Hierarchy of immunosuppressive strength ... - Wiley Online Library

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Luigi Dolcetti et al.

DOI 10.1002/eji.200939903

Eur. J. Immunol. 2010. 40: 22–35

Frontline

Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF Luigi Dolcetti1, Elisa Peranzoni1, Stefano Ugel1, Ilaria Marigo1, Audry Fernandez Gomez2, Circe Mesa2, Markus Geilich3, Gregor Winkels3, Elisabetta Traggiai4, Anna Casati5,6, Fabio Grassi5,6 and Vincenzo Bronte1 1 2 3 4 5 6

Istituto Oncologico Veneto, Padova, Italy Center of Molecular Immunology, Havana, Cuba Miltenyi Biotec GmbH, Bergisch Gladbach, Germany Istituto Giannina Gaslini, Dipartimento di Pediatria II, Genova, Italy Institute for Research in Biomedicine, Bellinzona, Switzerland Dipartimento di Biologia e Genetica per le Scienze Mediche, Milan University, Milano, Italy

CD11b1/Gr-11 myeloid-derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD81 T-cells. Two major MDSC subsets were recently shown to play an equal role in MDSC-induced immune dysfunctions: monocytic- and granulocytic-like. We isolated three fractions of MDSC, i.e. CD11b1/Gr-1high, CD11b1/ Gr-1int, and CD11b1/Gr-1low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b1/Gr-1int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD81 T-cell activation, while CD11b1/Gr-1high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b1/Gr-1int but not CD11b1/Gr-1high cells were also immunosuppressive in vivo following adoptive transfer. CD11b1/Gr-1low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM-CSF was necessary to induce preferential expansion of both CD11b1/Gr-1int and CD11b1/Gr-1low subsets in the spleen of tumor-bearing mice and mediate tumorinduced tolerance whereas G-CSF, which preferentially expanded CD11b1/Gr-1high cells, did not create such immunosuppressive environment. GM-CSF also acted on granulocyte–macrophage progenitors in the bone marrow inducing local expansion of CD11b1/Gr-1low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor-released GM-CSF.

Key words: GM-CSF . Immunosuppression . Myeloid-derived suppressor cells subsets . Tolerance

Supporting Information available online

Correspondence: Dr. Vincenzo Bronte e-mail: enzo.bronte@unipd.it

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

These authors contributed equally to this work.

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Introduction The tentative nomenclature of myeloid-derived suppressor cells (MDSC) summarizes the unifying characteristics that, so far, allow MDSC identification, i.e. their myeloid origin and immunosuppressive properties, in face of clear heterogeneity of these cells [1]. In mouse, MDSC were circumscribed to CD11b and Gr-1 double positive cells; however, when other markers are considered a general agreement is hard to reach. MDSC were described as F4/80int, CD11clow, MHCII /low, Ly6C1, expressing the early myeloid differentiation marker ER-MP58 and CD31 [2, 3]. IL-4Ra was recently described by our group as a functional marker of immunoregulatory CD11b1 cells since IL-4Ra1 cells, that show a prevalent monocytic morphology, presented clear immunosuppressive properties to the detriment of CD81 T cells both in vitro and in vivo, while IL-4Ra /CD11b1 cells, which comprised granulocytes at various differentiation stages, were poorly immunosuppressive [4]. Competence of MDSC to produce and respond to IFN-g was also a critical parameter that discriminated CD11b1/Gr-11 cells with immunosuppressive behavior [4]. Other groups have also focused on a monocytic immunosuppressive CD11b1/CD1151 subset [5]. More recently CD11b1 cells were fractionated in two distinct populations of PMN-MDSC and mononuclear-MDSC on the basis of the differential expression of Ly6G and Ly6C markers. Through a microbead-based sorting technique, Movahedi et al. showed that, in two different lymphoma models, the two subsets inhibited in vitro CD81 T-cell proliferation with almost the same potency; both fractions were positive for IL-4Ra and blockade of IFN-g abolished the immunosuppressive phenotype [6]. Another study analyzed a conspicuous number of tumor models in three different mouse strains and, based upon FACS sorting of CD11b1Ly6G Ly6C1 and CD11b1Ly6G1Ly6Clow subsets, achieved similar results; as a matter of fact, both MDSC subsets inhibited in vitro Ag-specific CD81 T-cell proliferation and ability to release IFN-g by means of different mechanisms: the granulocytic subset appealed to ROS production whereas the monocytic fraction exerted immunosuppression mainly via NO synthase [7]. The effort in MDSC subset definition is not a barren exercise, since it tends to address two very important and different issues: in the first place, it has the potential to reduce MDSC heterogeneity and eliminate the elements that do not directly contribute to the main functional properties of MDSC; on the other hand, and more importantly, this process could place MDSC more directly into the hematopoietic phylogenetic tree. From this perspective a great attention has been paid to the study of tumorderived soluble factors (TDSF) that drive myeloid maturation/ differentiation. It is accepted that MDSC expansion and activation are likely controlled by different TDSF [4, 8]. In particular two cytokines have been repeatedly associated with an in vivo expansion of CD11b1/Gr-11 cells: GM-CSF and G-CSF [3, 9, 10]. However, it is not clear whether these two cytokines have the same ability to activate the immunoregulatory program of MDSC. Therefore, we address two main issues of MDSC biology: defining the relative abundance and function of different subsets inside


the macro-population of CD11b1/Gr-11 cells, and clarifying the contribution of different cytokines to immunosuppression potency within each subset.

