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Chain in T Lymphocytes1
* Tumor Immunology Program, Stanley S. Scott Cancer Center, and
Department of Pathology, Louisiana State University, Health Sciences Center, New Orleans, LA 70112; and
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15261.
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Abstract |
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-chain (CD3) and have an impaired proliferation and a decreased cytokine production. The aim of this work was to test whether activated macrophages could modulate extracellular levels of L-arginine and alter T cell function, and to determine which metabolic pathway was responsible for this event. The results show that macrophages stimulated with IL-4 + IL-13 up-regulate ASE I and cationic amino acid transporter 2B, causing a rapid reduction of extracellular levels of L-arginine and inducing decreased expression of CD3 and diminished proliferation in normal T lymphocytes. Competitive inhibitors of ASE I or the addition of excess L-arginine lead to the re-expression of CD3 and recovery of T cell proliferation. In contrast, inducible nitric oxide synthase or ASE II failed to significantly reduce the extracellular levels of L-arginine and modulate CD3 expression. These results may provide new insights into the mechanisms leading to T cell dysfunction and the down-regulation of CD3 in cancer and chronic infectious diseases. |
Introduction |
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up-regulates iNOS exclusively, whereas IL-4, IL-10 and IL-13 induce ASE I (8, 9). The mitochondrial isoform ASE II is not significantly modulated by Th1 or Th2 cytokines (7, 10).
An increase in circulating ASE and a decrease in L-arginine levels has been described in patients and rodents after liver transplantation or trauma (11, 12, 13) and is accompanied by a marked T cell dysfunction characterized by diminished proliferation and decreased production of cytokines (14). The infusion of high doses of L-arginine results in a recovery of T cell proliferation and an increase in the number of CD4+ cells (15, 16, 17). However, the mechanisms by which low levels of L-arginine impair T cell function have remained unclear. We recently reported that Jurkat cells cultured in the absence of L-arginine have diminished proliferation and decreased expression of the -chain (CD3) (18, 19), the principal signal-transduction element of the TCR (20). CD3 down-regulation induced by L-arginine starvation was not caused by apoptosis, but rather through posttranscriptional mechanisms leading to decreased CD3 mRNA stability (19). The depletion of other nonessential amino acids such as L-glutamine does not alter CD3 expression (18, 19). However, the effect of L-arginine depletion on normal T cells and the mechanisms that may limit the availability of L-arginine during an immune response are unclear. Using a coculture system, we studied whether one or more of the enzymatic pathways that metabolize L-arginine in macrophages, namely iNOS, ASE I, or ASE II, could regulate the availability of L-arginine and possibly modulate CD3 expression and T cell function. The results demonstrate that macrophages producing ASE I and expressing the cationic amino acid (CAT) transporter 2B rapidly reduce the extracellular concentration of L-arginine and cause the down-regulation of CD3 in activated T lymphocytes, whereas iNOS and ASE II do not induce these changes.
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Materials and Methods |
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RPMI 1640 without L-arginine or with 150 µM L-arginine (Invitrogen, Carlsbad, CA) were used to culture Jurkat cells, T cells, and peritoneal macrophages (PM). The normal physiological concentration of L-arginine in serum ranges between 50 and 150 µM. Media were supplemented with 4% FCS (Hyclone, Logan, UT), 25 mM HEPES (Gibco-Invitrogen), 4 mM L-glutamine (Biowhittaker, Walkersville, MD), and 100 U/ml penicillin/streptomycin (Invitrogen). In some experiments, 2 mM exogenous L-arginine (Sigma-Aldrich, St Louis, MO) was added to cocultures containing IL-4 + IL-13 or IFN--stimulated PM and T cells. Murine rIL-4, rIL-13, and rIFN- (R&D Systems, Minneapolis, MN) were titrated for their ability to induce the expression of iNOS or ASE I (data not shown). The optimal concentrations needed to induce ASE I or iNOS were 50 U/ml IL-4 plus 50 ng/ml IL-13 and 100 U/ml IFN-, respectively. Inhibitors of ASE and/or iNOS included N-hydroxy-L-arginine (NOHA; 100 µM), an inhibitor of iNOS and ASE (Calbiochem, San Diego, CA), the specific ASE inhibitor N-hydroxy-nor-L-arginine (Nor-NOHA, 50 µM), and the NOS inhibitor L-N-iminoethyllysine (L-NIL, 5 µg/ml). Hydrogen peroxide scavenger catalase (Roche Diagnostics, Indianapolis, IN) was used at a final concentration of 200 U/ml. Analogs of L-arginine purchased from Calbiochem were used at 1 mM and included L-monomethyl-L-arginine (L-NMMA), sym-NG,NG-dimethyl-L-arginine (L-SDMA), N-nitro-L-arginine (L-NNA), and N-nitro-L-arginine methyl ester (L-NAME). L-Lysine (Sigma-Aldrich) was used at 1 mM.
