-arginine depletion blunts antitumor t-cell responses by ... · speciﬁc energy metabolic pathways...

Microenvironment and Immunology

L-Arginine Depletion Blunts Antitumor T-cellResponses by Inducing Myeloid-DerivedSuppressor CellsMatthew Fletcher1, Maria E. Ramirez1, Rosa A. Sierra1, Patrick Raber1,2, Paul Thevenot1,Amir A. Al-Khami1, Dulfary Sanchez-Pino1,Claudia Hernandez1, DorotaD.Wyczechowska1,Augusto C. Ochoa1,3, and Paulo C. Rodriguez1,2

Abstract

Enzymatic depletion of the nonessential amino acid L-Arginine(L-Arg) in patients with cancer by the administration of a pegy-lated form of the catabolic enzyme arginase I (peg-Arg I) hasshown some promise as a therapeutic approach. However, L-Argdeprivation also suppresses T-cell responses in tumors. In thisstudy, we sought to reconcile these observations by conducting adetailed analysis of the effects of peg-Arg I on normal T cells.Strikingly, we found that peg-Arg I blocked proliferation and cell-cycle progression in normal activated T cells without triggeringapoptosis or blunting T-cell activation. These effects were associ-ated with an inhibition of aerobic glycolysis in activated T cells,but not with significant alterations in mitochondrial oxidativerespiration, which thereby regulated survival of T cells exposed topeg-Arg I. Further mechanistic investigations showed that the

addition of citrulline, a metabolic precursor for L-Arg, rescuedthe antiproliferative effects of peg-Arg I on T cells in vitro.Moreover, serum levels of citrulline increased after in vivoadministration of peg-Arg I. In support of the hypothesis thatpeg-Arg I acted indirectly to block T-cell responses in vivo, peg-Arg I inhibited T-cell proliferation in mice by inducing accu-mulation of myeloid-derived suppressor cells (MDSC). MDSCinduction by peg-Arg I occurred through the general controlnonrepressed-2 eIF2a kinase. Moreover, we found that peg-ArgI enhanced the growth of tumors in mice in a manner thatcorrelated with higher MDSC numbers. Taken together, ourresults highlight the risks of the L-Arg–depleting therapy forcancer treatment and suggest a need for cotargeting MDSC insuch therapeutic settings. Cancer Res; 75(2); 275–83. �2014 AACR.

IntroductionTherapeutic modulation of specific cellular metabolic path-

ways in solid and hematologic malignancies represents a majorstrategy for the development of new treatments (1–4). Metabo-lism of the nonessential amino acid L-Arginine (L-Arg) plays acentral role in several biologic systems including the modulationof immune responses and tumor growth (5). We and othersdescribed the therapeutic benefit of depleting L-Arg in hemato-logic and solid tumors through a pegylated form of the human L-Arg–metabolizing enzyme arginase I (peg-Arg I; refs. 6–10).Pegylation of arginase I increased its stability in vivo, withoutaltering its activity (7). Moreover, peg-Arg I was effective against

malignant cells that depended upon exogenous L-Arg for growth(auxotrophic) and did not induce significant toxic side effects (3).The antitumor effect induced by peg-Arg I was mediated by theinduction of malignant cell apoptosis (6) and controlled throughthe phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2a) and the expression of general control nonrepressed2 eIF2a kinase (GCN2; ref. 11). Although peg-Arg I was highlyeffective in controlling the growth of leukemic T cells (6, 11), itseffect on normal T cells remains unclear. Initial in vitro studiesshowed that L-Arg starvation blocked proliferation of activatednormal T cells (12–14). In addition, we found that peg-Arg Idelayed development of GVHD and increased burden of Listeriamonocytogenes (15, 16), both conditions linked to impaired T-cellfunction. However, the mechanisms by which peg-Arg I couldimpair T-cell responses in vivo and how normal activated T cellsmaintain survival under L-Arg starvation remain unknown.

Specific energy metabolic pathways regulate the activationand proliferation of normal T cells. Production of ATP andreactive oxygen species (ROS) from the mitochondria control theinitial T-cell–activation phase, whereas aerobic glycolysis mod-ulates proliferation and effector T-cell functions (17–21).Although specific energymetabolic programming regulates globalfunction of T cells, it remains unknown the effect of L-Arg in themodulation of energy metabolism.