Results Intensity of Gr-1 staining identifies CD11b1 cells with functional heterogeneity Splenic CD11b1 cells from mice bearing either C26-GM, 4T1, or MCA203 tumors were enriched in two subsets that could be distinguished on the basis of Gr-1 marker brightness, Gr-1high and Gr-1int, by means of immunomagnetic sorting, optimized in a multistep separation protocol (Supporting Information Fig. 1A). Apart from mouse strain derivation and histological differences, C26-GM, 4T1, and MCA203 tumors were chosen because they span the observed spectrum of tumor-induced changes in both CD11b1 cell accumulation and Gr-1 marker distribution: spleens in 4T1 tumorbearing mice presented the highest proportion of CD11b1 cells, with abundance of Gr-1high subset (46.07%71.13% CD11b1/Gr-1high and 14.31%70.95% CD11b1/Gr-1int/low of total viable splenocytes), whereas MCA203 sarcoma induced a lower amount of total CD11b1 cells with a greater balance between the two subsets (15.6677.08% CD11b1/Gr-1high and 9.2173.39% CD11b1/ Gr-1int/low). C26-GM tumor originated an intermediated condition (21.5774.89% CD11b1/Gr-1high and 11.1271.58% CD11b1/ Gr-1int/low, representative plots are shown in Supporting Information Fig. 1B, as ‘‘original fraction’’). MDSC subsets obtained were added, at different numbers, to peptide- (mixed leukocyte peptide cultures, MLPC) or alloantigenstimulated lymphocytes (MLR) to evaluate their in vitro ability to restrain the generation of functional CTL (Fig. 1). In all tumor models considered, the Gr-1int subset presented a comparable suppressive influence when added to both MLPC and MLR and the suppressive effect was proportional to MDSC abundance (Fig. 1). On the contrary, the immunosuppressive potency of Gr-1high subset was both assay- and tumor-dependent, since Gr-1high fraction was slightly suppressive in C26-GM tumor model in MLPC, while the same cells lost their suppressive properties beyond a MDSC concentration of 6% in MLR. Gr-1high subset of MCA203 tumor model exerted weak suppression at highest MDSC percentages that was rapidly lost by decreasing MDSC abundance in co-cultures, whereas 4T1 Gr-1high subset increased rather than suppressed CTL generation in both MLPC and MLR (Fig. 1). In general, thus, Gr-1high subset exerted a marginal suppressive influence at high cell numbers. An alternative enrichment protocol, initially tested but then discarded, is described for comparison in Supporting Information Figs. 1B and 2. Although the aim was identical, i.e. separating Gr-1high from Gr-1int, the second protocol did not allow to separate a fraction of MDSC that was consistently suppressive in all tumors. Such results were likely due to the efficiency of separation protocol as shown in Supporting Information Fig. 1B. These results might explain some differences with previously published data based on MDSC enrichment through anti-Gr-1-conjugated beads.

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Figure 1. Gr-1int are more suppressive than Gr-1high MDSC on Ag-stimulated CD81 T cells. Gr-1high and Gr-1int MDSC subsets enriched from the spleens of C26-GM, 4T1, and MCA203 tumor-bearing mice were tested for their immunosuppressive properties by MLPC and MLR. MDSC subsets, syngeneic to responder splenocytes were added to the cultures in proportions of 0.75, 1.5, 3, 6, and 12% of total MLPC or MLR culture cellularity. MLR cultures were composed of responder splenocytes and an equal amount of allogeneic g-irradiated stimulator splenocytes, whereas MLPC cultures were composed of responder transgenic splenocytes (antigen-specific) and syngeneic WT g-irradiated splenocytes as feeder cells, in the presence of specific peptide. The extent of MDSC suppression was assessed after 5 days of coculture by means of a 5-h 51Cr release assay. Data are expressed as percent of lytic units 30 (L.U.30) of control cultures lacking MDSC (mean7SE, n 5 3 experiments).

