Proliferating Helper T Cells Require Rictor/mTORC2 Complex to Integrate Signals from Limiting Environmental Amino Acids

Abstract

Antigen-stimulated T cells require elevated importation of essential and non-essential amino acids to generate large numbers of daughter cells necessary for effective immunity to pathogens. When amino acids are limiting, T cells arrest in the G₁ phase of the cell cycle, suggesting that they have specific sensing mechanisms to ensure sufficient amino acids are available for multiple rounds of daughter generation. We found that activation of mTORC1, which is regulated by amino acid amounts, was uncoupled from limiting amino acids in the G₁ phase of the cell cycle. Instead, we found that Rictor/mTORC2 has an essential role in T cell amino acid sensing. In the absence of Rictor, CD4⁺ T cells proliferate normally in limiting arginine or leucine. Our data suggest that Rictor/mTORC2 controls an amino acid-sensitive checkpoint that allows T cells to determine whether the microenvironment contains sufficient resources for daughter cell generation.

Keywords: T helper cells; amino acid; immunology; immunosuppression; mTOR complex (mTORC).

FIGURE 1.

Kinetic responses of T cells to amino acid limitation. A, design of the MLR co-culture platforms. The optimal APC:T cell ratio differs, as defined by CFSE labeling experiments designed to induce maximal T cell proliferation in a 72-h period. B and C, viable cell numbers of CD4⁺ or CD8⁺ T cells recovered at 24, 48, or 72 h using the optimized MLR ratios in A. D, cell size differences between CD4⁺ and CD8⁺ T cells following exposure to antigen in the MLR co-culture systems. The time point of the first division, defined by parallel CFSE dye dilution assays, is shown by the dotted line. % of Max, percentage of maximum. E, T cell proliferation is dependent on threshold amino acid concentrations. DO11.10 CD4⁺ T cells were used in an MLR assay where the medium was adjusted to contain 10, 1, or 0% of the normal concentration of arginine, leucine, or lysine in RPMI medium. FSC-A, forward scatter area. F, microarray analysis of canonical mRNAs induced by TCR and co-stimulation stimulation (48-h time point) in purified CD4⁺ T cells. Shown are T cells in medium containing 1% of the normal concentration of amino acids, in both the presence and absence of antigen. Data in B–E are representative of 3–5 experiments. All MLR experiments also contain a separate CFSE control experiment to measure proliferation. Data in F represent individual 3 experiments (each column). One of the samples cultured in 1% Arg was excluded for quality control reasons.

FIGURE 2.

mTORC1 regulation in T cells in low amino acid environments. A and B, time course of pS6, S6, or p-mTOR in gated CD4⁺ cells from an MLR assay. The dotted line indicates the time zone of the first division defined by the independently performed CFSE dye dilution assay (B). C, time course of p-4E-BP1 in gated CD4⁺ T cells as in A where cultures were in normal RPMI or RPMI with 1% arginine or 1% leucine. D, time course of pS6 in gated CD4⁺ T cells as in C. E, quantification of flow data from the experiment in panels C and D. Experiments were done 3 independent times for arginine starvation. Panel B is the internal CFSE control experiment for data in panel A. Data in panel E is a summary figure from a single representative experiment.

FIGURE 3.

Loss of Rictor in T cells bypasses the amino acid depletion signal that blocks entry into cell cycle. A, quantification of the limiting amount of arginine needed for cell cycle entry. CD4⁺ T cells were stimulated in the MLR assay where arginine was titrated from 10 to 0% of the normal RPMI concentration. CD4⁺ proliferation was assessed at 72 h. B, CD4⁺ T cells from control or T cell-specific Rictor-deficient mice on a DO11.10 background were used in an MLR assay where arginine was 4, 3, 2, or 1% of the normal RPMI concentration. CD4⁺ T cell proliferation was assessed at 72 h. C, summary data of 4 independent experiments comparing proliferation in 1% arginine between control cells (WT) and Rictor-deficient CD4⁺ T cells (KO). ***, p = 0.0005 two-tailed t test. D, as in B, but using limiting leucine. Data are representative of two independent experiments.