Regulation of arginase I activity and expression by both PD-1 and CTLA-4 on the myeloid-derived suppressor cells

Abstract

An elevated number of Gr-1(+)CD11b(+) myeloid-derived suppression cells (MDSCs) has been described in mice and human bearing tumor and associated with immune suppression. Arginase I production by MDSCs in the tumor environment may be a central mechanism for immunosuppression and tumor evasion. In this study and before, we found that Gr-1(+)CD11b(+) MDSCs from ascites and spleen of mice bearing ovarian 18D carcinoma express a high level of PD-1, CTLA-4, B7-H1 and CD80 while other co-stimulatory molecules, namely CD40, B7-DC and CD86 are not detected. Further studies showed that PD-1 and CTLA-4 on the Gr-1(+)CD11b(+) MDSCs regulated the activity and expression of arginase I. The blockage and silencing of PD-1, CTLA-4 or both PD-1 and CTLA4 molecules could significantly reduce arginase I activity and expression induced with tumor-associated factor. Similar results were also observed while their ligands B7-H1 and/or CD80 were blocked or silenced. Furthermore, CD80 deficiency also decreased the arginase I expression and activity. Antibody blockade or silencing of PD-1, CTLA-4 or both reduced the suppressive potential of PD-1+CTLA-4+MDSCs. Blockade of PD-1, CTLA-4 or both also slowed tumor growth and improved the survival rate of tumor-bearing mice. Thus, there may exist a coinhibitory and costimulatory molecules-based immuno-regulating net among MDSCs.

Fig. 1

PD1+CTLA-4+MDSCs derived from ascites of mice bearing ovarian 1D8 carcinoma express arginase I. a Expression of PD1 and CTLA-4 on the surface of murine carcinoma-associated Gr1+MDSCs cells. A1, A2 MDSCs from ascites of mice bearing ovarian 1D8 carcinoma. A3, A4 Gr1+cells from naive mouse splenic cells. b Isolated PD1+CTLA-4+Gr1⁺CD11b⁺MDSCs from ascites of mice bearing ovarian carcinoma (Giemsa staining). PD-1+CTLA-4+MDSCs were sorted from the ascites of mice with ovarian 1D8 carcinoma using PE-labeled anti-Gr1 and FITC-labeled anti-CD11b using flow cytometry and the sorted cells were stained using Giemsa stain kit (Thermo Fisher Scientific, USA) according to the manufacture’s procedure. c Murine ovarian carcinoma supernatants promoted the arginase I activity and expression by PD-1+CTLA-4+MDSCs. Arginase activity (C1) and transcription level (C2) of arginase I in PD-1+CTLA-4+MDSCs upon exposing to different concentration of murine ovarian carcinoma supernatant and mrIL-13 (positive control). Cell contr. CD11b+Gr1+ cells from naive mouse splenic cells, MDSCs PD1+CTLA-4+MDSCs from the ascites of mice bearing ovarian carcinoma, Tu. Sup. tumor supernatants from ovarian 1D8 carcinoma after culturing for 24 h

Fig. 2

Blockade of PD-1, CTLA-4 or their ligands decreases arginase I activity and expression by ovarian carcinoma-associated MDSCs. a Blockade of PD-1, CTLA-4 or both reduced arginase I activity and expression. Anti-PD-1, CTLA-4 or both antibodies were, respectively, added into the culture of PD-1+CTLA4+MDSCs in the conditioned medium and then their arginase I activity and transcription level were analyzed after 24 h. b Blockade of B7-H1 and CD80 decreased the arginase I activity and transcription. Similarly, Anti-CD80, B7-H1 or both antibodies were added into the culture of PD-1+CTLA4+MDSCs in the conditioned medium and then their arginase I activity and transcription level were analyzed after 24 h. The isolated PD-1+CTLA4+MDSCs were plated at 1 × 10⁶ cells/well in 24-well tissue culture plates and stimulated with equal amount of tumor supernatants (25% V/V). Following stimulation, cells were washed with PBS and lysed in lysis buffer described in “Materials and methods”. Tu. Sup. Tumor supernatants from ovarian 1D8 carcinoma after culturing for 24 h

