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. 2002 Dec;107(4):452-60.
doi: 10.1046/j.1365-2567.2002.01526.x.

Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division

Geon Kook Lee  1 , Hyeon Jin Park, Megan Macleod, Phillip Chandler, David H Munn, Andrew L Mellor
Affiliations

Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division

Geon Kook Lee et al. Immunology. 2002 Dec.

Abstract

Cells expressing indoleamine 2,3-dioxygenase (IDO), an enzyme which catabolizes tryptophan, prevent T-cell proliferation in vitro, suppress maternal antifetal immunity during pregnancy and inhibit T-cell-mediated responses to tumour-associated antigens. To examine the mechanistic basis of these phenomena we activated naïve murine T cells in chemically defined tryptophan-free media. Under these conditions T cells expressed CD25 and CD69 and progressed through the first 12 hr of G0/G1 phase but did not express CD71, cyclin D3, cdk4, begin DNA synthesis, or differentiate into cytotoxic effector cells. In addition, activated T cells with their growth arrested by tryptophan deprivation exhibited enhanced tendencies to die via apoptosis when exposed to anti-Fas antibodies. Apoptosis was inhibited by caspase inhibitor and was not observed when T cells originated from Fas-deficient mice. These findings suggest that T cells activated in the absence of free tryptophan entered the cell cycle but cell cycle progression ceased in mid-G1 phase and T cells became susceptible to death via apoptosis, in part though Fas-mediated signalling. Thus, mature antigen-presenting cells expressing IDO and Fas-ligand may induce antigen-specific T-cell tolerance by blocking T-cell cycle progression and by rapid induction of T-cell activation induced cell death in local tissue microenvironments.

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Figures

Figure 1
Figure 1
T-cell proliferation in tryptophan-deficient medium. (a) Splenocytes from BM3, A1 and DES mice were co-cultured with irradiated splenocytes from CBK (BM3, DES) or CBA male (A1) mice in RPMI (Trp+) and tryptophan-deficient (Trp−) medium. Proliferation was assessed by thymidine incorporation after 72 hr as described in the Materials and methods. (b) Thymidine incorporation by activated BM3 T cells over time. (c) Effect of tryptophan (•) concentration or combined isoleucine/leucine (▪) concentrations on thymidine incorporation by activated A1 T cells 48 hr after stimulation. Assays shown are representative of at least two separate experiments. Outcomes were assayed in triplicate and standard deviations were <5% from the mean values shown.
Figure 2
Figure 2
Characterization of activated T cells. BM3 T cells were cultured with CBK splenocytes (a) or activated with anti-CD3 monoclonal antibody (b) in tryptophan-free (heavy lines) and tryptophan-sufficient, RPMI-1640 (fine lines) media for 24 and 48 hr. (a) Cells were stained with anti-CD8, anti-(TCR) clonotypic antibody (Ti-98), and antibodies against CD25, CD69 and CD71 and analysed by flow cytometer. Histograms show the expression of the activation markers on gated CD8+ Ti-98+ BM3 T cells. Markers indicate staining intensity of non-activated control BM3 cells. (b) BM3 cells were cultured for 24 (lanes 1, 2) or 48 (lanes 3, 4) hours in tryptophan-sufficient (lanes 1, 3) or tryptophan-free (lanes 2, 4) medium. Lanes 5 and 6 are positive controls for cdk4 (LS.174 cells) and cyclin D3 (Jurkatt cells), respectively. Cell lysates were fractionated by gel electrophoresis and then immunoblotted with anti-cyclin D3 (upper panel) and anti-cdk4 antibodies (centre panel) as described in the Materials and methods. Anti-cdk4 immunoprecipates were also immunoblotted with anticyclin D3 antibody (lower panel). Results shown are representative of two independent experiments.
Figure 3
Figure 3
Analysis of T-cell cycle arrest point in tryptophan-free media. BM3 T cells were activated by anti-CD3 and anti-CD28 antibodies in tryptophan-free medium and tryptophan was added to cultures at the times indicated by the arrowheads. Thymidine incorporation was assessed in triplicate at the time-points shown on the graph (plotted as the start of the 4-hr pulse period with [3H]thymidine). Top panel indicates G0/G1 phase (white bar), S phase (black bar) and G1/S transition period (grey bar) for BM3 T cells cultured in RPMI-1640. Results are representative of three separate experiments. Standard deviations from the mean values shown were <10%.
Figure 4
Figure 4
Tryptophan is required for effector cell differentiation. (a) Chromium release assays conducted in triplicate for antigen-activated BM3 T cells cultured in tryptophan-deficient and RPMI-1640 media after 48 hr. T cells were incubated with 51Cr-labelled EL-4 target cells at different effector : target cell ratios for 6 hr when target cell lysis was determined by standard procedures. Standard deviations were <10% from mean values shown. (b)Flow cytometric analyses of activated BM3 T cells stained with 1B11 antibody after culture in tryptophan-free (heavy line) and RPMI-1640 (fine line) media. Dotted line indicates staining profile of non-activated BM3 T cells. Results are representative of four separate experiments.
Figure 5
Figure 5
Tryptophan deprivation induces sensitivity to Fas (CD95)-mediated cell death by apoptosis. (a) BM3 T cells activated by anti-CD3 and anti-CD28 antibodies in the presence or absence of immobilized anti-CD95 (anti-Fas) antibodies were cultured in tryptophan-free (Trp−) and RPMI-1640 (Trp+) media, stained with Annexin V and analysed by flow cytometry (heavy lines). Fine lines indicate unstained control cells. (b) Mean fluorescent intensities over time for T cells from BM3 (▪, □) or CBA (•, ○) mice activated in tryptophan-sufficient (▪, •) or tryptophan-free (□, ○) and stained with anti-Fas (CD95) antibody. Data are representative of two experiments (c) Annexin V staining expressed as mean percentage of Annexin V+ cells in each treatment group after subtracting background staining detected in control (non-activated) cultures (5–10%). Data acquired after longer culture periods (65 hr) or after culturing splenocytes from B6-lpr (Fas-deficient) mice for 18 hr were analysed in the same way. Data were compiled from six separate experiments, including the results shown in (a). Statistically significant outcomes (Wilcoxon rank-sum test) were obtained when T cells were activated in tryptophan-free versus tryptophan-sufficient media with all three antibodies present and when T cells were activated in tryptophan-free media plus anti-CD3/anti-CD28 antibodies, with versus without addition of anti-CD95 antibody (P < 0·05). (d) Flow cytometric analyses of activated (anti-CD3 and anti-CD28) BM3 cells treated with anti-CD95 antibody in the presence and absence of caspase inhibitor following culture in tryptophan-deficient (Trp−) and RPMI-1640 (Trp+) media. Cells were stained with propidium iodide as described in the Materials and methods.
Figure 6
Figure 6
RT-PCR analyses of IDO gene transcription in splenocytes. (a) RNA extracted from spleens of CBA mice as described in the Materials and methods after 24 hr in culture; total splenocytes (lane 1); non-adherent splenocytes (lane 2), three separate preparations of adherent cells (lanes 3–5). Lane 6, no RNA; lane 7, epididymis RNA. (b) RNA samples extracted from adherent splenocytes after culture for the number of hours indicated. Asterisk indicates RNA samples from non-adherent splenocytes after 18 hr culture. (c) RNA samples from CD11c+- and CD11b+-enriched cell populations selected by magnetic cell sorting after overnight (18 hr) culture. Samples were screened using primers for IDO and Fas ligand sequences and PCR products were of the size expected for mRNA transcripts.
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