ml) in the presence of IgG1 isotype control or anti–human IL-6R (50 μg/ml), anti–human IL-12p40 (50 μg/ml), and anti–human IFNAR1 (50 μg/ml) for 3 days; n = 9. (A) Expression of CD38 and CD127 on CD8 + T cells. Right: Percentage of CD38 + CD127 − CD8 + T cells. (B and C) CD8 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (B) Expression of granzyme B and IFN-γ. Right: Percentage of granzyme B + IFN-γ + CD8 + T cells. (C) Expression of perforin and IFN-γ. Right: Percentage of perforin + IFN-γ + CD8 + T cells. [(D) and (E)] MFIs of GluCy5 (D), TMRM, and MTDR (E) in CD8 + T cells were presented. [(F) and (G)] CD4 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (F) Expression of IL-21 and IL-2. Right: Percentage of IL-21 + IL-2 + CD4 + T cells. (G) Expression of CD38 and CXCR5 on CD4 + T cells. Right: Percentage of CD38 + CXCR5 − CD4 + T cells. Data in graphs represent mean ± SEM. Significance was tested paired Student’s t test [(A) to (G)].

Article Snippet: A total of 0.5 million isolated CD4 + T cells or CD8 + T cells was stimulated with plate-coated anti-CD3 (10 μg/ml; BioXCell, catalog no. BE0001-2) and anti-CD28 (BioXCell, catalog no. BE0291), rhIL-6 (100 ng/ml), rhIFN-α 2 (100 ng/ml), rhIL-12 (100 ng/ml), or the combination of rhIL-6, rhIFN-α 2 , and rhIL-12 for 5 days.

Techniques: Cell Culture, Control, Expressing