MDSC subset phenotype MDSC Gr-1high and Gr-1int subsets were then tested for the differential expression of various surface markers (Fig. 2A). As expected, CD11b marker was present in all the cells and Gr-1 intensity did discriminate cells with higher and lower brightness. Interestingly, even though anti-Gr-1 mAb binds to both Ly6G and Ly6C, Ly6G marker showed a distribution similar to Gr-1, whereas differences in Ly6C were less evident among different fractions. This seems to support the idea that anti-Gr-1 mAb binds to Ly6C with minor avidity when compared with Ly6G, as originally suggested [11]. However, even if some groups have concluded that anti-Gr-1 mAb preferentially binds to either Ly6G or Ly6C, many discrepancies about epitope recognition and competition between Ly6C and Ly6G molecules have been reported [12, 13], and Gr-1 expression has been largely used to distinguish monocyte subsets [14]. The F4/80 and CD68 markers, both associated with a monocyte/macrophage phenotype, were principally expressed by the Gr-1int fraction. Besides CD11c, which was recently described in a subset of tumor-conditioned DC with immunosuppressive properties [15], we also exploited other markers that were associated


Eur. J. Immunol. 2010. 40: 22–35

to tumor-infiltrating CD11b1 myeloid subpopulations, such as angiopoietin receptor Tie2 [16], adhesion molecule CD31, glycolipid Ag-presenting CD1d, and transferrin receptor CD71 [6]. We were not able to distinguish the two fractions based on a single marker (Fig. 2A). We also tested the competence of MDSC subsets to produce both IFN-g and IL-13 (Fig. 2B) by means of cytokine intracellular staining. Gr-1high subset produced more IFN-g than Gr-1low subset, both as percentage of positive cells and MFI; IL-13 production showed no significant differences between the two subsets. MDSC subsets also showed differences in enzymes that have been associated with immunosuppressive properties of these cells (Fig. 2C): both Arg1 and Nos2 transcripts were upregulated in Gr-1int subset compared with Gr-1high subset, Arg2 and Nos3 were consistently down-regulated in Gr-1int subset, whereas Nos1 isoform had an opposite trend. Cyba and Cybb, subunits of NADPH oxidase, which had been reported to be involved in MDSC-related immunosuppression as source of reactive oxygen species [7, 17], were both down-regulated in Gr-1int compared with Gr-1high subset. Moreover, Nos2 protein was detected only in Gr-1int, while it was absent in Gr-1high subset; Arg1 protein was not detectable in either two subsets. As previously described, both proteins were present in tumor-infiltrating CD11b1 cells [4].

Isolation of Gr-1low MDSC Lack of expression of IL-4Ra in Gr-1int cells (Fig. 2A) was unexpected in light of our previous results showing that the cells with highest IL-4Ra expression had low Gr-1 levels [4]. This led us to hypothesize that cells with lowest Gr-1 levels might escape the second purification step of the previously described protocol. As an extension and further refinement of the first protocol, the negative fraction depleted from Ly6G- and Gr-1-based sorting was enriched in a third subset of CD11b1/Gr-1low cells (Fig. 3A, first row). In this new protocol the first purification steps were also adjusted, resulting in better results in terms of purity in the C26-GM and 4T1 models. However, the Gr-1low fraction from MCA203 tumorbearing mice was partially contaminated by CD11c1 cells (Fig. 3A, fourth row), congruently with the loss of immunosuppressive activity in this subset (Fig. 3B). With this exception, however, immunosuppressive power on Ag-stimulated CD81 T cells followed an inverse relationship with Gr-1 brightness in C26-GM and 4T1 tumor models (Fig. 3B). MDSC subset morphology was evaluated by common May-Gru ¨nwald-Giemsa staining: Gr-1high subset (Fig. 3B, first row) was mainly composed by mature polymorphonuclear cells, while Gr-1int subset presented a more heterogeneous pattern comprising monocytes and myeloid precursors with ‘‘doughnut’’-shaped nuclei [18]; Gr-1low, at least in C26-GM and 4T1 tumor models, comprised mainly cells with a prevalent monocytic morphology. As previously shown [4], the Gr-1low fraction isolated from the spleen of C26-GM tumor-bearing mice expressed the highest levels of IL-4Ra chain. However, distribution of IL-4Ra chain cannot allow to discriminate the different subsets in all the tumor models.

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Figure 2. Phenotype and properties of Gr-1int and Gr-1high MDSC subsets. (A) MDSC subsets enriched from the spleens of C26-GM, 4T1, and MCA203 tumor-bearing mice were characterized for the expression of various surface and intracellular (CD68) markers. Data are percentage of maximum; filled histograms are the isotype controls. (B) Gr-1high and Gr-1int subsets, obtained from the spleen of C26-GM tumor bearing-mice were tested for spontaneous IFN-g and IL-13 production by intracellular staining. One representative experiment out of four is shown in the upper panels; filled histograms are isotype controls. Pooled data (mean7SE; n 5 4) of either percentage of positive cells (middle panels) or integrated MFI (lower panels) are also shown; pr0.05 (Student’s t-test). (C) The subsets described in (B) were characterized by relative real-time PCR for the expression of Arg isoforms, Nos isoforms, and Cyba/Cybb subunits of NADPH. Data (upper panels) are expressed as logarithmic twofold increase (Gr-1low versus Gr-1high) of gene expression normalized to GAPDH housekeeping gene expression (mean7SD, n 5 3 experiments). Arg1 and Nos2 protein expression in the Gr-1high and Gr-1int subsets from the spleen and in total CD11b1 cells from the spleen and the tumor infiltrate of C26-GM tumorbearing mice was evaluated by Western blot (lower panels).