Cell lines and mice
Jurkat T cells, a CD4+ cell line (clone E6-1) (American Type Culture Collection, Manassas, VA) were used as an indicator cell line because they undergo a rapid loss of CD3 in the absence of L-arginine. Female C57BL/6 mice (6 wk old; Harlan Sprague-Dawley, Indianapolis, IN) were used to isolate PM and T cells from spleen. PM were isolated by injection of 2 ml of thioglycolate (BD Biosciences, San Jose, CA) into the peritoneal cavity of mice for 3 days. Mice were then sacrificed, and PM were isolated by washing the peritoneum with HBSS. After 2 days of culture in RPMI with 4% FBC, unattached cells were washed off, and attached cells were used in cocultures with Jurkat or anti-CD3-plus anti-CD28-stimulated mouse T cells.
T cell isolation and Ag stimulation
T cells were isolated from spleen of mice by T cell enrichment columns (R&D Systems) according to the manufacturer’s specifications. T cell purity (CD3+) ranged between 89 and 95%. The cycle of internalization and re-expression of the TCR-CD3 complex was induced by T cell stimulation with 1 µg/ml anti-CD3 plus 500 ng/ml anti-CD28 (BD PharMingen, San Diego, CA). Cells were stimulated in the absence of L-arginine and added to the cocultures 24 h after stimulation. T cell proliferation was tested by CFSE (Molecular Probes, Eugene, OR). Briefly, T cells were stained with 1 µM CFSE and then stimulated with anti-CD3 plus anti-CD28 and cocultured with PM. Fluorescence was tested by flow cytometry after 96 h.
Cocultures in Transwells (Boyden chambers)
PM were cultured in six-well plates in RPMI containing 150 µM L-arginine and stimulated with murine rIL-4 (50 U/ml) and murine rIL-13 (50 ng/ml), or murine rIFN- (100 U/ml) for 24 h. One million normal T cells stimulated with anti-CD3 plus anti-CD28 in the absence of L-arginine for 24 h were cultured in the top chamber of a Transwell system containing 0.4-µm pores (BD Biosciences, Franklin Lakes, NJ). PM were detached using trypsin-EDTA (Invitrogen), and the expression of protein and RNA for CAT-2B, ASE I, ASE II, and iNOS was tested by Western blot and Northern blot. Cytoplasmic extracts from PM were used to test ASE activity. Supernatants from stimulated PM were used to measure NO production using the Griess reagent method (Molecular Probes). In addition, the extracellular concentration of L-arginine was tested in supernatants by HPLC (Dr. G. Wu, Texas A&M University, College Station, TX) as described previously (21).
Abs and probes
CD3
and CD3 expression was detected by flow cytometry in murine T lymphocytes and Jurkat cells using anti-mouse CD3-FITC (BD PharMingen), anti-mouse CD3-PE (Santa Cruz Biotechnologies, Santa Cruz, CA), and anti-human CD3-FITC, and anti-human CD3-PE (Beckman-Coulter, Miami, FL), respectively. Rat IgG1-FITC, rat IgG2-PE (BD PharMingen) and mouse IgG1-FITC, mouse IgG1-PE (Beckman-Coulter) were used as isotype controls for mouse and human T cell detection, respectively. mAbs against iNOS (Santa Cruz Biotechnologies), ASE I (BD Transduction Laboratories, Lexington, KY), and ASE II (a gift of Dr. S. Morris, Jr.) were used for Western blots. To test the expression of CAT transporters mRNA, specific amplification products from RT-PCR of CAT-1, CAT-2A, CAT-2B, and CAT-3 were purified from agarose gels and used as probes to detect CAT expression by Northern blot. Murine full-length cDNA for GAPDH (1.6 kb; Clontech, Palo Alto, CA) was used as housekeeping control.
