In this study, we evaluated the nature of tumor-associated MDSC by comparing the phenotype and function of MDSC isolated from spleen and tumor sites from the same mice. It is known that MDSC can differentiate into MΦ and DC (Kusmartsev and Gabrilovich, 2003
). Therefore, it was important to assure that we are indeed comparing cells with the same phenotype. We sorted MDSC based on the expression of Gr-1 and CD11b, two markers which are considered hallmarks of MDSC. MDSCs from the tumor site and spleen had similar morphology and phenotype. Expression of the macrophage cell marker F4/80 was slightly higher on tumor MDSC than on spleen cells. However, such rather minor phenotypic differences contrasted with profound differences in MDSC function. As was reported previously (Corzo et al., 2009
), spleen MDSC contain a high level of ROS and a relatively modest level of NO and arginase I activity (although it was still elevated in comparison with Gr-1+
cells from naive mice). In striking contrast, tumor MDSC had no increase in ROS over naive Gr-1+
IMC but a very high level of NO and arginase I. These biochemical disparities translated into fundamental differences in their ability to suppress T cells. Tumor MDSCs were not only more potent inhibitors of antigen-specific T cell functions than spleen MDSCs but also, in contrast to spleen MDSCs, suppressed nonspecific T cells. Our experiments with a direct transfer of spleen MDSC to the tumor microenvironment demonstrated that 4 h was sufficient to cause dramatic changes in MDSC activity. These experiments also indicate that observed differences were indeed specific for the relative MDSC population and not caused by possible contamination of MΦ because the phenotype of MDSC was not changed within 4 h after transfer (unpublished data). Our recent study has found that in spleens, granulocytic CD11b+
MDSCs produce a substantially higher level of ROS and a lower level of NO than monocytic CD11b+
cells (Youn et al., 2008
). It was possible that the composition of these MDSC subsets could be different in spleens and tumors, which would explain the differences in functional activity of MDSC. However, the results of the experiments argue against this explanation.
Thus, this study demonstrated a dual role played by MDSC in immune suppression in cancer depending on their location. This may contribute to the phenomenon described previously in tumor-bearing mice. Although T cells from peripheral lymphoid organs of these mice did not respond to tumor-associated antigen, they nevertheless retained the ability to respond to nonspecific stimuli (Radoja et al., 2000
; Yang and Lattime, 2003
). Recent years have provided ample evidence supporting an important role of ROS in spleen MDSC-mediated suppression of T cells (Sinha et al., 2005
; Kusmartsev et al., 2008
; Markiewski et al., 2008
; Youn et al., 2008
; Mougiakakos et al., 2009
). ROS was specifically implicated in antigen-specific T cell tolerance mediated by MDSC (Nagaraj et al., 2007
; Hardy et al., 2008
However, a very different situation is observed with T cells isolated directly from tumors. Tumor-infiltrating lymphocytes displayed a profound defect in their function that could be overcome only after culture of these cells in the presence of antigen-presenting cells and IL-2. One possible explanation of these differences could be that the tumor microenvironment contains a large number of different suppressive factors that are not present in spleens. Our data demonstrate that the tumor microenvironment can convert MDSCs into nonspecific suppressor cells by up-regulating proteins involved in the metabolism of l
-arginine. These enzymes (iNOS and arginase I) are known to be actively involved in T cell suppression (Bronte and Zanovello, 2005
; Rodríguez and Ochoa, 2008
). Importantly, they do not require antigen-specific contact between MDSC and T cells to inhibit their function.
