Multiparametric Analysis of Antigen-Specific T Cells

Peripheral mouse blood cells and the splenocytes (fresh or stimulated) were stained with indicated combinations of antibodies plus H-2D^b/AFP₂₁₂ or H-2K^b-D^b/AFP₄₉₉ tetramers. For tetramer staining after re-stimulation with peptide in the media, the stimulated cells need 12 hours rest to recover the TCR. DAPI was added to exclude dead cells. Intracellular staining of IFNγ was conducted as described (24 (link)) after the cells were re-stimulated with peptides in the presence of GolgiStop for 3.5 hours. Events were collected on the LSR II and the data was analyzed using FCS Express III software. The antibodies used in this study include antibodies to mouse CD8 (Clone: 53-6.7, Biolegend), CD45.1 (Clone: A20, Biolegend), CD45.2 (Clone: 104, BD Bioscience), CD44 (Clone: 1M7, Biolegend), CD62L (Clone: MEL-14, Biolegend), CD127 (IL-7Ra) (Clone: A7R34, Biolegend), PD-1 (Clone: 29F.1A12, Biolegend), CCR7 (Clone: 4B12, Biolegend), CD122 (IL-2Rβ) (Clone: TM-β1, Biolegend), Sca-1 (Clone: D7, Biolegend,), CD95 (Clone: SA367H8, Biolegend), IFN-γ (Clone: XMG1.2, Biolegend), Bcl-2 (Clone: BCL/10C4, Biolegend), H-2^K b/H-2D^b (Clone: 28-8-6, Biolegend), TCRβ (Clone: H57-597, BD Bioscience). The Annexin-V and DAPI (4,6-diamidino-2 phenylinole dilactate) were from Biolegend.

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Wu S., Zhu W., Peng Y., Wang L., Hong Y., Huang L., Dong D., Xie J., Merchen T., Kruse E., Guo Z.S., Bartlett D., Fu N, & He Y. (2017). The antitumor effects of vaccine-activated CD8+ T cells associate with weak TCR signaling and induction of stem-like memory T cells. Cancer immunology research, 5(10), 908-919.

Publication 2017

CD62L Sca-1 IFN-γ Bcl-2 H-2K b/H-2Db Annexin-V DAPI

Annexin v Antibodies Bcl 2 Cd122 Cd44 Cd62l Cells Clone Dapi Ifn γ Il 7ra Intracellular Mouse Peptide Peptides re Peripheral blood cells Sca 1 Tcrβ Tetramer

Corresponding Organization : Zhujiang Hospital

Other organizations : UPMC Hillman Cancer Center

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Variable analysis

independent variables

Peripheral mouse blood cells and the splenocytes (fresh or stimulated)

dependent variables

Expression of surface markers (CD8, CD45.1, CD45.2, CD44, CD62L, CD127 (IL-7Ra), PD-1, CCR7, CD122 (IL-2Rβ), Sca-1, CD95)
Expression of intracellular markers (IFN-γ, Bcl-2)
Binding of H-2Db/AFP212 or H-2Kb-Db/AFP499 tetramers

control variables

DAPI (to exclude dead cells)
Unstimulated cells (for comparison with stimulated cells)

positive controls

Cells re-stimulated with peptides in the presence of GolgiStop for 3.5 hours (for intracellular IFN-γ staining)

negative controls

Unstimulated cells (for comparison with stimulated cells)

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