Flow cytometry
Flow cytometry analysis was performed as previously described (18). Briefly, 5 x 105 Jurkat cells or T lymphocytes were washed once with Dulbecco’s phosphate-buffered saline 1x (D-PBS) and resuspended in 200 µl of D-PBS containing 1 µg of anti-CD3-FITC or isotype control. Cells were incubated for 15 min at 4°C, washed with D-PBS, and resuspended in 200 µl of D-PBS containing 500 µg/ml digitonin plus 1 µg of anti-CD3 or 1 µg of isotypic control. Cells were incubated for 8 min at 4°C after which they were washed and resuspended in 400 µl of D-PBS. Fluorescence acquisition and analysis were done using a Coulter-EPICS XL flow cytometer (Beckman-Coulter) with a 488 nm argon laser. Data are expressed as mean channel fluorescence intensity.
Northern blot
Two million PM stimulated with IL-4 + IL-13 or IFN- were used for RNA extraction using lysis with TRIzol (Invitrogen, Carlsbad, CA) following the manufacturer’s specifications. Four micrograms of total RNA from each sample were electrophoresed under denaturing conditions, blotted onto Nytran membranes (Schleicher & Schuell, Keene, NH), and cross-linked by UV irradiation. Membranes were prehybridized at 42°C in ULTRAhyb buffer (Ambion, Austin, TX) and hybridized overnight with 1 x 106 cpm/ml 32P-labeled probe. Probes for detection of CAT transporters and GAPDH mRNA were labeled by random priming using a RediPrime Kit (Amersham, Little Chalfont, UK) and [
-32P]dCTP (3000 Ci/mmol; NEN Life Science Products, Boston, MA). Membranes were washed and subjected to autoradiography at -70°C using Kodak Biomax-MR films (Eastman Kodak, New Haven, CT) and intensifying screens.
Extracts and Western blot analysis
Cells were resuspended in lysis buffer (50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 1 mM NaVO4, and 0.5% Triton) containing 50 µg/ml aprotinin, 50 µg/ml leupeptin, 100 µg/ml trypsin-chymotrypsin inhibitor, and 2 mM PMSF. Lysates were centrifuged at 3000 x g for 10 min at 4°C. The expression of ASE I, ASE II, and iNOS was detected by immunoblot using PM extracts. GAPDH was used as housekeeping protein. Cytoplasmic extracts were electrophoresed in 12 or 8% Tris-glycine gels (Novex, San Diego, CA), transferred to polyvinylidene difluoride membranes, and immunoblotted with the appropriate Abs. The reactions were detected using the ECL kit (Amersham).
ASE activity assay
Cell lysates (2 µg) from PM stimulated with IL-4 + IL-13 or IFN- were tested for ASE activity by measuring the production of L-ornithine and urea. In brief, cell lysates from PM stimulated with IL-4 + IL-13 or IFN- in the presence or absence of NOHA or Nor-NOHA were added to 50 µl of Tris-HCl (50 mM; pH 7.5) containing 10 mM MnCl2. This mixture was heated at 55–60°C for 10 min to activate ASE. The hydrolysis reaction from L-arginine to L-ornithine was identified by a colorimetric assay after the addition of ninhydrin solution and incubation at 37°C for 1 h. In addition, the hydrolysis reaction from L-arginine to urea was detected with diacetyl monoxime (Sigma-Aldrich).
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Results |
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Previous reports have shown that the expression of iNOS and ASE I in murine macrophages is differentially regulated by Th1 and Th2 cytokines (6, 7, 8). PM stimulated with IL-4 + IL-13 up-regulate ASE I but not ASE II or iNOS, whereas those stimulated with IFN- increase iNOS expression and NO production, as measured by the production of nitrites, and decrease ASE I expression (Fig. 1, A and B). Kinetic studies showed that IL-4 + IL-13 stimulation increases ASE I mRNA expression in the first 4 h (data not shown), with an increase in ASE I protein starting at 12 h, whereas the mitochondrial isoform ASE II remains unchanged (Fig. 1C). Activation of PM by Th1 or Th2 cytokines also had different effects on the extracellular levels of L-arginine. PM stimulated with IL-4 + IL-13 rapidly reduced L-arginine in the tissue culture medium to levels below 15 µM in the first 12 h of culture (Fig. 1D) (p < 0.005). In contrast, PM stimulated with IFN- produced only a moderate decrease in L-arginine, similar to unstimulated PM.
