Induction of memory-like CD8+ T cells and CD4+ T cells from human naive T cells in culture

Abstract

Memory T cells are crucial players in vertebrate adaptive immunity but their development is incompletely understood. Here, we describe a method to produce human memory-like T cells from naive human T cells in culture. Using commercially available human T-cell differentiation kits, both purified naive CD8+ T cells and purified naive CD4+ T cells were activated via T-cell receptor signaling and appropriate cytokines for several days in culture. All the T-cell activators were then removed from the medium and the cultures were continued in hypoxic condition (1% O2 atmosphere) for several more days; during this period, most of the cells died, but some survived in a quiescent state for a month. The survivors had small round cell bodies, expressed differentiation markers characteristic of memory T cells and restarted proliferation when the T-cell activators were added back. We could also induce memory-like T cells from naive human T cells without hypoxia, if we froze the activated T cells or prepared the naive T cells from chilled filter buffy coats.

Keywords: ex vivo differentiation; CD4; CD8; memory T cell; naive T cell.

Graphical Abstract

Fig. 1.

Activated CD8⁺ T cells in either normal or hypoxic culture. Naive CD8⁺ T cells derived from a healthy donor were cultured in human T-activator CD3/CD28 and IL-2 containing medium for 8 days. (A) in 20% O₂ condition and (B) in 1% O₂ condition. The 7-AAD-negative cells in the area gated as P1 in the FSC/SSC panel were considered as living cells. The expression pattern of CD45RA, CD62L, and CD127 of living cells were analyzed by FACS. The numbers on the FACS-plot panel mean the frequencies (%) of population of cells. We repeated this experiment four times.

Fig. 2.

The scheme of ‘the Death Assay’. Naive T cells activated for 5–15 days were transferred into activator-free medium and cultured for 5–10 days more in ether 20% or 1% O₂ culture condition. Starvation of activating signals induces the cell death of activated T cell. The survivors after this procedure might be the memory T cells.

Fig. 3.

Activated CD8⁺ T cells survived in 8 days of the Death Assay in hypoxia. Naive CD8⁺ T cells activated for 8 days were transferred into activator-free medium and cultured for 8 days more in 1% O₂ condition. (A) cells preactivated in 20% O₂ condition and (B) cells preactivated in 1% O₂ condition. The 7-AAD-negative cells in the area gated as P1 in the FSC/SSC panel were considered as living cells. T_SCM; Stem cell memory T cell, T_CM; Central memory T cell, T_EM; Effector memory T cell. Arrows showed the expected positions of each memory T cell. The numbers on the FACS-plot panel mean the frequencies (%) of population of cells. We repeated this experiment three times.

Fig. 4.

CD8⁺ T cells in the Death Assay in hypoxia were quiescent. Naive CD8⁺ T cells were cultured in activating medium in 20% O₂ condition for 8 days. Cells were stained with CFSE and divided into two groups. One group was transferred into activator-free medium and the Death Assay was carried out in 1% O₂ condition. The other group continued activation culture in 1% O₂ condition as a control. FACS analysis was carried out on day 1 and day 6, and the number of cell division was estimated by the counts of CFSE stain. The scale on the vertical axis is an integer and the scale on the horizontal axis is logarithmic. The experiment was carried out one time.

Fig. 5.

Long-term living CD8⁺ T cells in the Death Assay in hypoxia could be reactivated. Naive CD8⁺ T cells were cultured in activating medium in 20% O₂ condition for 7 days. Then cells were transferred into activator-free medium and the Death Assay was carried out in 1% O₂ condition. On day 27 in the Death Assay in hypoxia, living cells were FACS-sorted. Cells were divided into two groups. Each 9.0 × 10³ cells were suspended with 0.2 ml of medium and seeded in a 96-well plate. One group continued the Death Assay in hypoxia for 6 days more. And the others were cultured in the activation medium for 6 days. Reactivation cultures were carried out in 1% O₂ condition, too. The phase-contrast microscopic images were shown in (A) the Death Assay in hypoxia on day 33, in (B) the reactivation culture on day 6. Black bars mean 100 μm. The experiment was carried out one time.

Fig. 6.

CD4⁺ Th1 cells survived in the Death Assay in hypoxia. Naive CD4⁺ T cells prepared from healthy donors were activated in CD4⁺ Th1 differentiation medium. (A) Cells activated in CD4⁺ Th1 differentiation medium for 8 days in 1% O₂ condition. (B) After the 8 days of activation culture, CD4⁺ Th1 cells were transferred into the activator-free medium and the Death Assay was carried out for 7 days in 1% O₂ condition. FACS analysis was carried out according to the method for CD8⁺ T cells. Arrows showed the expected positions of each memory T cell. The 7-AAD-negative cells in the area gated as P1 in the FSC/SSC panel were considered as living cells. 93.9% of the survived CD4⁺ Th1 was CD127 positive. The survivors were, T_CM; 73.5%, T_EM; 10.5%, T_SCM; 15.4%. The numbers on the FACS-plot panel mean the frequencies (%) of population of cells.

Fig. 7.

CD4⁺ Th2 cells survived in the Death Assay in hypoxia. Naive CD4⁺ T cells prepared from healthy donors were activated in CD4⁺ Th2 differentiation medium. (A) Cells activated in CD4⁺ Th2 differentiation medium for 10 days in 1% O₂ condition. (B) After the 10 days of activation culture, CD4⁺ Th2 cells were transferred into the activator-free medium and the Death Assay was carried out for 7 days in 1% O₂ condition. FACS analysis was carried out according to the method for CD8⁺ T cells. The 7-AAD-negative cells in the area gated as P1 in the FSC/SSC panel were considered as living cells. 96.6% of the survived CD4⁺ Th2 was CD127 positive. The survivors were, T_CM; 93.9%, T_EM; 1.4%, T_SCM; 4.6%. The numbers on the FACS-plot panel mean the frequencies (%) of population of cells. We repeated this experiment twice.

Fig. 8.

Frozen activated-T cells could survive in the Death Assay in 20% O₂. (A) Naive CD8⁺ T cells prepared from healthy donors were activated for 8 days in 20% O₂ condition. The activated cells were stored at −80°C. Cells were defrosted and assessed in the Death Assay in 20% O₂ condition for 7 days. (B) Naive CD4⁺ T cells prepared from healthy donors were activated for 10 days in Th2 differentiation medium in 20% O₂ condition. The activated cells were stored at −80°C Cells were defrosted and assessed in the Death Assay in 20% O₂ condition for 7 days. The 7-AAD-negative cells in the area gated as P1 in the FSC/SSC panel were considered as living cells. The numbers on the FACS-plot panel mean the frequencies (%) of population of cells. We repeated the both experiments three times.

Fig. 9.

Naive T cells prepared from chilled FBCs endured the Death Assay in 20% O₂. Naive CD8⁺ T cells and naive CD4⁺ T cells were prepared from the chilled FBCs. (A) Naive CD8⁺ T cells were activated for 5 days in 20% O₂ condition. The activated cells assessed in the Death Assay in 20% O₂ condition for 8 days. (B) Naive CD4⁺ T cells were activated for 5 days in Th2 differentiation medium in 20% O₂ condition. The activated cells were assessed in the Death Assay in 20% O₂ condition for 8 days. The 7-AAD-negative cells in the area gated as P1 in the FSC/SSC panel were considered as living cells. The numbers on the FACS-plot panel mean the frequencies (%) of population of cells. We repeated this experiment twice.