Expression of Cationic Amino Acid Transporter 2 Is Required for Myeloid-Derived Suppressor Cell-Mediated Control of T Cell Immunity - PubMed (original) (raw)

Expression of Cationic Amino Acid Transporter 2 Is Required for Myeloid-Derived Suppressor Cell-Mediated Control of T Cell Immunity

Cansu Cimen Bozkus et al. J Immunol. 2015.

Abstract

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells that expand during benign and cancer-associated inflammation and are characterized by their ability to inhibit T cell immunity. Increased metabolism of l-Arginine (l-Arg), through the enzymes arginase 1 and NO synthase 2 (NOS2), is well documented as a major MDSC suppressive mechanism. Therefore, we hypothesized that restricting MDSC uptake of l-Arg is a critical control point to modulate their suppressor activity. Using murine models of prostate-specific inflammation and cancer, we have identified the mechanisms by which extracellular l-Arg is transported into MDSCs. We have shown that MDSCs recruited to localized inflammation and tumor sites upregulate cationic amino acid transporter 2 (Cat2), coordinately with Arg1 and Nos2. Cat2 expression is not induced in MDSCs in peripheral organs. CAT2 contributes to the transport of l-Arg in MDSCs and is an important regulator of MDSC suppressive function. MDSCs that lack CAT2 have significantly reduced suppressive ability ex vivo and display impaired capacity for regulating T cell responses in vivo as evidenced by increased T cell expansion and decreased tumor growth in Cat2(-/-) mice. The abrogation of suppressive function is due to low intracellular l-Arg levels, which leads to the impaired ability of NOS2 to catalyze l-Arg-dependent metabolic processes. Together, these findings demonstrate that CAT2 modulates MDSC function. In the absence of CAT2, MDSCs display diminished capacity for controlling T cell immunity in prostate inflammation and cancer models, where the loss of CAT2 results in enhanced antitumor activity.

Copyright © 2015 by The American Association of Immunologists, Inc.

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Figures

Figure 1

Figure 1

Cat2 is coordinately induced with Arg1 and Nos2 in MDSC. CD11b+Gr-1+ cells were isolated from bone marrow of naïve wild type mice and cultured with or without GM-CSF, IL-13 and IFN- γ. mRNA was isolated after 3 days of culture (A) or every 24 hour during culture (n=4 mice, *** p=0.0005, ** p=0.0027, * p=0.0211, *** p=0.0009) (B). Arg1, Nos2 and Cat2 gene expression were analyzed by qPCR. Data are representative of at least 3 independent experiments. For all experiments errors bars indicate ±SEM.

Figure 2

Figure 2

Cat2 is induced in functionally active MDSC. CD11b+Gr-1+ and CD11b+Ly6ChighLy6G- (M-MDSC), CD11b+Ly6ClowLy6G+ (G-MDSC) cells were isolated by FACS from ascites and spleen of RM1 i.p. tumor bearing Cat2+/+ (WT) mice 6 days after tumor implantation or from the spleens of naïve mice. The percentage of CD11b+Gr-1+ cells were demonstrated under SSC/FSC gate (n=4, *** p=0.0002) (A). Percentages of CD11b+Ly6ChighLy6G- (M-MDSC) and CD11b+Ly6ClowLy6G+ (G-MDSC) were demonstrated under CD11b+ gate. Data are pooled from 6 independent experiments (C). mRNA was freshly isolated and analyzed by qPCR for Arg1, Nos2 and Cat2 expression (B and D). Data are representative of at least 3 independent experiments. For all experiments errors bars indicate ±SEM.