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down-regulation
Jurkat cells have been shown to rapidly lose CD3 in the absence of L-arginine (19). Therefore, we tested whether L-arginine reduction by macrophages had any effect on CD3 expression in Jurkat cells and syngeneic normal T cells. PM stimulated with IL-4 + IL-13 induced a rapid decrease in the expression of CD3 in cocultured Jurkat cells (separated by 0.4-µm pore size Transwells), which was dependent on the number of PM and the time in culture (Fig. 2). This effect coincided with the increase in ASE I expression (Fig. 1C) and the reduction in the extracellular levels of L-arginine (Fig. 1D). In contrast, PM stimulated with IFN- or unstimulated PM did not alter CD3 expression (Fig. 2B). Control cultures of Jurkat cells (without PM) with IL-4 + IL-13 or IFN- did not show changes in CD3 expression (data not shown).
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. However, the absence of this amino acid prevents the normal re-expression of CD3 after the stimulation with anti-CD3 plus anti-CD28 (Fig. 3A). Similarly, the coculture of stimulated T cells with PM activated with IL-4 + IL-13 in Transwells (0.4-µm pores), also resulted in a decreased expression of CD3 chain (Fig. 3B). In contrast, T cells cocultured with resting PM or PM stimulated with IFN- displayed the normal cycle of internalization and re-expression of CD3 within 48 h, as did T cells cultured in RPMI containing 150 µM L-arginine. Resting T cells did not lose CD3 in the absence of L-arginine. A similar effect has been seen in normal human T lymphocytes where the absence of arginine impairs the re-expression of chain by blocking its synthesis.4
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. The addition of ASE inhibitors NOHA or Nor-NOHA, or the excess of L-arginine in the cultures, partially prevented the reduction of L-arginine in the tissue culture medium of IL-4 + IL-13-stimulated PM (Table I). When these inhibitors or exogenous L-arginine were added into cocultures of stimulated T cells and IL-4 + IL-13-activated PM, there was a normal cycle of internalization and re-expression of CD3 in T cells (Fig. 3C). In contrast, the iNOS inhibitor L-NIL did not prevent the prolonged CD3 down-regulation. Catalase was included in some control cultures because previously published data had shown that hydrogen peroxide released by activated macrophages and neutrophils could induce a decrease in CD3 expression (22, 23). Catalase, however, did not prevent the decrease of CD3 in T cells induced by IL-4 + IL-13-stimulated PM.
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We also tested whether the loss of CD3 induced by ASE I had any effect on T cell proliferation. As shown in Fig. 4, stimulated T cells cultured in RPMI with 150 µM L-arginine or cocultured with resting PM showed a normal proliferation as seen by the reduction (halving) of CFSE fluorescence (23.63 and 22.04%, respectively) and the appearance of additional fluorescent peaks of proliferating T cells. In contrast, stimulated T cells cultured in the absence of L-arginine or cocultured with IL-4 + IL-13-activated PM failed to proliferate. However, if the ASE inhibitor Nor-NOHA was added to the cocultures of T cells and IL-4 + IL-13-activated PM, T cells showed active proliferation (20.123%), suggesting that ASE I not only depleted L-arginine and induced a decreased CD3 expression but also impaired T cell proliferation. NOHA and Nor-NOHA did not alter ASE I up-regulation after IL-4 + IL-13 stimulation (Fig. 5A) but instead blocked intracellular ASE activity (Fig. 5, B and C).
