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. 2009 Jul 21;106(29):12055-60.
doi: 10.1073/pnas.0903919106. Epub 2009 Jun 30.

Infectious tolerance via the consumption of essential amino acids and mTOR signaling

Stephen P Cobbold  1 , Elizabeth Adams, Claire A Farquhar, Kathleen F Nolan, Duncan Howie, Kathy O Lui, Paul J Fairchild, Andrew L Mellor, David Ron, Herman Waldmann
Affiliations

Infectious tolerance via the consumption of essential amino acids and mTOR signaling

Stephen P Cobbold et al. Proc Natl Acad Sci U S A. .

Abstract

Infectious tolerance describes the process of CD4(+) regulatory T cells (Tregs) converting naïve T cells to become additional Tregs. We show that antigen-specific Tregs induce, within skin grafts and dendritic cells, the expression of enzymes that consume at least 5 different essential amino acids (EAAs). T cells fail to proliferate in response to antigen when any 1, or more, of these EAAs are limiting, which is associated with a reduced mammalian target of rapamycin (mTOR) signaling. Inhibition of the mTOR pathway by limiting EAAs, or by specific inhibitors, induces the Treg-specific transcription factor forkhead box P3, which depends on both T cell receptor activation and synergy with TGF-beta.

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Conflict of interest statement

Conflict of interest statement: S.P.C. and H.W. are shareholders in TolerRx Inc. and receive royalties for CAMPATH antibody sales. S.P.C. is a shareholder and adviser to BioAnaLab Ltd. A.L.M. has intellectual property interests in the therapeutic use of IDO and IDO inhibitors and receives consulting income from NewLink Genetics Inc.