Accumulation of myeloid-derived suppressor cells (MDSC), aheterogeneous population of immature myeloid cells expressingCD11bþGr1þ, is a hallmark of chronic inflammation and amajormediator for the induction of T-cell suppression in various tumors(22, 23). MDSCs block T-cell responses through the metabolism

1Stanley S. Scott Cancer Center, Louisiana State University HealthSciences Center, New Orleans, Louisiana. 2Department of Microbiol-ogy, Immunology and Parasitology, Louisiana State University HealthSciences Center, New Orleans, Louisiana. 3Department of Pediatrics,Louisiana State University Health Sciences Center, New Orleans,Louisiana.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M. Fletcher and M.E. Ramirez contributed equally to this article.

Corresponding Author: Paulo C. Rodriguez, Stanley S. Scott Cancer Center,Louisiana State University Health Sciences Center, 1700 Tulane Ave Room-910,New Orleans, LA 70112. Phone: 504-210-3324; Fax: 504-599-0911; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-1491

�2014 American Association for Cancer Research.

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of L-Arg by the enzymes arginase I and inducible nitric oxidesynthase (iNOS), which promote L-Arg depletion and productionof peroxynitrite, respectively (24, 25). Although the role of L-Argmetabolism on the T-cell suppression induced by MDSC is wellunderstood, the effect of the deprivation of L-Arg in the accumu-lation and function of MDSC remains unknown.

Because the potential contradictory effect of L-Arg depletion asan antitumor therapy and as a mechanism for inhibition ofimmune responses, we aimed to understand the effects of peg-Arg I on normal T cells. Our results show the regulatory effect ofpeg-Arg I on T-cell proliferation and the ability of T cells to resistpeg-Arg I through de novo L-Arg synthesis. Moreover, L-Arg depri-vation induced the accumulation ofMDSC,which inhibited T-cellproliferation in mice. These results support the novel role ofMDSC in the regulation of T-cell responses by L-Arg starvationand suggest the need to therapeutically target MDSC in peg-ArgI–based therapies.

Materials and MethodsMice and cells

C57BL/6 mice were purchased from Harlan Laboratories.CD45.1þ, GCN2�/�, and anti-OVA257-264 (siinfekl) OT-1 micewere from The Jackson Laboratories. Lewis lung carcinoma cells(3LL) were obtained in 2012 from the ATCC and injected s.c. intothe mice (26). 3LL cells were periodically tested (last-test May2014) and validated to be mycoplasma-free, using an ATCC kit.All mice studies were achieved using an approved InstitutionalAnimal Care and Use Committee protocol from Louisiana StateUniversity-Health Sciences Center, New Orleans, LA. T cells wereisolated from spleens and lymph nodes of mice using T-cell-negative isolation kits (Dynal, Life Technologies). Then, T cellswere activated using 0.5 mg/mL plate bound anti-CD3 plus anti-CD28 (26).MDSCswere isolated from spleens ofmice usingGr-1selection kits (Stem Cell Technologies). Purity for cell isolationsranged from 90% to 99%.

Antibodies and reagentsDetailed description of antibodies, methodologies for flow

cytometry and fluorescence, and statistical analysis are in theSupplementary Methods. O'-methylpolyethylene-glycol (PEG)5,000 mw (Sigma-Aldrich) was covalently attached to human-recombinant arginase I (AbboMax) or bovine serum albumin(BSA; Sigma-Aldrich) in a 50:1 molar ratio (7). Pegylated-BSA(peg-BSA) was used as control for peg-Arg I.

Adoptive T-cell transferMice were treated with peg-Arg I or peg-BSA every 2 days

starting the day before the T-cell transfer. CD45.2þ mice wereadoptively transferred with 5 � 106 CD45.1þ/OT-1 T cells, fol-lowed by immunization s.c. with 0.5 mg siinfekl peptide inincomplete Freud's Adjuvant. Four days later, mice were injectedi.p with 200 mg 5-bromo-2-deoxyuridine (BrdUrd; BD Bio-sciences), and BrdUrd incorporation measured 24 hours laterusing the APC-BrdU Kit (BD Biosciences). For studies usingdepletion of MDSC, mice were treated with 200 mg anti–Gr-1antibody (RB6-8C5) or IgG control twice a week, starting the daybefore the adoptive transfer. For MDSC proliferation, mice weretreated with peg-Arg I every other day for 7 days, after whichBrdUrd uptake into CD11bþ Gr-1þ cells was tested. Analysis ofnuclear DNA content was achieved using the CycleTEST-DNA Kit

(BD Biosciences). T-cell apoptosis was tested using Annexin V–FITC Apoptosis Detection Kit (BD Biosciences). Staurosporine (1mmol/L) was added 24 hours prior the apoptosis analysis aspositive control.

