Journal: Open Medicine

Article Title: HLA-DRB5 promotes immune thrombocytopenia via activating CD8 + T cells

doi: 10.1515/med-2024-0955

Figure Lengend Snippet: Expressions of HLA-DRB5 and MHC-II in peripheral blood of ITP mice. (a) and (b) ITP murine models were constructed for 15 days. PLT count in peripheral blood was measured every 2 days from the first day, and HLA-DRB5 mRNA expression in peripheral blood was measured by qRT-PCR from the first day (Days 1, 2, 3, 5, 7, 9, 11, 13, 15). (c) and (d) After the ITP modeling for 15 days, analysis of HLA-DRB5 mRNA expression in peripheral blood was performed by qRT-PCR. (e)–(g) Western blot was used to measure HLA-DRB5 protein expression in peripheral blood of ITP murine models. Relative expression was normalized to GAPDH. Data from all triplicate experiments were expressed as mean ± standard deviation. Comparison between two groups was carried out using independent-samples t -test. ** p < 0.01, *** p < 0.001, vs Control. Abbreviation: ITP, immune thrombocytopenia; HLA-DRB5, human leukocyte antigen class II heterodimer β5; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction.

Article Snippet: Then, primary antibodies against HLA-DRB5 (21702-1-AP, 30 kDa, Proteintech, Chicago, IL, China), MHC-II (ab139365, 29 kDa; Abcam, Cambridge, UK) and GAPDH (ab9485, 36 kDam; Abcam, UK) were used to incubate the membranes at 4°C overnight.

Techniques: Construct, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Comparison, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Open Medicine

Article Title: HLA-DRB5 promotes immune thrombocytopenia via activating CD8 + T cells

doi: 10.1515/med-2024-0955

Figure Lengend Snippet: Effects of HLA-DRB5 knockdown on PLT count and expressions of MHC-II, CD80 and CD86 in peripheral blood of ITP mice. (a) ITP murine models were constructed for 15 days, during which sh-HLA-DRB5 adenovirus or negative control was injected into mice by tail vein once a week for 2 weeks. After the ITP modeling for 15 days, analysis of HLA-DRB5 mRNA expression in peripheral blood was performed by qRT-PCR. (b) and (c) Western blot was used to measure HLA-DRB5 protein expression in peripheral blood of ITP murine models. Relative expression was normalized to GAPDH. (d) PLT count in peripheral blood was measured every 2 days from the first day. (e)–(g) QRT-PCR was utilized to detect MHC-II, CD80 and CD86 mRNA expressions in peripheral blood of ITP murine models. Relative expression was normalized to GAPDH. Data from all triplicate experiments were expressed as mean ± standard deviation. Comparison among multiple groups was carried out using one-way analysis of variance. * p < 0.05, *** p < 0.001, vs Control; +++ p < 0.001, vs ITP + vector. Abbreviation: sh-HLA-DRB5, short hairpin RNA targeting HLA-DRB5.

Article Snippet: Then, primary antibodies against HLA-DRB5 (21702-1-AP, 30 kDa, Proteintech, Chicago, IL, China), MHC-II (ab139365, 29 kDa; Abcam, Cambridge, UK) and GAPDH (ab9485, 36 kDam; Abcam, UK) were used to incubate the membranes at 4°C overnight.

Techniques: Knockdown, Construct, Negative Control, Injection, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Comparison, Control, Plasmid Preparation, shRNA

Journal: Open Medicine

Article Title: HLA-DRB5 promotes immune thrombocytopenia via activating CD8 + T cells

doi: 10.1515/med-2024-0955

Figure Lengend Snippet: Effects of HLA-DRB5 knockdown on the population of PBMCs with MHC-II + , CD80 + , CD86 + and CD8 + in ITP mice. (a)–(d) ITP murine models were constructed for 15 days, during which sh-HLA-DRB5 adenovirus or negative control was injected into mice by tail vein once a week for 2 weeks. Fluorescent analysis of MHC-II, CD80 and CD86 in PBMCs of ITP mice was performed using fluorescence-labeled antibodies in combination with fluorescent microscopy (magnification: ×200, scale bar: 100 μm). (e)–(g) Flow cytometry combined with fluorescence-labeled CD8 and CD4 antibodies was performed to detect the percentage of CD8 + T cells and CD4 + T cells in PBMCs of ITP mice. Data from all triplicate experiments were expressed as mean ± standard deviation. Comparison among multiple groups was carried out using one-way analysis of variance. * p < 0.05, *** p < 0.001, vs Control; + p < 0.05, +++ p < 0.001, vs ITP + vector. Abbreviation: MHC-II, major histocompatibility complex II; PBMCs, peripheral blood mononuclear cells.

Article Snippet: Then, primary antibodies against HLA-DRB5 (21702-1-AP, 30 kDa, Proteintech, Chicago, IL, China), MHC-II (ab139365, 29 kDa; Abcam, Cambridge, UK) and GAPDH (ab9485, 36 kDam; Abcam, UK) were used to incubate the membranes at 4°C overnight.

Techniques: Knockdown, Construct, Negative Control, Injection, Fluorescence, Labeling, Microscopy, Flow Cytometry, Standard Deviation, Comparison, Control, Plasmid Preparation, Immunopeptidomics