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A significant reduction in L-arginine levels is found in the serum of patients and rodents in the first 24 h after liver transplantation and trauma and appears to be caused by a rapid increase in circulating ASE (11, 12). Therefore, the decrease in L-arginine levels induced by IL-4 + IL-13-stimulated PM could be the result of ASE I released into the tissue culture media. Interestingly, there was no significant ASE activity in the supernatants from the cultures of IL-4 + IL-13-stimulated PM (data not shown). Therefore, we tested whether L-arginine uptake by PM could in part explain the depletion of extracellular L-arginine. PM stimulated with IL-4 + IL-13 showed a rapid increase in L-[3H]arginine uptake at 12 and 24 h, which was significantly higher than PM stimulated with IFN- or nonstimulated PM (p < 0.005) (Fig. 6A). L-Arginine is transported across the cell membrane by the cationic amino acid transporter Y+ family of receptors (CAT). The increase in the L-[3H]arginine uptake and the decreased extracellular levels of L-arginine coincided with an increased CAT-2B mRNA expression in IL-4 + IL-13-stimulated PM (Fig. 6B). In contrast, there was no increase in CAT-2B mRNA in PM stimulated with IFN-. Furthermore, L-arginine analogs L-NMMA and L-SDMA and the amino acid L-lysine, which competitively inhibit CAT-2B (24), allowed a partial recovery of CD3 expression in stimulated T lymphocytes cocultured with IL-4 + IL-13-activated PM (Fig. 6C). Instead, other L-arginine analogs, not transported by CAT-2B such as L-NNA and L-NAME (24), failed to recover CD3 expression. Stimulation with IL-4 + IL-13 or IFN- did not induce significant changes in the expression of other CAT transporters including CAT-1 and CAT-2A. In addition, CAT-3, which is mostly expressed in brain (25), was not detected in resting and activated PM (data not shown).
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Discussion |
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expression, are unable to proliferate, and have a decreased cytokine production (18, 19).4 However, it was unclear how L-arginine availability might be regulated during an immune response. We therefore asked which of the metabolic pathways that use L-arginine in macrophages could modulate the availability of L-arginine and whether this would alter the expression of CD3 chain and proliferation in normal T cells.
Our initial data confirmed previous observations demonstrating the reciprocal regulation of iNOS and ASE I in macrophages by Th1 and Th2 cytokines (6, 7, 8, 9). Stimulation of PM with IFN- induced the expression of iNOS, whereas IL-4 plus IL-13 stimulation increased ASE I. In contrast, the isoform ASE II was not regulated by these cytokines. The coculture experiments demonstrated that PM expressing ASE I but not iNOS and ASE II rapidly decrease extracellular L-arginine concentrations to levels below 15 µM. A similar decrease in L-arginine levels had been previously reported by Chang et al. (29) and Que et al. (30) using an in vitro model with ASE I-transfected cell lines. In addition, the coordinated expression of CAT proteins that transport L-arginine from the extracellular microenvironment into the cell appears to play an important role in the regulation of extracellular levels of L-arginine. This particular carrier system is characterized by its high affinity for basic amino acids, its independence from Na+, and the ability of substrate on the opposite (trans) side of the membrane to increase transport activity (31). CAT genes have been recently cloned and designated CAT-1, CAT-2A, CAT-2B, and CAT-3. Whereas CAT-1, CAT-2B, and CAT-3 are high affinity (KM 100 µmol/L) transporters for L-arginine, CAT-2A is an alternatively spliced variant of CAT-2B that possesses low affinity for L-arginine (KM 1 to 2 mmol/L) (32). In accordance with Louis et al. (10), our data show that PM stimulated with IL-4 + IL-13 up-regulate the expression of CAT-2B, displaying a similar kinetics to ASE I (Figs. 1C and 6B). Furthermore, inhibitors of CAT-2B, L-NMMA, L-lysine, and L-SDMA partially allowed the re-expression of CD3 in T cells cocultured with IL-4 + IL-13-activated PM (Fig. 6C), suggesting a possible role of CAT-2B in the regulation of extracellular L-arginine and CD3 expression. Although L-NMMA can also inhibit iNOS activity, we have used L-SDMA and L-lysine, which do not inhibit iNOS at the concentration used (1 mM) (24). In addition, iNOS inhibitors L-NIL, L-NNA, and L-NAME, which are not transported by CAT-2B failed to recover CD3 expression after T cell stimulation. L-NAME has also been reported to be an ASE inhibitor in vitro and in vivo (33); however, we did not observe an inhibitory effect on ASE activity assay at the concentrations used (data not shown). Stimulation of PM with IL-4 + IL-13 did not induce major changes in the expression of CAT-1 and CAT-2A in PM (data not shown). In addition, CAT-3, which is mostly expressed in the brain (25), was not detected in PM-unstimulated or activated with IL-4 + IL-13 or IFN-.
