Abstract
Human antigen-specific CD8+ T-cell clones are valuable tools for dissecting CD8+ T-cell responses against antigens derived from infectious agents, cancer and self antigens. Here we describe a protocol for isolating human antigen-specific CD8+ T cells. This protocol uses surface capture of IFNγ to identify antigen responsive cells that are then cloned by single-cell sorting. Here we use CD8+ T-cell responses to influenza matrix protein (MP) as an example, but this approach can be applied to any antigen specificity.
Keywords: Cloning, CD8, CTL, Human, T cell
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Cloning MP58-66 specific, IFNγ+ CD8+ T cells. The gating strategy for MP58-66 specific, IFNγ+ CD8+. A. Gating on lymphocytes based on forward (FSC) and side scatter (SSC); B. Exclusion of dead cells by propidium iodide staining, viable cells are PI negative; C. Exclusion of doublets by FSC-Height (FSC-H) and FSC-width (FSC-W) gating strategy; D. IFNγ+ CD8+ T cells when PBMC are cultured without peptide; E. The same gate with PBMC cultured with MP58-66 and re-stimulated with the same peptide. Figure 2. Screening of antigen specificity for growing CD8+ T-cell clones. HLA-A2+ EBV cells were pulsed with either MP58-66, or without peptide. A sample of each growing clone was washed and incubated with these APCs overnight. The next day the concentration of IFNγ in the supernatant is determined by ELISA. Data represents mean ± standard deviation of duplicate cultures for each clone. The clones that secreted IFNγ in response to MP58-66 were selected for further expansion. Figure 3. Re-screening of antigen specificity after expansion of CD8+ T-cell clones. Clones were incubated with HLA-A2+ EBV cells pulsed with, or without, MP58-66. After overnight incubation the IFNγ in the supernatant was measured by ELISA. The data represent the means ± standard deviation of triplicate samples for each clone.
Recipes
Acknowledgments
We thank Eleanora Tresoldi for technical assistance and blood donors for providing blood samples for research. SIM is supported by The Australian National Health and Medical Research Council (NHMRC, APP1061961, APP1087341) and a JDRF Career Development Award 5-CDA2014210-A-N. St Vincent’s Institute of Medical Research receives support from the Operational Infrastructure Support Scheme of the Government of Victoria. The authors have no conflict of interest to declare.
References
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