Tolerogenic activity of adoptively transferred MDSC subsets Although interesting to dissect primarily functional properties of various subsets, in vitro assays might not be sufficient to recapitulate the ability of MDSC to mediate in vivo tolerance. Since a homogeneously immunosuppressive Gr-1low fraction could not be isolated from all the tumors, we focused our next in vivo studies on Gr-1high and Gr-1int fractions. Immunomagnetic sorting allowed us to enrich enough cells for MDSC adoptive transfer in mice that had previously received HA-specific CD81 T cells and were then immunized with HA emulsified in incomplete Freund adjuvant (IFA) (as shown in Supporting


Information Fig. 3). Lymph node cells were tested in ELISPOT assay to assess the number of HA-specific IFN-g producing cells. Whereas whole Gr-11 sorted population induced a slight decrease of specific IFN-g spots, Gr-1high population increased rather than decreased the immune response. Only transfer of Gr-1int subset produced a statistically significant suppression (p 5 0.039), halving the number of IFN-g specific spots compared with control mice immunized but not transferred with MDSC (Fig. 4A); the fall in numbers of transferred Thy1.11/CD81 T cells in mice that received Gr-1int cell subset showed that the principal immunoregulatory effect of MDSC could be referred to an elimination of Ag-specific CD81 T cells (Fig. 4B, p 5 0.049).

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Figure 3. Gr-1low cell fraction isolated from the spleen of tumor-bearing mice retains elevated immunosuppressive activity. (A) Gr-1hi, Gr-1int, and Gr-1low subsets obtained from the spleen of C26-GM, 4T1, and MCA203 tumor-bearing mice were evaluated for CD11b versus Gr-1 expression (upper row, contour plots) and for the expression of various surface markers within the CD11b1 gated population (central panels, open histograms) against their matched isotype controls (filled histograms). (B) Staining with May-Gru¨nwald Giemsa to evaluate subset morphology and a 5-h 51Cr release assay performed on 5-day MLPC cultures in the presence of decreasing numbers of MDSC (filled circles), ranging from 12 to 0.75% of total MLPC culture cellularity by 1:2 serial dilution, to evaluate the immunosuppressive properties of the different subsets. Data are expressed as mean percent of L.U.30 obtained in control cultures (open circles, n 5 3 replicates from one experiment representative of two).

Knocking down GM-CSF in 4T1 tumors alters MDSC subset ratios and reduces tumor-induced tolerance To investigate the in vivo role of tumor-released GM-CSF on MDSC subset distribution and function, we transfected 4T1 tumor cells with a short hairpin RNA (shRNA) plasmid directed against GMCSF mRNA and isolated some clones displaying low cytokine production. The 4V clone showed a significant, though not complete, reduction in GM-CSF production respect to its parental line, but other important MDSC-inducing cytokines, like G-CSF


and IL-6 [19], were not affected in their expression (Fig. 5A). This clone summarizes the properties of three independently selected clones (Supporting Information Fig. 4). When s.c. inoculated into BALB/c mice, 4V variant showed a growth curve similar to parental 4T1 tumor for the first 10 days following subcutaneous inoculation (Supporting Information Fig. 4) but a reduced accumulation of splenic MDSC compared with mice inoculated with the parental line (see below). Moreover, CD11b1 cells sorted from the spleens of 4V tumor-bearing mice were less suppressive on a per cell basis than their 4T1 counterpart (Fig. 5B).

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Figure 4. Gr-1int MDSC inhibit priming of Ag-specific CD81 T cells in vivo. (A) Gr-1high and Gr-1int subsets, enriched from the spleen of C26GM tumor-bearing mice, were tested for their ability to induce tolerance in mice adoptively transferred with HA-specific CD81 CTL and immunized with HA512–520-IFA. (A) ELIspot counts for IFN-g producing cells in lymph node cultures stimulated with HA512–520 peptide (mean7SE after subtraction of the number of spots obtained in control cultures; nZ7 mice/group; data are pooled from two independent experiments.) (B) Total numbers of HA-specific CD81/Thy1.11 T cells were evaluated in lymph nodes by flow cytometry. pr0.05, Student’s t-test.