The molecular mechanisms involved in the control of gene expression by amino acid deprivation have been extensively studied in yeast. However, the effect of starvation of different amino acids in mammalian cells is less clear (34). Recent publications have suggested a close correlation between the availability of certain amino acids and the immune response. Munn et al. (35) described that tryptophan metabolism by macrophages producing indoleamine 2,3-dioxygenase inhibits T cell proliferation (35). This group also suggested that tryptophan starvation induced cell cycle arrest in normal T lymphocytes and sensitized activated T cells to apoptosis before cell division (36). The absence of other amino acid such as leucine have been associated with an increase in the synthesis and stability of mRNA for CHOP (37). This gene encodes a transcription factor that interacts with CCAAT/enhancer-binding proteins family, which in turn may inhibit the normal proliferation of cells (38). Therefore, essential amino acids appear to induce changes in T cells that ultimately inhibit their normal proliferation. Our data show that metabolism of the nonessential amino acid L-arginine can also control T cell function through the modulation of CD3 expression. In Jurkat cells, the decrease of CD3 appears to be caused by decreased CD3 mRNA stability (19), associated with de novo synthesis of a protein that releases a ribonucleoprotein complex bound to the 3'-untranslated region of CD3 mRNA (P. Rodriguez, unpublished data). In normal human T cells, L-arginine starvation appears to alter CD3 re-expression by specific decrease in CD3 translation.4
How low must L-arginine levels be to alter CD3 chain expression and T cell function, and can these levels occur in vivo? In vitro data show that concentrations below 40 µM cause the rapid decrease of CD3 chain in Jurkat cells and impair the re-expression of this chain in stimulated T cells (P. Rodriguez and A. Zea, unpublished results). Consistent with this, rodents and patients with trauma or undergoing liver transplantation have a rapid decrease in circulating L-arginine levels to concentrations below 40 µM (11, 14), paralleled by poor T cell function (14). In addition, Ichihara et al. (39) recently demonstrated the trauma patients may lose CD3, although they did not associate this change with L-arginine reduction. Furthermore, transgenic mice that overexpress ASE I in their enterocytes have a selective depletion of L-arginine in serum, concomitant with a poor thymic development (40). However, de Jonge et al. (41) showed that the major dysfunction in these transgenic mice occurs in B cell maturation rather than T cell development. This observation is not contrary to our data because, as shown in Fig. 3, L-arginine depletion affects only activated T cells and not resting T lymphocytes. It is likely that most T cells in these mice are in a resting state and therefore are not susceptible to the effects of L-arginine depletion in vivo.
ASE I is also produced by several tumors including gastric, colon, breast, and lung cancers (42, 43, 44, 45, 46). Most reports have associated the increased ASE expression by tumor cells with the need to produce polyamines to sustain the rapid malignant cell proliferation (29). Our data also suggest that the increase in ASE I expression may have as a secondary effect the local (or systemic) reduction of L-arginine levels resulting in the decreased expression of CD3 and inhibition of proliferation in activated T cells, allowing tumors to escape the immune response. A recent report by Bronte et al. (47) suggested that ASE I produced by myeloid suppressor cells isolated from mice bearing the C26-GM tumor impaired T cell function. Our preliminary data in murine tumor models suggest that ASE I production in tumor-bearing mice can modulate CD3 expression in vivo (P. Rodriguez, manuscript in preparation). It is possible that L-arginine levels may be decreased locally in certain tumors, at sites where ASE I is being produced by tumor cells or infiltrating APC. This might explain the observation that CD3 is more profoundly decreased in tumor-infiltrating T cells than in circulating T lymphocytes (48).