[3-3H]-glucose uptakeGlucose uptake was tested after pulsing activated T cells cul-

tured with peg-BSA or peg-Arg I (48 hours) with 1 mCi/mLGlucose-[3-3H] (Perkin Elmer Life Sciences). Eight hours later,T cells were washed in PBS, collected, and tested for radioactivity(counts per minute).

Extracellular flux analysisOxygen consumption rates (OCR) and extracellular acidifica-

tion rates (ECAR)weremeasured using the XF-24-extracellular fluxanalyzer (Seahorse Bioscience). In brief, 5 � 105 activated T cellstreated for 24 to 72 hours with peg-Arg I or peg-BSAwere plated inXF-24 cell culture plates coated with 15 mg CellTak (BD Bio-sciences) in XFmedia (DMEM containing 11mmol/L glucose and100 mmol/L sodium pyruvate). For OCR, T cells were analyzedunderbasal conditions and in response to10mmol/Loligomycin,1mmol/L fluorocarbonyl-cyanide-phenylhydrazone (FCCP), and 1mmol/L rotenone (Sigma-Aldrich). For ECAR, T cells were plated inXF media lacking glucose and monitored under basal conditionsand in response to10mmol/L glucose, 10mmol/L oligomycin, and100 mmol/L 2-Deoxy-D-glucose (2-DG; Sigma-Aldrich).

siRNA transfection of primary T cellsArgininosuccinate synthetase (ASS-1) siRNA, nontargeting

control siRNA, and FAM-labeled nontargeting control siRNAwereobtained from Thermo Scientific (Accell-SMARTpool). ActivatedT cells (2 � 105) were transfected (2 mmol/L siRNA) using AccellsiRNA delivery media plus 2% FBS, following the vendor's pro-tocol. Transfection efficiency ranged from 90% to 95% andviability from 95% to 99%. Peg-Arg I (1 mg/mL) or peg-BSA (1mg/mL) were added 12 hours after transfection.

ResultsEffects of peg-Arg I on T-cell proliferation and activation

Our previous results showed that peg-Arg I blocked malignantT-cell proliferation through the induction of cellular apoptosis;however, the effect of peg-Arg I on normal T cells remains unclear(6, 11). To test this, we first investigated the effect of peg-Arg Ion the proliferation of CFSE-labeled activated T cells. A dose-dependent inhibition of T-cell proliferation was found afterthe addition of peg-Arg I, but not with control peg-BSA (Fig.1A). The antiproliferative effect triggered by peg-Arg I was simi-larly noted in both CD4þ and CD8þ T cells and was associatedwith a lower IFNg production (Supplementary Fig. S1A and S1B).To validate our results in vivo, we tested the effect of peg-Arg I in theproliferation of anti-OVA257-264 (siinfekl) transgenic CD8þ OT-1cells. Thus, CD45.2þ mice were adoptively transferred withCD45.1þ CD8þ OT-1 cells, followed by immunization withsiinfekl peptide. Mice were injected i.p. with peg-Arg I or peg-BSA the day before T-cell transfer and then every two days. Asignificant decrease in CD45.1þ OT-1 cell proliferation, as testedby BrdUrd uptake, was found in immunized mice treated withpeg-Arg I, compared with those injected with peg-BSA (Fig. 1B).To evaluate a potential T-cell toxicity side effect, we measured T-cell subsets in spleens of na€�ve mice treated for a week with

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increasing concentrations of peg-Arg I or peg-BSA. Peg-Arg Itreatment was well tolerated and did not significantly alter thepercentages of CD3þ, CD4þ, and CD8þ T cells in spleen (Sup-plementary Fig. S2A–S2C). Therefore, peg-Arg I blocked prolifer-ation of activated T cells, but did not affect homeostatic na€�ve T-cell numbers.

To better understand the mechanisms by which peg-Arg Iblocked T-cell proliferation, we tested the effect of peg-Arg I in theinduction of T-cell apoptosis and cell-cycle progression. A similarlow expression of the apoptosis marker Annexin V was found inactivated T cells treated with peg-Arg I or peg-BSA (Fig. 1C).Conversely, upregulation of Annexin V was noted in T cells treated

with the apoptosis-inducer staurosporine. Furthermore, an arrest intheG0–G1 phase of the cell cycle, which correlatedwith a decreasedexpression of cyclin D3 and cdk4, was found in activated T cellscultured with peg-Arg I, compared with peg-BSA–treated T cells(Fig. 1D and E). We next evaluated the effect of peg-Arg I on T-cellactivation by testing the expression of the early activation T-cellmarkers CD25 and CD69 and the production of IL2. Similarupregulation of CD25 and CD69 and comparable percentages ofIL2þ cells were found in activated T cells treated with increasingdoses of peg-Arg I or peg-BSA (Fig. 1F and G). Also, in agreementwith the results showing the activation-independent antiprolifera-tive effect induced by peg-Arg I, we found that addition of peg-Arg I