Figure 5. Silencing of GM-CSF in 4T1 mammary carcinoma results in reduction of the immunosuppressive activity of splenic MDSC. (A) Cytokine production by 1 106 4T1 and 4V cells as determined by ELISA after 48-h culture. Data are mean7SE of three independent experiments; pr0.001 (Student’s t-test) (B) The immunosuppressive properties of total CD11b1 cells isolated from the spleen of 4T1 (filled circles) and 4V (open circles) tumor-bearing mice were evaluated in a 51 Cr release cytotoxicity assay after 5-day MLPC culture. Data are expressed as percent of L.U.30 obtained in control cultures (n 5 3 experiment mean7SE).

By comparing Gr-1 versus CD11b staining we were able to identify the three Gr-11 subsets in the spleens of mammary carcinoma-bearing mice (Fig. 6A). 4V tumor growth was accompanied by significant reduction in splenic accumulation of Gr-1int and Gr-1low but not Gr-1high cells, when compared with 4T1 tumor-bearing mice (p 5 0.002, 0.001, and 0.154, respectively; Fig. 6A, middle histograms). When gating was restricted to CD11b1 cells (Fig. 6A, histogram on the right), the Gr-1int subset was significantly reduced in mice bearing 4V tumor, (p 5 0.003), as well as the Gr-1low subset (p 5 0.04), whereas Gr-1high subset increased significantly (p 5 0.002). We also analyzed the

phenotype of CD11b1 cells accumulated in the spleen of mice bearing the GM-CSF competent and deficient mammary carcinomas. Figure 5 in Supporting Information shows a partial characterization focusing on the most relevant changes. There was a statistically significant difference for the two experimental groups in the following Ag combinations: F4/801Gr-11 (p 5 0.014), CD11c1Gr-11 (p 5 0.008), and MHC II1Gr-11 (p 5 0.037), suggesting that CD11b1 cells in 4V tumor-bearing mice likely comprise more mature monocytes and DC. Moreover, MFI of several markers was augmented in splenocytes from 4V respect to 4T1 tumor bearers (data not shown). All the GM-CSF-


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Figure 6. Lack of GM-CSF release from mammary carcinoma cells reduces accumulation of Gr-1int/low MDSC and overcomes tumor-induced tolerance. (A) 4T1 and GM-CSF-silenced 4V cells were injected into syngeneic mice and CD11b and Gr-1 expression on isolated splenocytes analyzed by flow cytometry. Gr-1 versus CD11b contour plots illustrate the gate setting (left panels). Gr-1high, Gr-1int, and Gr-1low frequencies are presented as mean1SE (n 5 8 mice) of either total splenocytes (central panels) or CD11b1 cells (right panels). (B) Induction of in vivo tolerance was evaluated in 4T1-, 4V-tumor-bearing and healthy BALB/c mice that were adoptively transferred with HA-specific CTL and immunized with IFAemulsified HA512-520 peptide. Percentages of CD81 HA-specific (Thy1.11) lymphocytes in ex vivo cultures of lymph nodes cells as determined by intracellular staining and cytofluorimetric analysis (left panel). ELISPOT counts for HA-specific IFN-g producing lymph node cells corrected for non-specific background (middle panel). Percentages of IFN-g producing, HA-specific CD81 T lymphocytes as determined by intracellular staining and cytofluorimetric analysis (right panels). All data in (B) are mean7SE; n 5 3 mice/group from one representative out of two independent experiments; pr0.01, pr0.001 (Student’s t-test). (C) Five mice in each group were injected with either saline alone (data not shown) or saline containing G-CSF, twice a day for 3 days. Splenocytes from G-CSF-treated mice were analyzed for CD11b and Gr-1 expression by flow cytometry, as described in (A). (D) Suppressive activity of total CD11b1 MDSC enriched from pooled spleens of G-CSF treated mice was evaluated in a 5-h 51Cr release assay performed on 5-day MLPC culture containing MDSC, from 24 to 0.8% of total MLPC cellularity by 1:3 serial dilution. Means of specific lysis in either control MLPC (open circle) or MLPC containing MDSC (filled circle) from triplicate cultures of one representative experiment (out of three independent) are shown.


