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Cd28 cd28.2

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CD28 (CD28.2) is a cell surface receptor expressed on T cells. It plays a critical role in T cell activation and costimulation.

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Lab products found in correlation

Cd279 pd1 clone eh12.2h7, by BioLegend (1 mentions) Cd3 clone sp34 2, by BD (1 mentions) Cd8 sk1, by BD (1 mentions) Cxcr5 mu5ubee, by Thermo Fisher Scientific (1 mentions) Cd95 dx2, by BD (1 mentions) Ki67 b56, by BD (1 mentions) Cd4 l200, by BD (1 mentions) Perforin pf 344, by Mabtech (1 mentions) Hla dr l243, by BioLegend (1 mentions) Tnfα mab11, by BD (1 mentions) Il 2 mq1 17h12, by BD (1 mentions) Pd 1 eh12.2h7, by BioLegend (1 mentions) Mopc 21, by BioLegend (1 mentions) Hyclone fetal bovine serum, by Thermo Fisher Scientific (1 mentions) Cd58 ts2 9, by BioLegend (1 mentions) Bw264 56, by Miltenyi Biotec (1 mentions) Cd3 okt3, by Thermo Fisher Scientific (1 mentions) Polyclonal goat anti mouse ig, by BD (1 mentions) Bw135 80, by Miltenyi Biotec (1 mentions) Perm buffer 3, by BD (1 mentions)

4 protocols using cd28 cd28.2

1

Multi-Marker Immune Profiling Protocol

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Antibodies used in this study are as follows: CD3 (clone SP-34–2; BD Biosciences), CXCR5 (MU5UBEE; eBioscience), GagCM9 tetramer (NIH tetramer core), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; clone EH12.2H7; BioLegend), CD8 (SK1; BD Bioscience), Ki67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), GrzB (GB11; BD Biosciences), perforin (Pf-344; Mabtech), FoxP3 (206D; BioLegend), HLA-DR (L243; BioLegend), IFNλ (B27; BD Biosciences), TNFα (MAb11; BD Biosciences), and IL-2 (MQ1- 17H12; BD Biosciences).
Rahman S.A., Yagnik B., Bally A.P., Morrow K.N., Wang S., Vanderford T.H., Freeman G.J., Ahmed R, & Amara R.R. (2021). PD-1 blockade and vaccination provide therapeutic benefit against SIV by inducing broad and functional CD8+ T cells in lymphoid tissue. Science immunology, 6(63), eabh3034.
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2

Detailed T-cell Activation Assay Protocol

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RPMI 1640 (31870074), Dynabeads Human T-Activator CD3/CD28 for T cell expansion and Activation kit (1132D) were purchased from Thermofisher Scientific. Hyclone Fetal bovine serum was obtained from Fisher Scientific. Poly-L-lysine solution 0.1% (w/v) in H2O was obtained from Sigma (25988-63-0). The following anti-human antibodies were purchased from Biolegend, CD62L (DREG-56), CCR7 (G043H7), CD3 (UCHT-1), CD4 (A161A1), CD8 (HIT8a), CD2 (RPA2.10), CD28 (CD28.2), PD-1 (EH12.2H7), CD11a (TS2/4), mouse IgG1 isotype control (MOPC-21), CD58 (TS2/9), mouse IgG1 isotype control (MG1-45; 401402), CD45 (HI30), CD127 (A019D5), HLA-A2 (BB7.2), CD45RO (UCHL1). The following anti-human antibodies were purchased from BD Bioscience, CD45RA (HI100), CD127 (HIL-7R-M21), CD58 (1C3), CD2 (CD2.1) clone was a gift of D. Olive (Aix Marseille University). The following anti-human antibodies were purchased from eBioscience, CD8 (OKT8) and CD28 (CD28.2). Flow cytometry mAbs were used between 1-5ug/ml depending on how many cells were staining (i.e. between 5x103- 1x106). The following antibodies were purchased from Cell Signaling, pPLCγ1(pY783), pLAT(pY171), pSFK (pY416) (D49G4) and used at dilutions recommended by the company. The following anti-mouse antibodies were purchased from Biolegend, CD4 (RM4- 5), CD3 (145-2C11), TCRβ (H57-97). TCRβ (H57-97) Fab was obtained from Bio X Cell.
Demetriou P., Abu-Shah E., Valvo S., McCuaig S., Mayya V., Kvalvaag A., Starkey T., Korobchevskaya K., Lee L.Y., Friedrich M., Mann E., Kutuzov M.A., Morotti M., Wietek N., Rada H., Yusuf S., Afrose J., Siokis A., Meyer-Hermann M., Ahmed A.A., Depoil D, & Dustin M.L. (2020). A dynamic CD2 rich compartment at the outer edge of the immunological synapse boosts and integrates signals. Nature immunology, 21(10), 1232-1243.
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3

