Monocytic MDSCs express myeloid precursor markers. Studies have shown that they possess a phenotype containing elements of both M1 and M2 macrophages. However, upon entering the tumor environment, MDSCs appear to polarize toward an M2 phenotype due to the presence of high amounts of M2 cytokines such as IL-4 and IL13 (Gordon and Martinez
; Umemura et al., 2008
). The mechanism behind MDSC differentiation and the signals that control this commitment and biological function in tumor-bearing hosts are not well understood. In this study, we found that MDSCs are immature macrophage precursors, which have the potential to differentiate into both the M1 and M2 phenotypes. This switch can be regulated through PIR-B signaling as the cells migrate from the bone marrow into peripheral organs.
We demonstrated that MDSCs from Lilrb3−/−
mice exhibit an M1, rather than M2, phenotype in the tumor environment. The M1 propensity of Lilrb3−/−
MDSCs is intrinsic and not affected by cytokine profile in the tumor microenvironment since the tumors from Lilrb3−/−
mice had higher amounts of IL-4 and IL-13 than those from wild type mice (data not shown). Splenic MDSCs from Lilrb3−/−
mice also exhibit an M1 phenotype and diminished immune suppression, as well as Treg cell activating ability. This phenomenon was not observed in bone marrow-derived MDSCs, indicating that in order for this phenotypic switch to occur, MDSCs must receive specific signals leading to a state of differentiation and maturity, which is not attainable within the bone marrow environment. We found that Lilrb3−/−
MDSCs expressed significantly higher amounts of PIR-A in tumor–bearing, but not naïve animals. PIR-A signaling may be inhibited by PIR-B, suggesting that in an inflammatory environment, such as that found in tumor bearing mice, PIR-B may dominantly regulate MDSC polarization. Constitutive cis binding between PIR-B and MHC class I on mast cells plays an essential role in the regulation of allergic responses (Masuda et al., 2007
). Therefore, the MHC class I expression on Gr-1+
MDSCs may regulate the activation of PIR-A and -B. When comparing the difference in MHC class I expression between bone marrow derived and splenic Gr-1+
MDSCs, we found that splenic, but not bone marrow derived PIR-B-deficient MDSCs, expressed a higher amount of MHC-I molecules than their wild-type counterparts. This is consistent with our hypothesis that in the absence of PIR-B signaling, splenic Lilrb3−/−
MDSCs have the propensity to acquire an M1 oriented phenotype due to cis binding of MHC class I to PIR-A. Recently, other ligands such as the Staphylococcus aureus has been reported to activate PIR signal (Nakayama et al., 2007
), which indicates MHC class I is not the exclusive ligand of PIRs, PIR signaling may also be triggered by different ligands, which need be further investigated.
Differentiation of macrophages to the M1 phenotype depends on activation of the TLR4 and IFN-γ pathways, which in turn activate the ERK, NFκB, and STAT1 pathways. Meanwhile, M2 differentiation depends on activation signals from IL-10, IL-13, and IL-4, which activate the STAT3 and STAT6 pathways (Mantovani and Sica
; Mantovani et al., 2002
). NF-κB and STAT1 signaling suppresses the activation of STAT3 and STAT6, while activations of STAT3 and STAT6 inhibit NF-κB and STAT1 signaling (Mantovani and Sica
). Interestingly, the inhibitory signaling of PIR-B can suppress the activation of STAT1, STAT3, and STAT6, while PIR-A signaling mainly activates the NF-κB and ERK pathways (Hu et al., 2007
; Kubagawa et al., 1999
The counterbalance of numerous activating and inhibitory factors will ultimately influence the differentiation of MDSCs toward the M1 or M2 phenotype. The activation state is dependent on the cytokine milieu present within the inflammatory environment. Since IL-10, IL-13, and IL-4 are constitutively expressed and are the predominant cytokines present in the tumor-bearing host, STAT3 and STAT6 are activated, subsequently activating PIR-B and SHP-1, and leading to the inhibition of STAT1. SHP-1 can also inhibit ERK phosphorylation (Nakata et al.
), thereby inhibting NF-κB (p65) phosphorylation (Chen and Lin, 2001
). A lack of inhibitory signaling from PIR-B resulted in the activation of PIR-A in MDSCs, leading to the activation of the NF-κB and ERK signaling pathways. The combined effect of activation of NF-κB, ERK, and STAT1 further suppressed STAT3 and STAT6 activation and favored M1 differentiation of MDSCs. Studies have shown that ITAM activation signals can activate the NF-κB, ERK, and Stat1 pathways (Hu et al., 2007
; Taylor and McVicar, 1999
), but that the p50 subunit of NF-κB inhibits NF-κB activation, and orchestrates M2 macrophage differentiation (Mantovani and Locati, 2009
; Porta et al., 2009
). These previous findings correlate closely with what we have observed and support our hypotheses.
Our previous studies suggest that MDSCs strongly activate Treg cells in tumor-bearing animals (Huang et al., 2006
; Pan et al., 2008
). In this study, we demonstrated that M2 phenotypic MDSCs are critical for both in vitro
and in vivo
Treg cell activation. Lilrb3−/−
MDSCs favor the M1, rather than M2 phenotype, which led to less antigen-specific Treg cell activation and angiogenesis in the tumor environment in an adoptive transfer model. Our findings may indicate an intrinsic relationship between MDSCs and Treg cells.
Furthermore, we have analyzed both the pro- and antitumor activities of cancer-related inflammation and how, by shifting the balance from M2 to M1 macrophages, we can elicit a protective antitumor response. Indeed, adoptive transfer of Lilrb3−/− MDSCs significantly retarded primary tumor growth, prolonged survival rate, and inhibited lung metastasis. This implies a possible role for therapeutic regulation of paired immunoglobin like receptors in cancer, autoimmune diseases, and transplantation. PIR-B blockade may prove useful in human therapeutics through the inhibition of Treg cell expansion and angiogenesis in cancer patients
Furthermore, ILT3, the human counterpart of PIR-B, and its soluble form, have been shown to be involved in Treg cell development and GVHD prevention (Vlad et al.
). Interestingly, peripheral blood CD14+
monocytes from cancer patients, which are phenotypically equivalent to the mouse monocytic MDSCs, expressed a higher amount of ILT3, but not ILT1 (activating receptor), when compared to the equivalent population from normal donors (Fig. S3
The PIR signaling pathway plays an essential role in immune suppression and activation through the regulation of MDSC-mediated immune responses. The proper control of PIR function may enable modulation of the innate immune response, an effect that carries great potential for therapeutic translation.