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silenced 4T1 subclones shared the in vivo behavior described so far; however, whereas the reduction in Gr-1int/low cell fractions was common to all 4T1 variants, growth of some clones were also accompanied by a decrease in Gr-1high cells, suggesting a more profound effect on myelopoiesis (Supporting Information Fig. 4). In order to evaluate tumor-induced tolerance, 4T1 and 4V tumor-bearing mice were transferred with HA-specific CD81 T lymphocytes and vaccinated 2 days later with IFA-emulsified HA-peptide (Supporting Information Fig. 6). Lymph nodes cells of tumor-bearing mice and control tumor-free mice were then stimulated ex vivo with HA peptide and analyzed, both by intracellular staining and ELISPOT, to evaluate IFN-g production by HA-specific CD81 T lymphocytes. As shown in Fig. 6B, the number of viable HA-specific T lymphocytes was strongly reduced in the lymph nodes of 4T1-bearing mice compared with 4V-bearing mice. These results correlated strictly with the reduction in number of IFN-g-releasing cells in ELISPOT assay observed in mice bearing 4T1 tumors but not in 4V tumor bearers (Fig. 6B, middle histograms). Interestingly, when the few Thy1.11 cells in lymph node of 4T1 tumor-bearing mice were analyzed for the intracellular IFN-g release, there was no difference with the other groups on a per cell basis (Fig. 6B, histogram on the right), suggesting that the few HA-specific CD81 T cells remaining in lymph nodes could still respond to antigenicstimulation and the bulk of the immunoregulation was mediated by the disappearance of Ag-specific lymphocytes (as reported in Fig. 4B). Since growth of 4V clone results in an increase in Gr-1high cell fraction among CD11b1/Gr-11 cells (Fig. 6A), we investigated the role of G-CSF, which was shown to drive the accumulation of proangiogenic CD11b1/Gr-11 cells in tumor-bearing hosts [9, 10]. We therefore administered this cytokine to normal, tumor-free BALB/c mice and checked for CD11b/Gr-1 distribution among splenocytes. We observed an accumulation of myeloid cells similar to that found in mice bearing the GM-CSFsilenced tumor (Fig. 6C). CD11b1 cells isolated from G-CSFtreated animals were completely unable to suppress generation of CTL even when used at very high concentrations (Fig. 6D), revealing how this cytokine alone is not sufficient for functional MDSC induction, differently from what happens with GM-CSF administration [3, 20].

Effect of exogenously administered and tumor-derived GM-CSF on bone marrow progenitors To analyze the influence of GM-CSF on bone marrow (BM) hematopoietic progenitors, we injected BALB/c mice either with carrier alone or pegylated GM-CSF. GM-CSF-injected mice displayed reduced BM cellularity (carrier: 51.6716.4 106, n 5 3; GM-CSF: 43.573.7 106, n 5 3; p 5 ns), and therefore the statistical analysis for absolute numbers of the various subsets is displayed. We scored lineage-negative (lin ) c-Kit1/Sca-1low (LKS ) cells as FcgRlowCD34low common myeloid progenitors (CMP), FcgRhighCD341 granulocyte–macrophage progenitors


(GMP) as well as FcgRlowCD34 megacaryocyte/erythroid progenitors (MEP) [21]. GM-CSF treated mice displayed significant increase in the percentage of GMP (carrier: 0.1270.02; GMCSF: 0.270.03; p 5 0.02, n 5 3). However, absolute numbers were not significantly different (Fig. 7A). As expected, CMP and MEP were both significantly reduced in GM-CSF treated mice as well as common lymphoid progenitors (CLP) defined as lin /c-Kitlow/IL-7R1 cells [22] (Fig. 7A). FACS sorting of BM cells at FACS with CD11b and Gr-1 Ab results in the recovery of metamyelocytes/granulocytes in the CD11b1/Gr-1high subset and monocytes in the CD11blowGr-1int subset, whereas promyelocytes/myelocytes (comprising cells with a characteristic doughnut-like nucleus) are confined to the CD11b1/Gr-1low fraction [18]. In GM-CSF treated mice, CD11b1 /Gr-1high cells were significantly increased, whereas CD11blow/ Gr-1int cells were not significantly different from control mice. Notably, CD11b1/Gr-1low cells were dramatically increased following treatment with GM-CSF (Fig. 7B). We performed the same analysis in mice bearing either the GM-CSF producing 4T1 tumor or the GM-CSF silenced 4V variant. Cell recoveries as well as relative representations of CMP, MEP, and CLP were not different between the two groups of animals (Fig. 7C). In contrast, GMP were significantly reduced and CD11b1/Gr-1low cells were significantly increased in 4T1 bearing mice (Fig. 7D). Altogether, these results suggest that GMCSF produced by tumor cells directly influences the differentiation of GMP and determines the accumulation of cells at the promyelocytes/myelocytes stage of differentiation.

Discussion We show in this manuscript that it is possible to distinguish MDSC subsets with enhanced immunoregulatory property in vitro and, most importantly, in vivo. These data are further supported by in vivo experiments either with cytokine-silenced tumors or cytokine administration that can allow us to propose GM-CSF as main driver of Gr-1int/low suppressive MDSC, whereas G-CSF preferentially induce Gr-1high cells with poor immunosuppressive activity. C26-GM and 4T1 tumor models showed a clear gradient of increased immunosuppression inversely related to Gr-1 expression, whereas MCA203 presented a peak of immunosuppression strength in coincidence with an intermediate Gr-1 brightness among CD11b1 cells. This most likely depends on the inability of the protocol to completely separate CD11b1/Gr-1low cells from other contaminating cells (likely DC and NK cells) in this tumor model. Gr-1int and Gr-1low subsets presented a prevalent monocytic morphology, according to the description of other groups [6, 7]; moreover, the Gr-1low subset in C26-GM tumor model showed the highest expression of IL-4Ra, as we originally described [4]. However, it must be pointed out that IL-4Ra is present in both PMN-like and monocyte-like MDSC, as recently confirmed in studies with human MDSC [23], and thus cannot be used to discriminate MDSC subpopulations in all tumors.