Figure 3

Figure 3

MDSC suppressive capacity is reduced in the absence of CAT2. CD11b+Gr-1+ cells were isolated from bone marrow of naïve mice and cultured with GM-CSF, IL-13 and IFN- γ. After 3 days cells were harvested and co-cultured with naïve OTI in the presence of SIINFEKL at indicated ratios for another 3 days. BrdU was added 6 hours before harvest. OTI proliferation was evaluated by measuring BrdU incorporation. Data are representative of 3 independent experiments (n=3-4/group, ** p=0.0057, *** p=0.0001, **** p<0.0001) (A). CD11b+Gr-1+ cells were isolated from tumor site and spleens of RM1 bearing WT and KO mice (n=3-5/group, pooled) 6 days after tumor implantation and co-cultured with preactivated OTIs for 18 hours. BrdU was added 6 hours before harvest. OTI proliferation was evaluated by measuring BrdU incorporation. Data are pooled from 5 and 3 independent experiments for tumor and for spleen, respectively (*** p=0.0002, **** p<0.0001) (B). CD11b+Gr-1+ cells from the ascites of RM1 i.p. tumor bearing Cat2+/+ (WT) and Cat2-/- (KO) mice (n=5/group, pooled) were co-cultured with naive OTI cells with SIINFEKL for 48 hours. BrdU and protein transport inhibitor were added 6 hours before harvest. OTI proliferation was evaluated by measuring BrdU incorporation (C) IFN-γ levels in OT-I cells were determined by flow cytometry for intracellular IFN-γ staining (D). Data are representative of 5 independent experiments. M-MDSC and G-MDSC were isolated by FACS from the ascites of RM1 i.p. tumor bearing WT and KO mice (n=3-5/group, pooled) 6 days after tumor implantation (E) and co-cultured with naive OTI cells with SIINFEKL for 48-72 hours. BrdU was added 6 hours before harvest. OTI proliferation was evaluated by measuring BrdU incorporation. Data are pooled from 5 independent experiments (* p=0.0312 at 0.5:1 and 0.0139 at 0.25:1) (F). For all experiments errors bars indicate ±SEM.

Figure 4

Figure 4

CAT2 mediates L-Arginine transportation in MDSC. Thioglycollate elicited peritoneal macrophages from Cat2+/+ (WT) and Cat2-/- (KO) mice (n=5/group) were activated with LPS and IFN-γ for 18h. L-Arginine transport was measured by using 6μCi/ml L-3[H]-Arg in Na+ containing buffer. For _N-_ethyl maleimide (NEM), cells were pretreated with 5 mM NEM for 5 min and washed with transport buffer before measuring uptake. Data are representative of 3 independent experiments. (**** p<0.0001) (A). CD11b+Gr-1+ cells were isolated from ascites of RM1 tumor bearing WT and KO mice and cultured over night with GM-CSF, IL-13 and IFN- γ. Total L-Arginine transport was measured by using 6μCi/ml L-3[H]-Arg in Na+ containing buffer. Data are pooled from 3 independent experiments. (n=13/group, * p=0.0184) (B) Data correspond to reduction of total L-3[H]-Arg transport when y+ system was blocked using 5 mM NEM in WT and KO MDSC. Data are pooled from 3 independent experiments (n=10/group, ** p=0.0016) (C). Data correspond to reduction of total L-3[H]-Arg transport when y+L system was blocked due to the presence of 5 mM L-Leu in transport media. Data are pooled from 4 independent experiments (n=13/group) (D). All error bars indicate ±SEM.

Figure 5

Figure 5

Cat2-/- MDSC have reduced NO production. CD11b+Gr-1+ MDSC (A) and CD11b+LyChighLy6G- (M-MDSC) and CD11b+LyClowLy6G+ (G-MDSC) subsets (B) were isolated from tumor site of RM1 bearing Cat2+/+ (WT) and Cat2-/- (KO) mice. Cells were cultured in the presence and absence of LPS and IFN-γ for 24 hours. Nitrite formation upon stimulation with LPS and IFN-γ was measured in a Griess Assay. Data are pooled from 2 independent experiments (n=5-9 mice/group for MDSC. ** p=0.0065, n=4/group, pooled for subsets. * p=0.0426). Thioglycollate elicited peritoneal macrophages from Cat2+/+ (WT) and Cat2-/- (KO) mice (n=4/group) were activated with LPS and IFN-γ for 18h. Nitrite formation was measured in a Griess Assay. Data are representative of at least 2 independent experiments (**** p<0.0001) (C). Cells from the ascites of WT and KO RM1 mice were cultured with LPS and IFN-γ overnight prior to intracellular staining of NOS2 for flow cytometry. Percentages of NOS2+ cells were demonstrated under CD11b+LyChighLy6G- (M-MDSC) and CD11b+LyClowLy6G+ (G-MDSC) gates (n=4-5/group). Data are representative of 3 independently performed experiments (D). Quantichrome Urea Assay Kit that measures urea and citrulline was utilized to measure arginase activity in CD11b+Gr-1+ cells freshly isolated from the bone marrow and ascites of WT and KO RM1 mice (n=4/group) with or without supplementing L-Arg. Data are representative of 3 independent experiments (E). Cells freshly isolated from the ascites of WT and KO RM1 mice were stained with anti-ARG1 antibody. ARG1 expression was detected by flow cytometry. Mean fluorescent intensity (MFI) of ARG1 expression was reported under CD11b+Gr-1+ gate (n=4/group). Data are representative of 3 independently performed experiments (F). WT and KO MDSC were used in a 48-hour suppression assay at 2:1 (MDSC:OTI) ratio in the presence or absence of NOS inhibitor, LNMMA (0.5 mM), ARG inhibitor, nor-NOHA (0.5 mM). BrdU was added 6 hours before harvest. OTI proliferation was evaluated by measuring BrdU incorporation. Data were pooled from 2 independent experiments (H). For all experiments errors bars indicate ±SEM.