Figure 1.Peg-Arg I arrests T-cell proliferationwithout inducing apoptosis or altering activation. A, CFSE-labeled T cellswere stimulatedwith anti-CD3/CD28 in the presence ofpeg-Arg I or peg-BSA. Percentages of proliferating T cellswere determined 72 hours later by flow cytometry. Values are from three experiments. B, CD45.1þ/OT-1 cellproliferationwasmonitored in spleens ofmice treatedwith peg-Arg I or peg-BSA (5mg/mouse, n¼ 10) usingBrdUrd, as described inMaterials andMethods. C andD,activated T cells were treated with 1 mg/mL peg-Arg I or peg-BSA for 72 hours, after which they were stained with Annexin V (C) or propidium iodide (D).T cells treated with 1 mmol/L staurosporine were used as positive apoptosis controls. E, representative experiment showing the expression of cyclin D3 and Cdk4 inT cells treatedwith 1 mg/mL peg-Arg I. F andG, percentages of T cells havingCD25, CD69 (F), or IL2 (G) after activation and culture for 48 hourswith increasing levelsof peg-Arg I or peg-BSA. H, peg-Arg I or peg-BSA (1 mg/mL) were added to stimulated T cells labeled with CFSE at 0 or 24 hours after plating, and proliferation wasdetected after 72 hours by flow cytometry. All data are expressed as mean � SEM from three experiments. �� , P < 0.001.

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Suppression of T-cell Responses by L-Arginine Depletion




after 24 hours of activation still inhibited T-cell proliferation (Fig.1H). Therefore, peg-Arg I blocks proliferation and cell-cycle pro-gression of T cells without leading to apoptosis or impairing theexpression of early activation mediators.

Effects of peg-Arg I on T-cell energy metabolic pathwaysSpecific energy metabolic pathways control the proliferation

and activation of normal T cells. Proliferating T cells are highlydependent on the production of energy through aerobic glycol-ysis, whereas mitochondrial oxidative respiration fuels T-cellactivation (20). Thus, we tested the effect of peg-Arg I on T-cellenergy metabolism using extracellular flux analysis. A significantdecrease in glycolysis, as measured by ECAR, was noted inactivated T cells treated with peg-Arg I, compared with T cellscultured with peg-BSA (Fig. 2A and Supplementary Fig. S3A andS3B). Moreover, a lower glucose-[3-3H] uptake and decreasedexpression of the glucose transporter Glut-1 were found in T cellscultured in the presence of peg-Arg I (Fig. 2B and C), comparedwith peg-BSA–treated T cells, suggesting that peg-Arg I blockedglycolysis in normal activated T cells.

Next, we evaluated the effect of peg-Arg I on mitochondrialoxidative respiration by measuring OCR. Activated T cells werecultured in the presence of peg-Arg I or peg-BSA for 24 hours, afterwhich basal OCRwas tested. A similar upregulation ofOCR levelswas noted in activated T cells cultured with peg-Arg I or peg-BSA,compared with nonactivated T cells (Fig. 2D). We then testedmitochondrial function by evaluating ATP-linked respirationusing the mitochondrial ATP synthase inhibitor oligomycin,maximal respiration using the uncoupling ionophore FCCP, andnonmitochondrial OCR using the electron transport chain inhib-itor rotenone. ATP-linked and nonmitochondrial OCR were sim-ilar in the peg-Arg I and peg-BSA–treated T cells, though the peg-Arg I–treated cells did have a slightly lower OCR reserved capacity(Fig. 2E). Furthermore, in agreement with the lack of effect of peg-Arg I on mitochondrial respiration, we noticed a similar produc-tion of mitochondrial ROS (MitoSOX) and mitochondrial bio-genesis (MitoTracker) in activated T cells cultured with peg-Arg Ior peg-BSA (Fig. 2F and G). Next, we elucidated whether energyproduction by mitochondrial respiration is needed for the sur-vival of T cells to peg-Arg I. Addition of oligomycin resulted in a