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Figure 7. Analysis of BM progenitors in GM-CSF treated or 4T1 or 4V tumor bearing mice. (A) and (C) Dot plot analysis of LKS cells for FcgR and CD34 expression (upper panels) from carrier (control) and GM-CSF (GM-CSF) injected mice (A), and from mice bearing 4T1 or 4V tumors (C). Gates corresponding to CMP (FcgRlowCD34low), GMP (FcgRhighCD341) and MEP (FcgRlowCD34) are displayed. Dot plot analysis of Lin cells stained with c-Kit and IL-7R Ab (lower panels), and the gate used to score CLP (c-KitlowIL-7R1 cells) is displayed. Numbers in the dot plots indicate the percentage of total BM cells in the corresponding gate in a representative staining. Absolute number (A) and percentage (C) of the gated progenitors are presented (pooled data from at least three mice/group, mean7SE, histograms on the right) (B) and (D) Dot plot analysis of BM cells, stained with CD11b and Gr-1 Ab, from carrier and GM-CSF-treated or 4T1- and 4V-tumor bearing mice (upper panels). Absolute number (B) and percentage (D) of the indicated subsets are presented (pooled data from at least three mice/group, mean7SE, histograms at the bottom). pr0.05; pr0.01; pr0.001 (Student’s t-test).

One of the major differences between our data and previously published results is that we could detect little immune suppressive activity in the Gr-1high fraction, which mostly comprises the so-called PMN-MDSC. These discrepancies might depend on several factors, such as strategies for subset separation, use of different functional assays to test immunosuppression, or time when tumor-bearing mice were analyzed (which could affect differential relative prevalence of MDSC subsets) [6, 7, 19]. None of previous studies, however, addressed in vivo immune dysfunctions associated with either presence or absence of different MDSC subsets so it is not clear how in vitro data relate to in vivo tumor-induced immune dysfunctions. In addition, it must be pointed out that, in general, PMN-MDSC were suppressive when high number of cells were added to the in vitro cultures containing Ag-stimulated T cells [7, 8]. We could indeed confirm, with some but not all tumor models, that Gr-1high cells might be as suppressive as whole CD11b1 cells in both MLR and MLPC, when high dose of cells were added to the cultures (Supporting information Fig. 7A). Interestingly, in C26-GM tumor model we and others have extensively showed that CD11b1 splenocytes require Arg and Nos combined enzyme activity to restrain T-cell


activation at low MDSC numbers [4, 24]; the synergistic and noncompetitive activation of Arg and Nos was actually suggested to represent an hallmark of MDSC distinguishing them from tumorassociated macrophages [19, 25]. However, in vitro suppression by high dose MDSC depended upon partially different pathways. Figure 7A in Supporting Information, in fact, shows that total CD11b1 population is susceptible to NGmonomethyl-L-arginine (L-NMMA), specific Nos inhibitor but not to Arg inhibitor Nw-hydroxy-nor-L-arginine. Gr-1high subset also responded to L-NMMA but to greater extent when compared with the total CD11b1 population. Additionally, while manganese (III) tetrakis (4-benzoic acid) porphyrin chloride and protoporphyrin IX zinc(II) – a scavenger of peroxynitrites and inhibitor of heme oxigenase, respectively – were both ineffective, catalase, scavenger of hydroxyl-peroxide, acted quite differently on either CD11b1 total population or Gr-1high subset since only immune suppression sustained by Gr-1high subset was partially recovered (Supporting Information Fig. 7A), in agreement with previous results [7]. Data on in vitro immune suppression by high dose MDSC must be considered with some caution. As previously reported in some

