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

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.

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.

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.

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.

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.

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.