Rapamycin increases the yield and effector function of human γδ T cells stimulated in vitro

Abstract

Clinical strategies to exploit Vγ2Vδ2 T cell responses for immunotherapy are confronted with short-term increases in cell levels or activity and the development of anergy that reduces the response to therapy with succeeding treatments. We are exploring strategies to increase the yield and durability of elicited Vγ2Vδ2 T cell responses. One approach focuses on the mammalian target of rapamycin (mTOR), which is important for regulating T cell metabolism and function. In Vγ2Vδ2 T cells, mTOR phosphorylates the S6K1 and eIF4EBP1 signaling intermediates after antigen stimulation. Rapamycin inhibited these phosphorylation events without impacting Akt or Erk activation, even though specific inhibition of Akt or Erk in turn reduced the activation of mTOR. The effects of rapamycin on the T cell receptor signaling pathway lead to increased proliferation of treated and antigen-exposed Vγ2Vδ2 cells. Rapamycin altered the phenotype of antigen-specific Vγ2Vδ2 cells by inducing a population shift from CD62L + CD69- to CD62L-CD69+, higher expression of CD25 or Bcl-2, lower levels of CCR5 and increased resistance to Fas-mediated cellular apoptosis. These changes were consistent with rapamycin promoting cell activation while decreasing the susceptibility to cell death that might occur by CCR5 or Fas signaling. Rapamycin treatment during antigen-stimulation of Vγ2Vδ2 T cells may be a strategy for overcoming current obstacles in tumor immunotherapy.

Fig. 1

mTOR is involved in TCR signaling pathway of Vδ2 T cells. a Freshly isolated PBMC contained 1–10% of Vδ2 T cells (3.12% in upper panel a); after 10–14 days of culture with IPP (15 μM) plus IL-2 (100 U/ml), the percentage of Vδ2 T cells was more than 90% (92.4% in lower panel a). b Vδ2 T cell lines (corresponding to lower panel a) were washed twice and cultured in fresh medium for 24 h before they were treated with rapamycin, U0126 (Erk inhibitor) or LY294002 (Akt inhibitor) for 1 h followed by the addition of IPP (15 μM) for 20 min. After stimulation, cells were collected for western blotting assay with specific antibodies. Data are representative of three independent experiments with cells from different donors

Fig. 2

Rapamycin alters the kinetics of IPP/IL2-induced Vδ2 T cell proliferation. PBMC were stimulated with IPP/IL2 and maintained in rapamycin at the doses indicated (between 0.05 and 5 nM). Cultures were maintained for 44 days with periodic addition of low-dose IL2. The proliferation kinetics of Vδ2 T cells was monitored by determining the frequency (a) and absolute cell numbers (b) of Vδ2 T cells every 3 days. Data are representative of three independent experiments with cells from different donors

Fig. 3

Vδ2 T cells expanded in the presence of rapamycin (Rapa-Vδ2 T cell) express higher levels of CD25 and Bcl-2, lower levels of CCR5 and resist apoptosis. PBMC were stimulated with IPP/IL2 and maintained in rapamycin at different doses (between 0.05 and 5 nM) as described in Fig. 2. On days 10 and 30, CD25 (a, e), CCR5 (b, f) or Bcl-2 (c, g) expression on Vδ2 T cells was measured by flow cytometry. Curves are color-coiled to indicate the amount of rapamycin used or to show the isotype control staining. The increase in mean fluorescence intensity (∆MFI) was calculated as: [MFI (specific mAb) – MFI (isotype control)]/MFI (isotype control). d, g The expanded Vδ2 T cells (day 30) were stimulated with anti-Fas antibody. After 4 h, cells were collected, stained with AnnexinV and evaluated by flow cytometry. The statistical significance compared with medium control was analyzed (e–h). *P < 0.05, **P < 0.005, ***P < 0.0001 (Student’s t test). Data are representative of three independent experiments (error bars, SD)

Fig. 4

Rapa-Vδ2 T cells have a higher proportion of CD62L⁻CD69⁺ cells and produce stronger TCR-dependent responses. a PBMC were stimulated with IPP/IL2 in the presence or absence of increasing doses of rapamycin (between 0.05 and 5 nM) as described in Fig. 2. On day 30, Vδ2 T cells were stained with antibodies for CD62L (marker of naïve cells) or CD69 (marker of activated cells) and analyzed by flow cytometry. b The expanded Vδ2 T cells (day 30) were stimulated with IPP. After 4 h, IFN-γ production and CD107a expression were detected by flow cytometry. Data are representative of three independent experiments with cells from different donors. c, d The cytotoxicity of expanded Vδ2 T cell (day 30) in the presence or absence of rapamycin (5 nM) against Daudi (c) or TU167 (d) was evaluated at different E:T ratios in triplicate. The statistical significance of specific lysis compared with medium control was analyzed (Student’s t test). *P < 0.05. Data are representative of two independent experiments

Fig. 5

Rapa-Vδ2 T cells express higher levels of APC molecules. PBMC were stimulated with IPP/IL2 and maintained in rapamycin at doses between 0.05 and 5 nM as described in Fig. 2. On days 10 and 30, MHC-II (a, d), CD80 (b, e) or CD86 (c, f) expression on Vδ2 T cells was measured by flow cytometry. Data are representative of three independent experiments with cells from different donors. The increase in mean fluorescence intensity (∆MFI) was calculated as: [MFI (specific mAb) – MFI (isotype control)]/MFI (isotype control). The statistical significance compared with medium control was analyzed (d–f). *P < 0.05, **P < 0.005, ***P < 0.0001 (Student’s t test). Data are representative of three independent experiments (error bars, SD)