Figure 2.Effects of peg-Arg I on T-cell energymetabolic pathways. A, T cellswere treatedwith 1mg/mLpeg-Arg I or peg-BSA for 24 to 72 hours. Then, ECARwasmeasured viaan extracellular flux analyser. B, glucose-[3-3H] uptake in activated T cells treatedwith peg-Arg I or peg-BSA for 48 hours. C, expression of Glut-1 was tested in T cellscultured in the presence of 1mg/mLpeg-Arg I or peg-BSA. D and E, OCR in activated T cells culturedwith peg-Arg I or peg-BSA for 24 hourswas analyzed under basalconditions (D) and in response to oligomycin, FCCP, and rotenone (E). F, activated T cells treated with peg-Arg I or peg-BSA for 48 hours were stained withMitoSOX and analyzed by flow cytometry. G, activated T cells treated with peg-Arg I or peg-BSA for 72 hours were stained with Mitotracker Green-FM and DAPIand imageswere captured at equal exposure. Voxel quantificationwas achieved usingMask analysis. H, Annexin V staining in activated T cells treatedwith peg-Arg I� oligomycin (100 nmol/L) for 48 hours. Results are expressed as mean � SEM from three experiments. Nonstatistical significant differences (ns), P > 0.05.�� , P < 0.001.

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significant increase of apoptosis in peg-Arg I–treated T cells, butnot in controls (Fig. 2H), suggesting the importance of themitochondrial ATP production in the survival of normal T cellsto peg-Arg I.

L-Arg synthesis from citrulline and T-cell resistance to peg-Arg IDe novo synthesis of L-Arg occurs from citrulline and is reliant

upon enzymes ASS-1 and argininosuccinate lyase (ASL; ref. 23).Because complete culture medium lacks citrulline, we askedwhether the addition of exogenous citrulline or L-Arg rescued theproliferation arrest induced by peg-Arg I. Addition of citrulline,but not L-Arg, restored proliferation of T cells cultured in thepresence of peg-Arg I (Fig. 3A). The lack of effect of the supple-mented L-Arg can be explained by its immediate metabolism bypeg-Arg I (6). Then, we compared the expression of ASS-1 andASLin activated T cells cultured with peg-Arg I or peg-BSA. A similarexpression of both enzymes was observed in T cells independentof the activation or the presence of peg-Arg I (Fig. 3B). To

understand the role of L-Arg synthesis in the proliferation of Tcells cultured with peg-Arg I and citrulline, we silenced theexpression of ASS-1 using siRNA (Supplementary Fig. S4). Silenc-ing of ASS-1 significantly impaired the ability of peg-Arg I–treatedT cells to proliferate in citrulline-supplemented media (Fig. 3C)without resulting in T-cell apoptosis (Fig. 3D). Because normal Tcells were resistant to peg-Arg I in the presence of citrulline, weevaluated the availability of citrulline in the serum of peg-Arg I–treated mice. A dose-dependent increase in citrulline levels wasnoted in the serum of mice treated with peg-Arg I, but not in peg-BSA controls (Fig. 3E), indicating the elevation of citrulline as asystemic response to acute L-Arg deprivation in vivo.

MDSCs mediate antiproliferative effects of peg-Arg I in vivoBecause the elevation of citrulline in peg-Arg I–treatedmice and

the ability of T cells to produce L-Arg from citrulline, we sought todetermine whether an indirect mechanism was mediating theimpaired T-cell proliferation after peg-Arg I treatment. To test the

Figure 3.Role of L-Arg synthesis in T cells in theeffects induced by peg-Arg I. A,activated CFSE-labeled T cells weretreated with peg-Arg I or peg-BSA(1 mg/mL) in media supplementedwith 2 mmol/L citrulline or L-Arg.Percentages of proliferating T cellswere evaluated by flow cytometry at 72hours. B, representative experiment forthe expression of ASS-1 and ASL in Tcells cultured in the presence of peg-Arg I or peg-BSA. C, activated CFSE-labeled T cells were transfected withASS-1 or nontargeting control siRNA (2mmol/L) � peg-Arg I or peg-BSA inmedia supplemented with citrulline for72 hours. T-cell proliferation wasmeasured by flow cytometry, withpooled results from three experiments(left) and a representative result(right). D, T cells from Cwere tested forAnnexin V expression by flowcytometry. E, serum collected frommice (n ¼ 5) treated with peg-Arg I orpeg-BSA for 12 hours. Levels ofcitrulline were established by high-performance liquid chromatography.Findings are expressed as mean� SEMfrom three experiments. Nonstatisticalsignificant differences (ns), P > 0.05.�� , P < 0.001.






