mTOR regulates memory CD8 T-cell differentiation

Abstract

Memory CD8 T cells are a critical component of protective immunity, and inducing effective memory T-cell responses is a major goal of vaccines against chronic infections and tumours. Considerable effort has gone into designing vaccine regimens that will increase the magnitude of the memory response, but there has been minimal emphasis on developing strategies to improve the functional qualities of memory T cells. Here we show that mTOR (mammalian target of rapamycin, also known as FRAP1) is a major regulator of memory CD8 T-cell differentiation, and in contrast to what we expected, the immunosuppressive drug rapamycin has immunostimulatory effects on the generation of memory CD8 T cells. Treatment of mice with rapamycin following acute lymphocytic choriomeningitis virus infection enhanced not only the quantity but also the quality of virus-specific CD8 T cells. Similar effects were seen after immunization of mice with a vaccine based on non-replicating virus-like particles. In addition, rapamycin treatment also enhanced memory T-cell responses in non-human primates following vaccination with modified vaccinia virus Ankara. Rapamycin was effective during both the expansion and contraction phases of the T-cell response; during the expansion phase it increased the number of memory precursors, and during the contraction phase (effector to memory transition) it accelerated the memory T-cell differentiation program. Experiments using RNA interference to inhibit expression of mTOR, raptor (also known as 4932417H02Rik) or FKBP12 (also known as FKBP1A) in antigen-specific CD8 T cells showed that mTOR acts intrinsically through the mTORC1 (mTOR complex 1) pathway to regulate memory T-cell differentiation. Thus these studies identify a molecular pathway regulating memory formation and provide an effective strategy for improving the functional qualities of vaccine- or infection-induced memory T cells.

Figure 1. Rapamycin enhances the number and quality of virus specific memory CD8 T cells

a, Kinetics of endogenous GP33 epitope specific CD8 T cells in PBMCs of LCMV-infected B6 mice treated with rapamycin (from day -1 to 30 post-infection; shaded area) (No Rapa, n=3 mice; Rapa Tx, n=6). b, Phenotypic analysis of endogenous DbGP33 tetramer positive cells in the spleen at day 36 post infection. c, GP33 epitope specific P14 transgenic memory CD8 T cells (day 34 post infection) were generated in the presence or absence of rapamycin, CFSE labeled and then adoptively transferred into naïve mice to monitor their homeostatic proliferation. CFSE dilution of P14 cells at 30 days post transfer is shown and the number represents percentage of memory cells that divided more than two times. d, Memory P14 cells derived from rapamycin treated or untreated mice were adoptively transferred and mice were challenged with vaccinia virus expressing the GP33 epitope (VVGP33). Kinetics of P14 cells in PBMCs after challenge and the total P14 cell numbers in spleen on day 30 post-infection are shown (No Rapa, n=4; Rapa Tx, n=6). Error bars indicate SEM.

Figure 2. Rapamycin treatment during T cell expansion phase increases the number of memory precursors

a, Kinetics of endogenous GP33 epitope specific CD8 T cells in PBMCs of LCMV-infected B6 mice treated with rapamycin (from day -1 to 8 post-infection; shaded area) (n=3~6; each time point). b, The average number of DbGP33 tetramer positive cells on day 36 post-infection in spleens of LCMV infected mice treated with rapamycin (No Rapa, n=9; Rapa Tx day -1~8, n=3). c, CD127, KLRG-1, and Bcl-2 expression on endogenous DbGP33 tetramer positive cells in PBMCs at 8 days post LCMV infection in B6 mice. Rapamycin was administered from day -1 to day 8 post infection. d, Phenotypic analysis of DbGP33 tetramer positive cells in spleens of LCMV infected mice (rapamycin treatment from day -1 to 8 post infection). Error bars indicate SEM.

Figure 3. Rapamycin treatment during effector to memory transition phase accelerates memory differentiation

a, Kinetics of endogenous GP33 epitope specific CD8 T cells in PBMCs of LCMV-infected B6 mice treated with rapamycin (from day 8 to 36 post-infection; shaded area) (No Rapa, n=9 mice; Rapa Tx, n=9). b, Phenotypic changes in endogenous DbGP33 tetramer positive CD8 T cells in spleen on day 36 post LCMV infection (n=12; each group). B6 Mice were treated with rapamycin during the effector to memory T cell transition period (days 8~35 post infection). c, CD62L negative day 8 P14 transgenic effector CD8 T cells were purified, labeled with CFSE, and then adoptively transferred into naïve mice. Half of these mice were treated with rapamycin after transfer and (d) CD62L conversion in the antigen specific CD8 T cells was analyzed longitudinally in the blood. e, CFSE profile and CD62L expression on antigen specific memory CD8 T cells in the spleen at day 27 after transfer of CD62L negative effector T cells. f, CD62L negative day 8 P14 transgenic effector CD8 T cells were adoptively transferred into naïve mice. These mice were treated with rapamycin for 25 days, and were challenged with VVGP33 on day 28 post transfer. At 5 days after challenge, P14 expansion in spleen (g) and viral titers in ovary (h) were analyzed (n=4~6; each group). Flow data were gated on CD8 T cells. Error bars indicate SEM.

Figure 4. mTOR acts intrinsically in antigen specific CD8 T cells through the mTORC1 pathway to regulate memory T cell differentiation

Specific genes (mTOR or raptor) were knocked down using a retrovirus based RNAi system. Retrovirus transduced LCMV specific P14 transgenic CD8 T cells (marked by GFP expression) were adoptively transferred into naïve mice, followed by LCMV infection. Phenotypic analysis of retrovirus transduced cells (GFP⁺) and nontransduced (GFP^-) P14 cells in the PBMCs was performed on days 14~16 post infection. a, and b, Each line shows expression of the indicated phenotypic markers on transduced and nontransduced antigen specific CD8 T cells in individual animals. a, mTOR RNAi. b, raptor RNAi. Same control data are shown in panels (a) and (b). c, FKBP12 RNAi expressing retrovirus- or control retrovirus-transduced P14 transgenic CD8 T cells (marked by GFP expression) were adoptively transferred into naïve mice, followed by LCMV infection. Half of the mice were treated with rapamycin throughout infection. Phenotypic analysis of retrovirus transduced cells (GFP⁺) and nontransduced (GFP^-) P14 cells in the PBMCs was performed on days 14~16 post infection.