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tel-00827710, version 1 - 29 May 2013 13. The starting number of cells is a critical point. It is required in order to calculate precursor frequency; and in most instances it is the determinant of the limit of sensitivity for the assay. For example, if you suppose your population to be around 10 -6 (meaning 1 cell into 10 6 CD8), you will need to start from at least 10 7 cells to maximize the possibility of achieving a well-defined multimer-positive population. Of note, during the enrichment procedure, cell loss is in the range of 10 – 30%. 14. Staining cells with two multimers sharing the same specificity but labeled in different colors permits a further decrease in the non-specific binders [21]. We and others strongly recommend to include dual labeling, especially if your aim is to detect ultra-rare populations of cells of variable avidity. Of course, if you want to pursue with enrichment with anti-PE microbeads, one of the two colors need to be PE. Otherwise, you can use any fluorochrome combination (up to 25) [21], as soon as you titrate both streptavidin and multimers before use, and be cautious with settings and compensations. 15. Concentration of multimers is another important parameter to consider. Optimal concentrations must be defined for each multimer by titrating on specific cell lines. In general, we recommend working at high concentrations, i.e. 10-20nM = 3-10µg/mL final concentration (NB: PE Tetramers ~ 500kDa; APC Tetramers ~ 350kDa). Of note, the receptor density of TCR on responding T cells is also critical. While not thoroughly validated, it may be of interest to evaluate exposure to the protein tyrosine kinase inhibitor dasatinib prior to staining and/or enrichment as a means of enhancing tetramer binding [31]. 16. For example, if you aim to stain and enrich T cells specific for CMV, Influenza A, and MART1 specific populations at the same time, you will put in the same tube, at the same incubation step, the following multimers: PE-CMV, PE-Cy7-CMV, PE-Flu, APC-Flu, PE-MART1, APC-Cy7-MART1 (see Note 22 and Figure 6A). 17. Temperature is a critical parameter in multimer staining. It is advisable to assess the effects of temperature (and time) for each individual system. In our assays, MHC I staining is performed at 4° 222
tel-00827710, version 1 - 29 May 2013 for 30 min. For staining at room temperature, be cautious with respect to internalization of multimers – which might interfere with the enrichment procedure. 18. Please note that the number of beads used here is not adjusted to the number of targeted cells but rather fixed at a high level, sufficient for enrichment of rare or common populations. 19. We recommend to systematically analyze the Depleted fraction, at least during assay optimization in order to evaluate cell loss during the procedure (usually 10-30% in our hands). . 20. All reagents should be titrated before use. 21. To ensure you can detect rare specific T-cell populations, it is important to start with sufficient number of cells (see Note 13), and acquire sufficient number of events [32]. 22. In the example provided above (see Note 16 and Figure 6), after gating on CD8 + PE-multimer- positive T cells (which will contain the four T cell specificities), CMV-specific T cells may be segregated based on PE-Cy7 positivity; Flu-specific cells will be stained with APC; and MART1- specific T cells will be APC-Cy7 labeled. With the increased number of available fluorescent channels (e.g., 18-parameter cytometer), one can theoretically combine up to 10 enriched specificities at the same time, the difficulty being the optimization of compensation settings. The quality of the enrichment procedure, and capacity of detecting rare events will in fact also depend on ones experience using the cytometer. 23. This calculation is based on the hypothesis that you recover all epitope-specific T cells while acquiring your Enriched sample and should therefore be considered as the lower limit of precursor frequency. However, this requires rigorous adherence to the protocol, and in particular the standardization of wash steps, and consistent acquisition of the Enriched sample. Mixing studies with known input numbers of monoclonal TCR transgenic cells in wild-type congenic mix, or T cell clones into HLA-mismatched PBMCs may be used to evaluate efficiency and establish in- house criteria [25,23]. Page 223 of 256
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tel-00827710, version 1 - 29 May 2013<br />
for 30 min. For staining at room temperature, be cautious with respect to internalization of<br />
multimers – which might interfere with the enrichment procedure.<br />
18. Please note that the number of beads used here is not adjusted to the number of targ<strong>et</strong>ed cells but<br />
rather fixed at a high level, sufficient for enrichment of rare or common populations.<br />
19. We recommend to systematically analyze the Depl<strong>et</strong>ed fraction, at least during assay optimization<br />
in or<strong>de</strong>r to evaluate cell loss during the procedure (usually 10-30% in our hands). .<br />
20. All reagents should be titrated before use.<br />
21. To ensure you can d<strong>et</strong>ect rare specific T-cell populations, it is important to start with sufficient<br />
number of cells (see Note 13), and acquire sufficient number of events [32].<br />
22. In the example provi<strong>de</strong>d above (see Note 16 and Figure 6), after gating on CD8 + PE-multimer-<br />
positive T cells (which will contain the four T cell specificities), CMV-specific T cells may be<br />
segregated based on PE-Cy7 positivity; Flu-specific cells will be stained with APC; and MART1-<br />
specific T cells will be APC-Cy7 labeled. With the increased number of available fluorescent<br />
channels (e.g., 18-param<strong>et</strong>er cytom<strong>et</strong>er), one can theor<strong>et</strong>ically combine up to 10 enriched<br />
specificities at the same time, the difficulty being the optimization of compensation s<strong>et</strong>tings. The<br />
quality of the enrichment procedure, and capacity of d<strong>et</strong>ecting rare events will in fact also <strong>de</strong>pend<br />
on ones experience using the cytom<strong>et</strong>er.<br />
23. This calculation is based on the hypothesis that you recover all epitope-specific T cells while<br />
acquiring your Enriched sample and should therefore be consi<strong>de</strong>red as the lower limit of precursor<br />
frequency. However, this requires rigorous adherence to the protocol, and in particular the<br />
standardization of wash steps, and consistent acquisition of the Enriched sample. Mixing studies<br />
with known input numbers of monoclonal TCR transgenic cells in wild-type congenic mix, or T<br />
cell clones into HLA-mismatched PBMCs may be used to evaluate efficiency and establish in-<br />
house criteria [25,23].<br />
Page 223 of 256