potential role of MDSC, CD8þ T cells from CD45.1þ OT-1 micewere adoptively transferred into cohorts of CD45.2þmice treatedwith peg-Arg I, peg-BSA, or PBS, which received repeated injec-tions with anti–Gr-1 or control IgG antibodies and were immu-nized with siinfekl. A significant restoration in OT-1 cell prolif-eration was detected in peg-Arg I–treated mice receiving anti–Gr-1, but not in those receiving IgG (Fig. 4A), suggesting that MDSCregulated T-cell suppression induced by peg-Arg I in vivo. Inaddition, a dose-dependent accumulation of CD11bþ Gr1þ cellsoccurred in spleens of mice treated with peg-Arg I, but not inthose treated with peg-BSA or PBS (Fig. 4B), which correlatedwith a higher CD11bþ Gr1þ cell proliferation, as tested byBrdUrd (Fig. 4C). Moreover, the increase in MDSC by peg-Arg I

treatment was the result of the expansion of granulocytic-MDSC (G-MDSC, CD11bþ Ly6Gþ Ly6Clow), but not monocyt-ic-MDSC (M-MDSC, CD11bþ Ly6Gneg Ly6Chigh; Fig. 4D).To test whether the increase in MDSC was also induced bydeprivation of other amino acids, we studied the effect ofasparagine-metabolizing enzyme peg-asparaginase. A similarincrease in G-MDSC, but not M-MDSC, was found in peg-asparaginase–treated mice compared with controls (Supple-mentary Fig. S5), supporting the effect of amino acid starvationon MDSC accumulation.

Next, we tested the ability of peg-Arg I–induced CD11bþ Gr1þ

cells to block T-cell responses in vitro. CD11bþGr1þ cells isolatedfrom peg-Arg I–treated mice, but not from those treated with

Figure 4.MDSCs mediate antiproliferative effects of peg-Arg I in mice. A, proliferation, as tested by BrdUrd, in CD45.1þ/OT-1 T cells adoptively transferred into CD45.2þmicethat received peg-Arg I or peg-BSA (1 mg/mouse), immunization with siinfekl, and treatments with anti–Gr-1 antibody or IgG, as described in Materials and Methods(n¼ 10 for each group). B, mice (n¼ 10) were injected i.p. with peg-Arg I, peg-BSA, or PBS every 2 days for 7 days. Then, percentages of CD11bþGr-1þwere tested inspleens by flow cytometry. C, proliferation of MDSC was determined by BrdUrd, as described in Materials and Methods. D, samples from B (0.5 mg/mouse)were tested for G-MDSC (CD11bþ Ly6Gþ Ly6Clow) and M-MDSC (CD11bþ Ly6Gneg Ly6Chigh). E, proliferation of CFSE-labeled T cells was measured 72 hours aftercoculture with different ratios of splenic CD11bþ Gr-1þ cells from peg-Arg I, peg-BSA, or PBS-treated mice. F, arginase I and iNOS in splenic CD11bþ Gr-1þ cells frommice treated with peg-Arg I, peg-BSA, or PBS. G, proliferation of CFSE-labeled T cells cocultured with splenic peg-Arg I–induced MDSC (1:1/2 ratio) after addition ofNN (200 mmol/L), L-NMMA (500 mmol/L), or L-Arg (2 mmol/L). H, 1 � 106 3LL cells were injected s.c. into mice (n ¼ 5), followed by peg-Arg I, peg-BSA, orPBS injections i.p. (1mg) every 3days. I, CD11bþGr-1þwithin spleens of peg-Arg I, peg-BSA, or PBS-treated 3LL-bearingmice (17 days). Results are expressed asmean� SEM from three experiments. Nonstatistical significant differences (ns), P > 0.05; �� , P < 0.01; �� , P < 0.001.

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peg-BSA or PBS, blocked proliferation of activated T cells in adose-dependent manner (Fig. 4E). In addition, MDSC from peg-Arg I–treated mice displayed increased expression of MDSC-inhibitory protein arginase I (Fig. 4F) and higher arginase activity(Supplementary Fig. S6). Also, the addition of arginase inhibitornor-NOHA, iNOS inhibitor L-NMMA, or exogenous L-Argblocked the increased regulatory effect of peg-Arg I–inducedMDSC (Fig. 4G), suggesting a role of L-Arg metabolism in theinhibitory functionofMDSC inducedbypeg-Arg I.Next,we testedthe effect of peg-Arg I in the growth of 3LL tumors, a model thatdepends upon MDSC (27, 28). An accelerated tumor growth wasfound in 3LL-bearing mice treated with peg-Arg I, compared withmice treated with peg-BSA or PBS (Fig. 4H), which correlatedwithhigher numbers of MDSC (Fig. 4I). Thus, our results support theprimary role of MDSC in the T-cell suppression induced by L-Argstarvation in vivo andwarrants caution in the use of peg-Arg I as anantitumor therapy without targeting MDSC accumulation.