Figures

Fig. 1.
Fig. 1.
The adoptive transfer of antigen specific, foxp3+ Tregs induces multiple amino acid enzyme depleting transcripts within skin grafts. (A) In vitro-generated Tregs (1 × 107 DBYT cells) were adoptively transferred to CBA.RAG1−/− recipient female mice 1 day before grafting with CBA.RAG1−/− male tail skin (n = 8; black bars). Control RAG1−/− recipients received similar grafts, but no DBYT cells (n = 4; white bars). After 6 days, grafts were harvested and analyzed for the expression of gene transcripts by low-density TaqMan RT-PCR array. Grafted samples were compared with normal tail skin from CBA.RAG1−/− (n = 4; gray bars). Data are shown as mean ± SD of log10(RQ) values, where n indicates the number of recipient skin graft samples tested in independent experiments. Transcripts significantly (P < 0.05) up-regulated either by syngeneic grafting alone or Tregs in male grafts are indicated. Note that FoxP3 detection within grafts showed a wide variation, because it was close to the limit of detection, but was negative in all mice not given DBYT cells. (B) Tregs (1 × 107 DBYT cells) were adoptively transferred to A1.RAG1−/− female recipient mice given a male CBA.RAG1−/− skin graft 1 day later. One group of grafted mice received only Tregs (white bars), and other groups also received 2 mg of either a neutralizing anti-TGF-β (gray bars) or an isotype-matched control (black bars) mAb. Another group received no Tregs as a control for ongoing graft rejection (to which all data were normalized: indicated by the horizontal line at RQ = 0). All grafts were harvested and analyzed 6 days later for the expression of gene transcripts by low-density TaqMan RT-PCR array. Data are shown as mean ± SD of log10(RQ) values for independent biological replicate samples from 6 individually grafted mice per group. Transcripts significantly (P < 0.05) up-regulated by Tregs in male grafts are indicated by *, and those significantly dependent on TGF-β are indicated by #.
Fig. 2.
Fig. 2.
Tregs induce amino acid-consuming enzymes in DCs as do CTLA4, IL-10, and TGF-β. (A–C) Immature bmDCs from female CBA/Ca mice were cultured either alone (white bars) or together (1:1 ratio) with male-specific TCR transgenic Tregs (A), similar, but conventionally activated, T cells (Tconv) (B), or Tr1D1 (a Tr1 cell clone) (C), both with (black bars) or without (striped bars) the cognate DBY antigen peptide, for 24 h at 37 °C. Samples were then analyzed for the expression of gene transcripts by low-density TaqMan RT-PCR array. * indicates gene transcripts significantly overexpressed only in the presence of T cells plus cognate peptide. (D) Splenic DCs (106/mL) from CBA/Ca (slashed and black bars) or CBA.IDO−/− (striped and gray bars) mice, as indicated, were cultured either with (slashed and striped bars) or without (black and gray bars) CTLA4-Ig (100 μg/mL) for 48 h, before analysis for the expression of gene transcripts by low-density TaqMan RT-PCR array. * indicates gene transcripts significantly overexpressed, in the presence of CTLA4-Ig, in either wild-type or IDO−/− T cells. (E) Gene transcripts were measured in immature DCs (white bars) or LPS-treated DCs that had been exposed to TGF-β (slashed bars), IL-10 (striped bars), or no additional cytokines (black bars) during their generation from bone marrow cultures, and analysis as above. * indicates gene transcripts up-regulated by TGF-β or IL-10 over and above that seen by LPS exposure alone. Data are shown as the mean ± SD log10(RQ) values of at least 3 independent biological replicates.
Fig. 3.
Fig. 3.
DCs can suppress T cell activation through specific depletion of many different amino acids in vitro. LPS-matured (gray bars) or TGF-β + LPS-treated (black bars) bmDCs were cultured overnight with LPS at 37 °C in either complete RPMI medium 1640 or medium with a limiting concentration of the amino acid of interest, with or without a specific enzyme inhibitor (Table S1), as indicated. Control samples of each medium were similarly incubated without any cells (white bars). After removal of DCs by centrifugation and 0.2-μ filtering, fresh medium lacking the amino acid of interest was added 1:1 to each sample. Medium completely lacking the amino acid of interest was used as a negative control for T cell proliferation. Purified A1.RAG1−/− CD4+ T cells were then stimulated by anti-CD3+CD28 beads in each sample of conditioned medium and proliferation measured at 48 h by 3H thymidine incorporation. The arrows indicate relief of suppression by the inhibitors (A–F) and any nonspecific toxicity of the inhibitor (F).
Fig. 4.
Fig. 4.
GCN2 is required for the survival of activated T cells during amino acid starvation, but not the control of proliferation. CD4+ T cells from C57BL/6 or C57BL/6.GCN2−/− mice, either naïve (A) or previously activated by plate bound anti-CD3 plus soluble CD28 for 48 h (B), were stimulated by anti-CD3+CD28 beads at 37 °C in the absence of individual EAAs. Cell samples were taken daily and analyzed by flow cytometry for the uptake or 7AAD and the fluorescent caspase product of FITC-VAD-FMK to indicate the proportion of dead (necrotic and apoptotic) cells. Plots show the geometric mean percentage of surviving cells (7AAD−Caspase−) for C57BL/6 wild type (square) compared with C57BL/6.GCN2−/− (triangles) T cells under conditions lacking individual branched-chain (P < 0.05; 2-way ANOVA), compared with nonbranched chain EAAs (P < 0.0001), respectively.
Fig. 5.
Fig. 5.
Inhibition of mTOR signaling during T cell activation, either by rapamycin or amino acid depletion, induces foxp3 in synergy with TGF-β. Naive CD4+ T cells from female A1.RAG1−/− mice were stimulated with syngeneic bmDC in the presence of 100 nM DBY peptide for 4 days at 37 °C under the conditions indicated. Cells were surface-stained for CD4 and after fixation and permeabilization for foxp3, p4E-BP1, and pS6 they were analyzed by flow cytometry. Dot plots are shown for foxp3 versus pS6 after gating on CD4+ cells within the live lymphocyte forward and side scatter gates. Percentages of foxp3+ for CD4+ T cells are indicated. pS6 staining positively correlated with p4E-BP1 staining.
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