L-arginine availability regulates T-lymphocyte cell-cycle progression

Abstract

L-arginine (L-Arg) plays a central role in several biologic systems including the regulation of T-cell function. L-Arg depletion by myeloid-derived suppressor cells producing arginase I is seen in patients with cancer inducing T-cell anergy. We studied how L-Arg starvation could regulate T-cell-cycle progression. Stimulated T cells cultured in the absence of L-Arg are arrested in the G0-G1phase of the cell cycle. This was associated with an inability of T cells to up-regulate cyclin D3 and cyclin-dependent kinase 4 (cdk4), but not cdk6, resulting in an impaired downstream signaling with a decreased phosphorylation of Rb protein and a low expression and binding of E2F1. Silencing of cyclin D3 reproduced the cell cycle arrest caused by L-Arg starvation. The regulation of cyclin D3 and cdk4 by L-Arg starvation occurs at transcriptional and posttranscriptional levels. Signaling through GCN2 kinase is triggered during amino acid starvation. Experiments demonstrated that T cells from GCN2 knock-out mice did not show a decreased proliferation and were able to up-regulate cyclin D3 when cultured in the absence of L-Arg. These results contribute to the understanding of a central mechanism by which cancer and other diseases characterized by high arginase I production may cause T-cell dysfunction.

Figure 1

Decreased proliferation in stimulated T cells cultured in the absence of l-Arg. (A) Freshly isolated human T cells (5 × 10⁵) stimulated with anti-CD3 plus anti-CD28 were cultured in the presence or the absence of l-Arg, and uptake of [³H]-thymidine was measured at 24, 48, 72, and 96 hours. (B) Stimulated T cells (5 × 10⁵) were labeled with 1 μM CFSE and cultured in the presence or the absence of l-Arg; fluorescence was measured at 72 and 96 hours. Unstimulated (NS) control cells were CFSE-labeled T cells cultured in medium containing l-Arg. (C) Stimulated T cells (5 × 10⁵) cultured in the absence of l-Arg for 24 hours were replenished with 2 mM l-lysine, l-glutamine, l-citrulline, or l-Arg, and incorporation of [³H]-thymidine was measured at 72 hours.

Figure 2

l-Arg starvation arrests T cells in G₀-G₁ phase of cell cycle. (A) Human T cells (5 × 10⁵) were stimulated with anti-CD3 plus anti-CD28 in the presence or the absence of l-Arg, and cell cycle was assessed at 48 and 72 hours by flow cytometry using propidium iodide. Percentages of T cells at each cell-cycle phase from 5 different experiments were determined. (B) Representative experiments from panel A. Unstimulated control T cells (NS, left row) were in medium containing l-Arg.

Figure 3

Impaired expression of cyclin D3 and cdk4 in stimulated T cells cultured in the absence of l-Arg. (A-B) Whole-cell lysates were obtained from 3 × 10⁶ activated T cells cultured in the presence or the absence of l-Arg for various lengths of time (hours) or from activated T cells cultured in the absence of l-Arg for 24 hours and then replenished with l-Arg at 48 or 72 hours. The expression of cyclin and cdks was tested by Western blotting. (C) Lysates from stimulated T cells cultured for 24 hours were prepared and 100 μg was immunoprecipitated with agarose-conjugated anti–cyclin D3 and anti-cdk4. Immunoprecipitates were then tested for cyclin D3 and cdk4 expression by Western blotting.

Figure 4

Activated T cells cultured in the absence of l-Arg have a decreased Rb phosphorylation and a decreased ability to phosphorylate Rb in vitro. (A) Whole-cell lysates obtained from stimulated T cells cultured for 24 hours in the presence or the absence of l-Arg were immunoblotted against Rb and vinculin. (B) Lysates from unstimulated or stimulated T cells cultured for 24 hours in the presence or the absence of l-Arg were prepared, and 100 μg was immunoprecipitated with agarose-conjugated anti–cyclin D3 and incubated with a reaction cocktail containing 0.2 μg GST-Rb and 10 μCi (0.37 MBq) [γ-³²P] ATP. (C) Similarly, 100 μg whole-cell lysates from stimulated T cells cultured for 24 hours in medium containing l-Arg was initially immunodepleted with an irrelevant protein G (NRS), or agarose-conjugated antibodies against cyclin D3, cdk4, or cdk6, and then immunoprecipitated with irrelevant protein G (NRS) or anti–cyclin D3 and tested for kinase activity.

Figure 5

Cyclin D3 silencing mimics l-Arg starvation effects on cell cycle. (A) Wild-type Jurkat cells (Jurkat) and Jurkat cells transfected with empty vector, nonrelated human sequence, or sh–cyclin D3 plasmid were tested for the expression of cyclin D3 RNA by Northern blot and cyclin D3 protein by Western blot. (B) Wild-type Jurkat cells or transfected cells were cultured in the presence or the absence of l-Arg and tested for proliferation after 48 hours by [³H]-thymidine incorporation.

Figure 6

l-Arg starvation impairs expression of cyclin D3 and cdk4 through transcriptional, posttranscriptional, and translational mechanisms. (A) RNA (5 μg) from nonactivated human T cells or T cells activated with anti-CD3 plus anti-CD28 and cultured in the presence or the absence of l-Arg was tested for cyclin D3, cdk4, and cdk6 RNA expression by Northern blot. (B) Nuclei obtained from activated T cells cultured in the presence and the absence of l-Arg were tested for cyclin D3, cdk4, and cdk6 transcriptional rate by run-on analysis as described in “Materials and methods.” (C) RNA stability was also tested in activated T cells cultured in the presence and the absence of l-Arg for 12 hours, after which Act D (5 μg/mL) was added and RNA collected after 3, 6, 12, 24, and 36 hours. RNA expression was then tested by Northern blot. (D) Nonactivated (NS, lane 1) and activated (lanes 2-3) T cells were cultured in the presence or the absence of l-Arg for 48 hours, after which cells were washed and pulsed with 250 μCi (9.25 MBq) [³⁵S] methionine for 3 hours. Lysates were immunoprecipitated using anti–cyclin D3 as described in “Materials and methods.”

Figure 7

T cells from GCN2 KO mice proliferate in the absence of l-Arg. (A) T cells (2 × 10⁵) were activated with bound anti-CD3 plus anti-CD28, and thymidine incorporation was tested after 48 hours (cpm ± SD). (B) Kinetics of expression of cyclin D3 and cdk4 in activated T cells (4 × 10⁶) from GCN2 knock-out mice and congeneic wild-type mice.