Peg-Arg I–induced MDSC accumulation is mediated by GCN2We aimed to determine themolecular mediators by which peg-

Arg I induced the accumulation of MDSC. Kinase GCN2 is a keymediator of the effects induced by amino acid starvation(11, 29, 30, 30–33). Therefore, we sought to examine the effectof GCN2 in the MDSC induction by peg-Arg I. An impairedaccumulation of MDSC was detected in peg-Arg I–treatedGCN2-deficient mice compared with controls (Fig. 5A), suggest-ing the key role ofGCN2 in the accumulation ofMDSCbypeg-ArgI. To determine whether GCN2 also mediated the inhibitoryfunction of peg-Arg I–induced MDSC, we compared on a per cellbasis the ability of CD11bþGr1þ cells fromwild-type andGCN2-deficient mice to impair T-cell proliferation and to express argi-nase I. An identical ability to suppress T-cell proliferation and asimilar expression of arginase I were found in sorted MDSC fromwild-type andGCN2-deficientmice treatedwith peg-Arg I (Fig. 5Band C), suggesting that GCN2 is involved in the accumulation ofMDSC, but not in the per-cell function of peg-Arg I–inducedMDSC.

DiscussionPrevious studies established the therapeutic benefit of peg-Arg I

in various preclinical malignancies (6–10). Moreover, peg-Arg I isundergoing evaluation in phase I/II trials for adults with livercarcinoma, with favorable safety profiles (34). Because L-Argdepletion is also an important mechanism for suppression ofT-cell responses in tumors (22), we sought to reconcile theseobservations by conducting a detailed analysis of the effects ofpeg-Arg I on normal T cells. Our findings show the regulatoryeffect of peg-Arg I onT-cell proliferation and the ability of T cells toresist peg-Arg I through L-Arg synthesis. In addition, we show thenew role of MDSC in the inhibition of T-cell responses by L-Argstarvation.

Our findings showed the ability of T cells to synthesize L-Arg denovo and indicated the elevation of serum citrulline as a systemicresponse to acute depletion of L-Arg. The increased citrullinesynthesis occurring under systemic low arginine levels has beenshown to be mediated by enterocytes using diet-based glutamine(35–39). Also, in agreement with the role of citrulline after L-Argdeprivation, injection of citrulline ameliorated toxic effects afterarginase therapy (20). Although our data suggest the role of L-Argsynthesis in the effects induced by peg-Arg I, we could not

specifically test the effect of citrulline on T cells or MDSC frompeg-Arg I–treated mice because of the lack of pharmacologicinhibitors against ASS-1 or ASL, the toxicity of ASS-1 deletion inmice (40, 41), and the unavailability of conditional ASS-1– orASL-deficient mice.

Peg-Arg I blocked malignant T-cell proliferation through theinduction of apoptosis (6), while it failed to trigger cell death innormal T cells. Survival of normal T cells to peg-Arg I wasmediated by ATP production in the mitochondria. The lack ofthis adaptive pathway in malignant cells could explain the differ-ences in apoptosis observed in malignant versus normal T cellstreatedwithpeg-Arg I. In fact, tumor cells aremostly dependent onaerobic glycolysis, whereas normal cells retain the ability to sensestress and maintain survival through mitochondrial-mediatedpathways (42). Our results also showed that peg-Arg I did notimpair activationprocesses ormitochondrial respiration inT cells.However, the role of T-cell activation in the low proliferationinduced by peg-Arg I is still unknown, in part because of the

Figure 5.Peg-Arg I–induced MDSC accumulation is mediated by GCN2. A, wild-typeand GCN2-deficient mice were injected i.p. with 1 mg of peg-Arg I, peg-BSA,or PBS every 2 days for 7 days. Then, splenic CD11bþ Grþ cells weredetermined by flow cytometry. B, proliferation of CFSE-labeled T cells aftercoculture with different ratios of splenic MDSC from control or GCN2�/� peg-Arg I–treated mice. C, arginase I in splenic CD11bþ Grþ cells fromwild-type orGCN2�/� mice treated with peg-Arg I, peg-BSA, or PBS. Results areexpressed as mean � SEM from three experiments. �� , P < 0.001.






difficulty to separating proliferation and activation processes.Previous research showed the importance of fatty acid oxidationandmitochondrial respiration on the proliferation of regulatory Tcells (2). Therefore, it is possible that regulatory T cells are lesssusceptible to peg-Arg I, which could also contribute to thetolerogenic effects induced by L-Arg starvation.

