Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cells

Abstract

The T cell compartment is phenotypically and functionally heterogeneous; subsets of naive and memory cells have different functional properties, and also differ with respect to homeostatic potential and the ability to persist in vivo. Human stem cell memory T (T(SCM)) cells, which possess superior immune reconstitution and antitumor response capabilities, can be identified by polychromatic flow cytometry on the basis of the simultaneous expression of several naive markers together with the memory marker CD95. We describe here a protocol based on the minimum set of markers required for optimal identification of human and nonhuman primate (NHP) T(SCM) cells with commonly available flow cytometers. By using flow sorters, T(SCM) cells can thereby be isolated efficiently at high yield and purity. With the use of the 5.5-h isolation procedure, depending on the number of cells needed, the sorting procedure can last for 2-15 h. We also indicate multiple strategies for their efficient expansion in vitro at consistent numbers for functional characterization or adoptive transfer experiments.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

Figures

Figure 1|

Gating strategy for the identification of human and rhesus TSCM cells. (a,b) Human and NHP PBMCs were stained as indicated in Table 1. In this example, both panels included anti-CD4 conjugated to Qdot 585 in addition to anti-CD8 conjugated to Pacific Blue in order to allow the simultaneous identification of CD4+ and CD8+ T cells. These T cells were identified by first gating on singlets (FSC-H versus FSC-A), live CD3+ T cells (CD3 versus Dump/AQUA ) and lymphocytes (SSC versus FSC). Naive-like T cells were defined as CD45RO–CCR7+ CD62L+ in humans and as CD45RA+ CCR7+ CD28+ in rhesus macaques. In these gated populations, TSCM cells express CD95, whereas TN cells are CD95–. In NHP CD8+ T cells, CXCR3 is coexpressed with CD95 and thus helps identify CD8+ TSCM cells but not CD4 + TSCM cells, as not all CD95+ TSCM in naive-like CD4+ cells express CXCR3+ (black arrow). Red straight arrows indicate the sequential gating strategy. The curved red arrows indicate the gate to be copied on gated ‘naive-like’ cells. (c) CD95 FMO control in human T cells. Dashed bars indicate the threshold for positivity for CD95 expression, whereas the diagonal red bar indicates the TSCM gate. (d) Identification of human antigen–specific TSCM cells. Human PBMCs were stained as indicated in Table 1. CD8+ T cells were identified as in a. T cells specific for the influenza matrix protein58–66 (Flu GL9) were identified using an MHC class I tetramer (blue dots overlaid on total CD8+ T cells in gray). In this case, frozen PBMCs were used, and CD27 replaced CD62L for the identification of TSCM cells. Blue straight arrows indicate the sequential gating strategy. FSC, forward scatter; SSC, side scatter.

Figure 2|

Reproducibility and robustness of three versus seven marker-based TN cell definitions for TSCM identification. (a) Naive-like T cells were defined as CD45RO–CCR7+ CD45RA+ CD62L+ CD27+ CD11adim CD127 (seven markers) or as CD45RO–CCR7+ CD62L+ (three markers). TSCM cells were subsequently identified as CD95+. The proportion among CD8+ and CD4+ is shown (n = 11). (b) Interuser and interexperiment variability of the strategy for the identification of human TSCM cells. Data were analyzed by the same user to minimize subjectivity in the gating procedure, **P < 0.01; ***P < 0.001.

Figure 3|

Co-staining strategies to improve the identification and isolation of human TSCM cells. (a) Human CD4+ and CD8+ naive-like cells were identified as described previously. The expression of CCR7 (left), CD58 (center) or CD122 (right) versus CD95 in human CD4+ and CD8+ T cells is shown. The gate identifies TSCM cells as depicted in Figure 1b. Numbers indicate the percentage of cells identified by the gates. (b) Expression of CD95 by CD4+ and CD8+ TSCM cells identified by the increased expression of CD58 and CD122. T cell subsets are defined as in Figure 1. Memory T cells are defined as positive for CD45RO.

Figure 4|

Flow cytometric sorting of TSCM cells. (a,b) Representative post-sort purity analyses of human (a) and NHP (b) CD8+ TSCM cells. TSCM cells are identified as described in Figure 1. Numbers indicate the percentage of cells in each gate.

Figure 5|

Differential response of T cell subsets to different stimuli in vitro. Human TN, TSCM, TCM and TEM cells were sorted as in Figure 1, stained with CFSE and stimulated with beads coated with CD3/CD2/CD28-specific antibodies for 6 d, 25 ng ml−1 IL-7 for 14 d or 25 ng ml−1 IL-15 for 10 d.