Fig. 3

Knockdown of PD-1, CTLA-4 or both decreases arginase I activity and expression. a PD-1 and CTLA4 siRNA reduced the level of PD-1 and CTLA-4, respectively, but not that of GAPDH transcript expression in the transfected ovarian carcinoma-associated PD-1+CTLA-4+MDSCs, as assessed by RT-PCR. b PD-1, CTLA4 or both siRNA but not non-specific siRNA, reduced the level of surface PD-1 and CTLA4 expression in transfected ovarian carcinoma-associated PD-1+CTLA-4+MDSCs, as assessed by flow cytometric analysis, respectively. B1, B2 Expression of PD-1 and CTLA-4 on the siRNA transfected ovarian carcinoma-associated PD-1+CTLA-4+MDSCs, respectively. Black line Isotype control, dark black line expression of PD-1 (B1) and CTLA-4 (B2) on ovarian carcinoma-associated PD-1+CTLA-4+MDSCs transfected with PD-1 and CTLA-4 specific siRNA, respectively, dotted line expression of PD-1 (B1) and CTLA-4 (B2) on ovarian carcinoma-associated PD-1+CTLA-4+MDSCs transfected by control mock siRNA. c Knockdown of PD-1, CTLA-4 or both reduced arginase I activity (C1) and level of transcription (C2). PD-1, CTLA-4 or both siRNA transfected PD1+CTLA-4+ MDSCs were cultured in the conditioned medium for 24 h and then lysed for analysis of arginase I activity. The total RNA was extracted for transcription assay according to the described protocol in “Materials and methods”. T. Sup. Conditioned medium containing 25% murine ovarian carcinoma supernatant (V/V), PD1siRNA, CTLA4siRNA and PD1+CTLA4siRNA, respectively, were PD1 siRNA, CTLA4 siRNA or both transfected PD1+CTLA4+MDSCs, Mock siRNA control siRNA transfected PD1+CTLA4+MDSCs

Fig. 4

CD80 deficiency decreases arginase I activity and expression by ovarian carcinoma-associated PD1+CTLA-4+ MDSCs. a The activity and expression of fresh CD11b+Gr1+ cells from wild type (WT) or CD80 deficiency (CD80−/−) spleen of mouse with (Tumor) or without ovarian carcinoma (Contr.). Activity (A1) and transcriptional level (A2) of arginase I were analyzed in WT mice or CD80−/− mice by the amount of urea released from cell extract and RT-PCR. b CD80−/− reduced the arginase activity (B1) and transcription (B2) induced using tumor supernatant and mrIL13. Contr. PD1+CTLA-4+MDSCs cells alone, Tu.Sup. PD1+CTLA-4+ MDSCs were cultured in conditioned medium containing 25% tumor supernatant, and mrIL13 PD1+CTLA-4+ MDSCs were cultured in 10 ng/ml IL13 medium

Fig. 5

Antibody blockade or siRNA silencing of PD-1 and CTLA-4 reduces the suppressive potential of PD-1+CTLA-4+ MDSCs. a Effect of pretreatment of PD-1+CTLA-4+ MDSCs using neutralizing antibody PD-1, CTLA-4 or both on the generation of IFNγ by VLP-specific splenocytes (VLP.SC). Supernatants of cultures were collected after 24 h and IFNγ was assayed by capture ELISA. Isotypic contr. Control antibody. b PD-1, CTLA-4 or both siRNA transfected MDSCs had the reduced suppression on VLP-induced generation of IFN-γ in VLP-specific splenocyte culture. PD-1+CTLA-4+ MDSCs were sorted from the ascites of mice with ovarian 1D8 carcinoma and then transfected using PD-1, CTLA-4 or both siRNA or mock siRNA and then added into VLP-specific splenocyte culture. PD-1+, CTLA-4+ or CTLA-4 and PD1 siRNA represented PD-1+CTLA-4+MDSCs, which were transfected with PD-1, CTLA-4 or both siRNA, respectively, mock siRNA PD-1+CTLA-4+ MDSCs transfected with control siRNA

Fig. 6

Blockade of PD-1 or CTLA-4 slows tumor growth and improves the survival rate of mice bearing tumor. a In vivo injection of neutralizing antibody to PD-1, CTLA-4 or both, significantly slowed and retarded tumor growth. b In vivo injection of neutralizing antibody to PD-1, CTLA-4 or both, significantly prolonged the survival rate. Mice (6/group) were injected using 1D8 cells (s.c. with 1 × 10⁷) and i.p. with 250 μg of control antibody or antibody specific for PD-1 and CTLA-4 or both on days −6, −4, −2 and +1 as previously described [20]

Fig. 7

Schematic illustration of a mechanism by which PD-1 and B7-H1 on MDSCs subvert antitumor immunity via regulating the activity and expression of arginase I. MDSCs were induced by tumor-derived factors such as VEGF, TGF-β, IL-6 and PEG2 etc. These tumor-associated MDSCs express higher level of PD-1, CTLA4 and also B7-H1 and CD80, which are ligands of PD-1 and CTLA-4, respectively. Both PD-1 and CTLA4 on the surface of MDSCs may upregulate the activity and expression of arginase I via binding with their ligands expressed on the MDSCs. These upregulated arginase I can inhibit the activation of CD4+ and CD8+ T cells to regulate immune responses. BMC Bone marrow cells