Figure 6

Figure 6

Cat2-/- MDSC have increased ROS formation. Cells from ascites of i.p. RM1 bearing WT and KO mice were analyzed for ROS levels. DCFDA signal was analyzed under the gates for CD11b+Gr-1+ (n=4/group)(A) and CD11b+LyChighLy6G- (M-MDSC) and CD11b+LyClowLy6G+ (G-MDSC) subsets (n=8/group, data are pooled from 2 independent experiments) (B). Signal intensity was represented as MFI. CD11b+Gr-1+ cells were isolated by FACS from the ascites of RM1 i.p. tumor bearing Cat2+/+ (WT) and Cat2-/- (KO) mice (n=5/group, pooled)6 days after tumor implantation and suppression assay was performed at 2:1 (MDSC:OTI) ratio in the presence of differing concentrations of ROS inhibitor, NAC (C). WT MDSC were used for suppression assay at indicated MDSC:OTI ratios with or without 10 mM NAC (D). Data are representative of at least 2 independent experiments. All suppression assays were performed by co-culturing MDSC with preactivated OTI cells for 48 hours. BrdU was added 6 hours before harvest. OTI proliferation was evaluated by measuring BrdU incorporation. For all experiments errors bars indicate ±SEM.

Figure 7

Figure 7

CAT2-/- MDSC display diminished capacity for controlling T-cell immunity in vivo. Cat2-/- mice were injected intradermally with 3× 106 EG7 lymphoma cells alone, 3× 106 EG7 with 104 activated OTIs, 3× 106 EG7 with 104 activated OTIs and 104 Cat2+/+ or Cat2-/- CD11b+Gr-1+ cells. Data are pooled from 2 independent experiments (n=5-6 mice/group). p values for the comparison of WT or KO MDSC received groups are 0.0001, 0.0003 and 0.0033 on Day 5, 8 and 11, respectively (A). Cat2+/+ and Cat2-/- mice were injected intradermally with 3× 106 EG7 lymphoma cells. 7 days after tumor inoculation, mice were intravenously injected with 106 activated OTI cells (n=6/group). Tumor growth was monitored. p values for the comparison of WT and KO mice that received OTI are 0.0072 and 0.0449 on Day 10 and 13, respectively. Tumor size was calculated as (W2× L) /2 [mm3]. Errors bars indicate ±SEM.

Figure 8

Figure 8

CAT2 modulates MDSC regulatory functions in an acute prostate inflammation model.

P

rostate

O

valbumin

E

xpressing

T

ransgenic (POET-3) mice (n=3) were injected with 5× 106 activated OTI cells to induce prostate inflammation. 5 days later spleen and prostate CD11b+Gr-1+ cells (under CD45+ gate) (A) were sorted by FACS. mRNA was freshly isolated from sorted cells and analyzed by qPCR for Arg1, Nos2 and Cat2 expression (B). Data are representative of at least 3 independent experiments (** p=0.038, **** p<0.0001). MDSC were isolated from inflamed prostates of Cat2+/+ and Cat2-/- POET-3 mice. (n=3/group, pooled) and co-cultured with preactivated OTI cells for 48 hours. BrdU was added 6 hours before harvest. OTI proliferation was evaluated by measuring BrdU incorporation (C). 5× 106 activated OTI cells were intravenously injected into POET-3 mice to induce inflammation. 2 days later, mice were adoptively transferred (i.p.) with 4× 106 Cat2+/+ and Cat2-/- bone marrow cells that were cultured 5 days with GM-CSF and IL-6.Cat2+/+ POET-3 received Cat2+/+ cells and Cat2-/- POET-3 received Cat2-/- or Cat2+/+ cells. Percentage of Thy1.1+ (OTI) cells in 5-day inflamed POET-3 prostate (D) and spleen (E) are represented under CD45+CD8+ gate. Each individual datum point represents a single mouse (***p<0.001, *p<0.05). Prostate (F) and spleen (G) cells were cultured with SIINFEKL and protein transport inhibitor for 8 hours and IFN-γ was detected by intracellular staining for flow cytometry. Data are representative for at least 2 independent experiments. Errors bars indicate ±SEM.

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