Up-regulation of arg1
by MDSC in the tumor site is a very rapid process and takes only several hours to occur. One of the major factors that distinguish the tumor microenvironment from lymphoid organs is hypoxia. It appears that hypoxia plays a critical role in the regulation of MDSC function by the tumor microenvironment. Our experiments have demonstrated that exposure of spleen MDSC to hypoxia could reproduce the effect of the tumor microenvironment on these cells by inducing a dramatic up-regulation of iNOS
, decreasing the expression of NOX2 and ROS and converting MDSC from antigen-specific to -nonspecific suppressors. How can hypoxia affect the function of MDSC? The major molecular mechanism of the hypoxia effect is mediated by the HIF-1 transcription factor. In hematopoietic cells, HIF-1α is the predominant oxygen-sensitive subunit (Simon et al., 2002
). Regulation of HIF-1 activity is mediated by posttranslational modification of the oxygen-dependent degradation (ODD) domain of HIF-1α. At oxygen levels >5%, hydroxylation of the proline residues 402 and 564 in the ODD of HIF-1α enables binding of the ubiquitination ligase von Hippel-Lindau tumor suppressor protein, which leads to degradation of HIF-1α by the proteosome. In contrast, at oxygen levels <5%, hydroxylation is inhibited leading to stabilization of HIF-1α. HIF-1α has been directly implicated in the up-regulation of iNOS
(for review see Yang et al., 2002
) and arginase
(Albina and Reichner, 2003
; Sica and Bronte, 2007
) in macrophages. HIF-1α has been shown to suppress oxidative phosphorylation and ROS production in mitochondria (Kim et al., 2006
; Papandreou et al., 2006
). Our data demonstrated that HIF-1α is directly responsible for conversion of MDSC in the tumor microenvironment to antigen-nonspecific suppressors of T cell function via up-regulation of arginase and NO.
Our data also indicate that hypoxia, primarily via HIF-1α, has a direct effect on MDSC differentiation. 2 d after adoptive transfer, >60% of MDSC in the spleen retained an immature phenotype, whereas the rest of the cells differentiated evenly into MΦ and DCs. In contrast, MDSC transferred into tumor site differentiated much more rapidly, with most of the cells acquiring the phenotype of MΦ. Experiments with MDSC culture in hypoxic conditions recapitulated these findings. Stabilization of HIF-1α with DFO reproduced this effect, suggesting that HIF-1α could be an important factor regulating the differentiation of MDSC to TAM. MDSC lacking HIF-1α did not differentiate into TAM within the tumor microenvironment or hypoxia but instead acquired markers of DCs.
Experiments with vaccination of HIF-1α–deficient tumor-bearing mice support an important role of HIF-1α in antitumor responses. Even without vaccination, mice that received HIF-1α–deficient BM cells demonstrated a significant delay in tumor progression. This effect was observed only 4–5 wk after tumor inoculation and could be possibly explained by the reconstitution of the lymphoid compartment by that time (6–7 wk after BM transfer). Stronger antitumor responses were not necessarily the result of improved function of myeloid cells because HIF-1α is known to negatively affect the function of T cells as well. Deletion of HIF-1α in T cells resulted in their activation in vitro and in vivo (Lukashev et al., 2006
; Thiel et al., 2007
). However, experiments with adoptive transfer of antigen-specific Pmel-1 T cells (which are HIF-1α+/+
) and vaccination of mice 1 wk after tumor inoculation allowed for better interpretation of the results. Our experiments indicated that mice that received HIF-1α–deficient BM developed stronger antitumor response than mice with WT HIF-1α BM. It is important to point out that those experiments, although suggestive, cannot definitively address the specific effect of HIF-1α deletion in MDSC because lack of HIF-1α in other myeloid cells (DC and MΦ) may also impact antitumor responses. More specific depletion experiments will be necessary to clarify this question.
Our study may suggest a model of MDSC differentiation and function in cancer. Expansion of IMCs in BM of tumor-bearing hosts, which is governed in large part by up-regulation of STAT3 (Nefedova et al., 2004
; Cheng et al., 2008
; Kujawski et al., 2008
), results in an accumulation of MDSC in peripheral lymphoid organs and in the tumor site. In lymphoid organs, MDSCs retain a high level of NOX2 and increased ROS levels. This is associated with a little increase in NO production and arginase I activity. As a result, these MDSCs produce peroxynitrite and exert their effect only via close cell–cell contact with activated antigen-specific T cells, which induce antigen-specific T cell tolerance. At the same time, these MDSCs fail to suppress antigen-nonspecific activation of T cells. In contrast, at the tumor site, MDSCs, as a result of the effect of hypoxia via HIF-1α, dramatically up-regulate expression of inos
, which is associated with down-regulation of both NOX2 expression and ROS production. Because of these changes, MDSCs acquire the ability to suppress antigen-nonspecific T cell functions, which contribute to the profound immune suppression observed within the tumor microenvironment. In addition, hypoxia via HIF-1α promotes differentiation of MDSC to immune suppressive TAM, which further supports the immune-suppressive network (Fig. S8
). Elucidation of this dual role of MDSC may not only help to understand the biology of tumor-associated immune suppression but also suggest that any therapeutic interventions should take into account the effect of microenvironment on the function of these cells.