We found that in addition to the potential suppression of T cellsby MDSC-mediated L-Arg deprivation, L-Arg starvation also pro-motes accumulation of MDSC, further suppressing antitumor T-cell responses. Accordingly, peg-Arg I mimicked T-cell suppres-sion induced by physical injury, a process alsomediated byMDSC(16). Although immune suppression could be a major limitationfor the use of peg-Arg I as a therapy in cancer, it may also lead tonovel therapies in transplantation and autoimmune diseases. Inthese settings, peg-Arg I could maintain a permanent influx ofMDSC to provide T-cell suppression. As such, peg-Arg I prolongedmice survival after bone marrow transplantation and preventeddevelopment of GVHD similar tomice receiving adoptive transferof MDSC (15). With regard to the use of peg-Arg I in differentcancers, peg-Arg I could be combined with chemotherapy agentsthat block MDSC accumulation and function, such as gemcita-bine (43), 5-fluorouracil (44), and sunitinib (45). Furthermore,we found a role ofGCN2 in the accumulation ofMDSC inpeg-ArgI–treatedmice. Accordingly, previous studies indicated the role ofGCN2 in the immune effects induced by L-asparaginase (30, 46).However, the intracellular mechanisms by which the depletion ofL-Arg and its sensing pathways promote MDSC accumulationremain unknown. Possible pathways could include the inductionof factors that promote MDSC accumulation, including VEGF,S100A8/A9, granulocyte-macrophage colony stimulating factor(GM-CSF), andG-CSF. Also, in agreement with the role of cellularstress in the regulation of myeloid cells function and accumula-tion, recent studies showed the effect of L-Arg starvation onmacrophage responses to infections (41) and the specific role ofstress mediators in the induction of VEGF, arginase I (47), andMDSC accumulation (48) in tumors.

In summary, our results show for thefirst time the role ofMDSCin the suppression of T-cell function caused by L-Arg starvation.

This increases the understanding of the effects of L-Arg deprivationin the regulation of antitumor immunity and suggests caution forthe use of peg-Arg I in malignancies without targeting MDSCaccumulation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design:M. Fletcher, M.E. Ramirez, A.C. Ochoa, P.C. RodriguezDevelopment of methodology: M. Fletcher, M.E. Ramirez, P. Raber, A.A.Al-Khami, D. Sanchez-Pino, C. Hernandez, P.C. RodriguezAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Fletcher, M.E. Ramirez, R.A. Sierra, P. Raber,P. Thevenot, A.A. Al-Khami, C.Hernandez,D.D.Wyczechowska, P.C. RodriguezAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Fletcher, M.E. Ramirez, R.A. Sierra, P. Raber,P. Thevenot, A.A. Al-Khami, D. Sanchez-Pino, C. Hernandez, A.C. Ochoa,P.C. RodriguezWriting, review, and/or revision of themanuscript:M. Fletcher, M.E. Ramirez,P. Raber, P. Thevenot, A.A. Al-Khami, C. Hernandez, P.C. RodriguezAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M.E. Ramirez, R.A. Sierra, C. Hernandez,P.C. RodriguezStudy supervision: P.C. Rodriguez

AcknowledgmentsThe authors thank Dr. Arnold Zea, PhD (Stanley S. Scott Cancer Center

HPLC facilities), for testing the citrulline levels in the sera from treated mice.

Grant SupportThis work was partially supported by Hope on Wheels Hyundai award (M.

Fletcher); NIH grant P20GM103501 subproject #3 and NIH-R21CA162133(P.C. Rodriguez); and R01OD016990, R01CA107974, and U54GM104940(A.C. Ochoa).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 20, 2014; revised October 22, 2014; accepted November 4,2014; published OnlineFirst November 18, 2014.

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2015;75:275-283. Published OnlineFirst November 18, 2014.Cancer Res Matthew Fletcher, Maria E. Ramirez, Rosa A. Sierra, et al. Inducing Myeloid-Derived Suppressor Cellsl-Arginine Depletion Blunts Antitumor T-cell Responses by

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