Activation of NF-κB in PHA-stimulated T cells

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PHA blasts derived from patients’ PBMCs were IL-2 starved 16 h before the assay. 106 cells were distributed in a 96-well V-bottom plate and stained on ice during 10 min with anti-CD2 (RPA2.10; eBioscience), CD28 (CD28.2; eBioscience), or CD3 (OKT3; eBioscience) mAb as indicated (5 µg/ml each). Cells were washed twice with cold medium, and a polyclonal goat anti–mouse Ig (BD) was added to each well (5 µg/ml) to cross-link activating receptors. 40 ng/ml PMA was used in separate wells as a positive control. After 20-min incubation at 37°C, cells were washed once with cold 1× PBS and then stained for 10 min on ice using the Aqua Dead Cell Marker kit (Thermo Fisher Scientific). Cells were fixed for 10 min at 37°C using Fix Buffer I (BD) and stained for 30 min with mAb against CD3 (BW264/56; Miltenyi Biotec), CD4 (M-T321; Miltenyi Biotec), and CD8 (BW135/80; Miltenyi Biotec). After extracellular marker staining, cells were permeabilized for 20 min at room temperatures using Perm Buffer III (BD) and stained for 3 h at room temperature with an IgG2b isotypic control or anti–NF-κB p65-(pS259)-PE (BD). Cells were subsequently acquired on a FACS Gallios flow cytometer and analyzed with FlowJo software (v10; Tree Star).
Wang Y., Ma C.S., Ling Y., Bousfiha A., Camcioglu Y., Jacquot S., Payne K., Crestani E., Roncagalli R., Belkadi A., Kerner G., Lorenzo L., Deswarte C., Chrabieh M., Patin E., Vincent Q.B., Müller-Fleckenstein I., Fleckenstein B., Ailal F., Quintana-Murci L., Fraitag S., Alyanakian M.A., Leruez-Ville M., Picard C., Puel A., Bustamante J., Boisson-Dupuis S., Malissen M., Malissen B., Abel L., Hovnanian A., Notarangelo L.D., Jouanguy E., Tangye S.G., Béziat V, & Casanova J.L. (2016). Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations. The Journal of Experimental Medicine, 213(11), 2413-2435.
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4

Isolation and Activation of CD8+ T Cells

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PBMCs were isolated from peripheral blood by Ficoll (Cat# P8900, Solarbio, China). Briefly, peripheral blood and PBS buffer were diluted at a ratio of 1:1 and fully mixed. Then, an equal volume of lymphocyte separation solution was added and the mixture was centrifuged at 500 g for 20 min at 4°C. The white membrane cells were carefully extracted and washed with PBS. Subsequently, the cells were centrifuged at 300 g for 10 min at 4°C. CD8+ T cells from PBMC of aGVHD patients and non-aGVHD patients were sorted using the CD8+ T cell isolation kit (Cat# 130-096-495; Miltenyi Biotec, Germany). The purity of the isolated cells was confirmed to be over 95% by flow cytometry. After incubation with CD3 (clone OKT3; Cat# 16-0037-85) and CD28 (CD28.2, 0.5 μg/mL; Cat# 16-0289-85) (eBioscience, USA) for 48 h in vitro, the sorted CD8+ T cells were further incubated with Galectin-9 recombinant protein (Cat# LG9-H5244, 5 μg/mL; ACRO Biosystems, USA) or PBS at 37°C for 24 h. After that, both the cells and culture supernatants were collected. Cell apoptosis was analyzed with flow cytometry, while cytokine levels in the supernatants were measured with ELISA.
Pang N., Yu M., Xu J., Yuan H., Chen G., Wang D., Han C., Wang W., Ding J, & Jiang M. (2023). The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT. Brazilian Journal of Medical and Biological Research, 56, e12997.
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