ASE I production in cancer may not be the only mechanism by which CD3 is decreased and T cell function impaired. Previous studies have also demonstrated that Fas-Fas ligand interactions and the production of hydrogen peroxide by macrophages or neutrophils may cause these changes (22, 23). Therefore, additional studies in tumor models in vivo and in cancer patients will help elucidate the contribution of these different mechanisms in the induction of T cell anergy and CD3 down-regulation in cancer and determine their possible impact on immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence to Dr. Augusto C. Ochoa, Stanley S. Scott Cancer Center, Louisiana State University, Health Sciences Center, 533 Bolivar Street, 455, New Orleans, LA 70112. E-mail address: aochoa{at}lsuhsc.edu
3 Abbreviations used in this paper: iNOS, inducible nitric oxide; ASE I, arginase I; ASE II, arginase II; CAT, cationic amino acid transporters; PM, peritoneal macrophages; NOHA, N-hydroxy-L-arginine; nor-NOHA, N-hydroxy-nor-L-arginine; L-NIL, L-N-iminoethyllysine; L-NMMA, L-monomethyl-L-arginine; L-SDMA, sym-NG,NG-dimethyl-L-arginine; L-NNA, N-nitro-L-arginine; L-NAME, N-nitro-L-arginine methyl ester.
4 A. Zea. L-Arginine modulates CD3 expression and function in activated T cells. Submitted for publication.
Received for publication January 13, 2003. Accepted for publication May 27, 2003.
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M. Munder, H. Schneider, C. Luckner, T. Giese, C.-D. Langhans, J. M. Fuentes, P. Kropf, I. Mueller, A. Kolb, M. Modolell, et al. Suppression of T-cell functions by human granulocyte arginase Blood, September 1, 2006; 108(5): 1627 - 1634. [Abstract] [Full Text] [PDF]
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K. C. McKenna and J. A. Kapp Accumulation of Immunosuppressive CD11b+ Myeloid Cells Correlates with the Failure to Prevent Tumor Growth in the Anterior Chamber of the Eye J. Immunol., August 1, 2006; 177(3): 1599 - 1608. [Abstract] [Full Text] [PDF]
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I. Kryczek, L. Zou, P. Rodriguez, G. Zhu, S. Wei, P. Mottram, M. Brumlik, P. Cheng, T. Curiel, L. Myers, et al. B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J. Exp. Med., April 17, 2006; 203(4): 871 - 881. [Abstract] [Full Text] [PDF]
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V. P. Makarenkova, V. Bansal, B. M. Matta, L. A. Perez, and J. B. Ochoa CD11b+/Gr-1+ Myeloid Suppressor Cells Cause T Cell Dysfunction after Traumatic Stress J. Immunol., February 15, 2006; 176(4): 2085 - 2094. [Abstract] [Full Text] [PDF]
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B. Huang, P.-Y. Pan, Q. Li, A. I. Sato, D. E. Levy, J. Bromberg, C. M. Divino, and S.-H. Chen Gr-1+CD115+ Immature Myeloid Suppressor Cells Mediate the Development of Tumor-Induced T Regulatory Cells and T-Cell Anergy in Tumor-Bearing Host Cancer Res., January 15, 2006; 66(2): 1123 - 1131. [Abstract] [Full Text] [PDF]
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P. Sinha, V. K. Clements, and S. Ostrand-Rosenberg Interleukin-13-regulated M2 Macrophages in Combination with Myeloid Suppressor Cells Block Immune Surveillance against Metastasis Cancer Res., December 15, 2005; 65(24): 11743 - 11751. [Abstract] [Full Text] [PDF]
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S. Rutella, F. Zavala, S. Danese, H. Kared, and G. Leone Granulocyte Colony-Stimulating Factor: A Novel Mediator of T Cell Tolerance J. Immunol., December 1, 2005; 175(11): 7085 - 7091. [Abstract] [Full Text] [PDF]
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P. C. Rodriguez, C. P. Hernandez, D. Quiceno, S. M. Dubinett, J. Zabaleta, J. B. Ochoa, J. Gilbert, and A. C. Ochoa Arginase I in myeloid suppressor cells is induced by COX-2 in lung carcinoma J. Exp. Med., October 3, 2005; 202(7): 931 - 939. [Abstract] [Full Text] [PDF]
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S. Kusmartsev, S. Nagaraj, and D. I. Gabrilovich Tumor-Associated CD8+ T Cell Tolerance Induced by Bone Marrow-Derived Immature Myeloid Cells J. Immunol., October 1, 2005; 175(7): 4583 - 4592. [Abstract] [Full Text] [PDF]
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 L. Di Costanzo, G. Sabio, A. Mora, P. C. Rodriguez, A. C. Ochoa, F. Centeno, and D. W. Christianson Crystal structure of human arginase I at 1.29-A resolution and exploration of inhibition in the immune response PNAS, September 13, 2005; 102(37): 13058 - 13063. [Abstract] [Full Text] [PDF]
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A. H. Zea, P. C. Rodriguez, M. B. Atkins, C. Hernandez, S. Signoretti, J. Zabaleta, D. McDermott, D. Quiceno, A. Youmans, A. O'Neill, et al. Arginase-Producing Myeloid Suppressor Cells in Renal Cell Carcinoma Patients: A Mechanism of Tumor Evasion Cancer Res., April 15, 2005; 65(8): 3044 - 3048. [Abstract] [Full Text] [PDF]
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C. M. Cham and T. F. Gajewski Glucose Availability Regulates IFN-{gamma} Production and p70S6 Kinase Activation in CD8+ Effector T Cells J. Immunol., April 15, 2005; 174(8): 4670 - 4677. [Abstract] [Full Text] [PDF]
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S. Kusmartsev and D. I. Gabrilovich STAT1 Signaling Regulates Tumor-Associated Macrophage-Mediated T Cell Deletion J. Immunol., April 15, 2005; 174(8): 4880 - 4891. [Abstract] [Full Text] [PDF]
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C. De Santo, P. Serafini, I. Marigo, L. Dolcetti, M. Bolla, P. Del Soldato, C. Melani, C. Guiducci, M. P. Colombo, M. Iezzi, et al. Nitroaspirin corrects immune dysfunction in tumor-bearing hosts and promotes tumor eradication by cancer vaccination PNAS, March 15, 2005; 102(11): 4185 - 4190. [Abstract] [Full Text] [PDF]
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M. Munder, F. Mollinedo, J. Calafat, J. Canchado, C. Gil-Lamaignere, J. M. Fuentes, C. Luckner, G. Doschko, G. Soler, K. Eichmann, et al. Arginase I is constitutively expressed in human granulocytes and participates in fungicidal activity Blood, March 15, 2005; 105(6): 2549 - 2556. [Abstract] [Full Text] [PDF]
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P. Sinha, V. K. Clements, and S. Ostrand-Rosenberg Reduction of Myeloid-Derived Suppressor Cells and Induction of M1 Macrophages Facilitate the Rejection of Established Metastatic Disease J. Immunol., January 15, 2005; 174(2): 636 - 645. [Abstract] [Full Text] [PDF]
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 V. Bansal, K. M. Syres, V. Makarenkova, R. Brannon, B. Matta, B. G. Harbrecht, and J. B. Ochoa Interactions Between Fatty Acids and Arginine Metabolism: Implications for the Design of Immune-Enhancing Diets JPEN J Parenter Enteral Nutr, January 1, 2005; 29(1_suppl): S75 - S80. [Abstract] [Full Text] [PDF]
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V. Bansal, P. Rodriguez, G. Wu, D. C. Eichler, J. Zabaleta, F. Taheri, and J. B. Ochoa Citrulline Can Preserve Proliferation and Prevent the Loss of CD3 {zeta} Chain Under Conditions of Low Arginine JPEN J Parenter Enteral Nutr, November 1, 2004; 28(6): 423 - 430. [Abstract] [Full Text] [PDF]
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P. C. Rodriguez, D. G. Quiceno, J. Zabaleta, B. Ortiz, A. H. Zea, M. B. Piazuelo, A. Delgado, P. Correa, J. Brayer, E. M. Sotomayor, et al. Arginase I Production in the Tumor Microenvironment by Mature Myeloid Cells Inhibits T-Cell Receptor Expression and Antigen-Specific T-Cell Responses Cancer Res., August 15, 2004; 64(16): 5839 - 5849. [Abstract] [Full Text] [PDF]
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D. E. Spaner Amplifying cancer vaccine responses by modifying pathogenic gene programs in tumor cells J. Leukoc. Biol., August 1, 2004; 76(2): 338 - 351. [Abstract] [Full Text] [PDF]
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J. Zabaleta, D. J. McGee, A. H. Zea, C. P. Hernandez, P. C. Rodriguez, R. A. Sierra, P. Correa, and A. C. Ochoa Helicobacter pylori Arginase Inhibits T Cell Proliferation and Reduces the Expression of the TCR {zeta}-Chain (CD3{zeta}) J. Immunol., July 1, 2004; 173(1): 586 - 593. [Abstract] [Full Text] [PDF]
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