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studies [26], high concentrations of CD11b1 cells enriched from the spleen of tumor-free mice could also induce some immunosuppression in vitro (Supporting Information Fig. 7B), a situation that contrasts normal immune responsiveness of tumor-free mice. Interestingly, while total CD11b1 cells showed a suppressive activity at very high doses, normal Gr-1high cells did not even at concentrations greater than 60% of total cells in cultures (Supporting Information Fig. 7B). These findings reinforce the concept that CD11b1Gr-1high cells are not suppressive, even when their numbers are substantially increased by G-CSF administration (Fig. 6D). A recent manuscript suggests that differences in MDSC subsets might also exist during inflammatory processes. IFN-g and LPS boost development and activation of MDSC while blocking further maturation of bone marrow precursors to DC [27]. By staining with anti-CD11b and anti-Gr-1 Ab, authors identified and separated different cell subsets but only Gr-1lowCD11bint Ly-6ChighSSClow and Gr-1highCD11bint cell fractions possessed suppressive activity on T lymphocytes. Gr-1highCD11bhigh neutrophils and Gr-1lowCD11bhighSSClow eosinophils were not suppressive. Gr-1highCD11bint cells also expressed F4/80, high levels of Ly6C, and low levels of CSF-1 receptor (CD115), and comprised cells with ring-shaped nuclei [27]. The suppressive subsets thus appear to be very similar to Gr-1int and Gr-1low subsets described in our manuscript, suggesting common features of immunoregulatory myeloid cells expanded under different pathological situations [28]. TDSF comprise a wide variety of signaling molecules driving myelopoiesis [29, 30]. Our data suggest that GM-CSF functions as a dominant factor promoting immunosuppressive pathways within the macro-population of CD11b1 cells. GM-CSF silencing resulted in the perturbation of MDSC subset distribution in the spleens of tumor-bearing mice (Fig. 6A) and also caused some changes in their phenotype (Supporting Information Fig. 5). GMCSF causes expansion of GMP present in the BM and increases substantially their differentiation toward CD11b1/Gr-1low cells, with a significant but less dramatic effect on CD11b1/Gr-1high cells (Fig. 7). These findings are mirrored by the decrease of BM CD11b1/Gr-1low cells when GM-CSF was silenced in 4T1 mammary carcinoma, accompanied by an increase in GMP. It is thus likely that prolonged release of GM-CSF by WT tumor cells might force a continuous expansion and differentiation of GMP with major increase of the promylocyte/myelocyte subset. Interestingly, GM-CSF silencing in mammary carcinoma induced MDSC subset distribution similar to G-CSF administration to normal, tumor-free mice (Fig. 6C). Considering that 4V tumor cells release the same amount of G-CSF of parental 4T1 cell line, we are tempted to speculate that inhibiting GM-CSF release by tumor cells probably paved the way to the action of G-CSF, promoting the expansion of a CD11b1/Gr-1high subset to the detriment of Gr-1low/int subpopulation. Although it was speculated that G-CSF could induce CD11b1/Gr-11 MDSC with proangiogenic activity [9, 10, 31], and G-CSF has been associated with skewing to Th2 type responses and promotion of T regulatory lymphocyte and tolerogenic DC differentiation [32],


connection of this cytokine to immunoregulatory MDSC has never been addressed. We show here that, indeed, G-CSF mobilizes CD11b1/Gr-1high cells devoid of immunosuppressive properties on CD81 T lymphocytes. Although GM-CSF is necessary for immunoregulatory program, we cannot exclude that G-CSF can influence either the phenotype or function of GM-CSF-driven MDSC. GM-CSF was reported to induce in vitro differentiation of BM cells into immunosuppressive CD11c Gr-1low CD11b1 CD311 ER-MP581 asialoGM11 F4/801 cells [33]. However, our data suggest that GM-CSF is a required, but not sufficient factor, to induce the in vitro maturation of BM precursor to a full MDSC phenotype (Marigo et al., submitted). The phenotype of cells with heightened immunoregulatory activity appeared to be CD11b1, Gr-1int/low, Ly6C1, F4/80low, IL-4Ra1, and CD681. Two issues require further comments: first, this population retains morphological heterogeneity, since it mainly comprises monocyte-like cells but cells with less mature phenotype can be clearly defined (with ‘‘doughnut’’ nuclei); second, CD115 (CSF-1/M-CSF receptor), which is required to identify classic monocytes [14], was barely detectable in freshly isolated Gr-1int/low cells but it could be seen after cells were rested for few hours, suggesting that the receptor was either downregulated for its continuous engagement in vivo or rather cells underwent activation/differentiation in vitro. It is thus possible that we can further dissect this population but we think that this cannot be achieved by a simple comparison of surface markers, which might be either down- or up-regulated just for the peculiar environment enforced by developing tumors, as CD115 staining seems to suggest. We are currently evaluating mRNA and microRNA expression profiles of CD11b1 cell subsets under resting condition and during tumor development.

Materials and methods Mice Eight-wk-old C57BL/6 (H-2b) and BALB/c (H-2d) mice were purchased from Harlan (San Pietro al Natisone, Italy). Transgenic mice on a BALB/c background expressing a Kd-restricted HA512-520 peptide-specific a/b TCR (CL4 mice) were a gift from L. Sherman (The Scripps Research Institute, La Jolla, CA, USA); pmel-1 TCR transgenic mice on a C57BL/6 background that express the Va1Vb13 TCR and specifically recognize the H-2Db restricted mouse and human gp10025–33 [34] were a gift from N. Restifo (Surgery Branch, National Institutes of Heath, Bethesda, MD, USA).

Cell lines and gene silencing CT26 is a carcinogen-induced, undifferentiated colon carcinoma, and 4T1 is a mouse mammary carcinoma cell line, all derived

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from BALB/c mice (H-2d). 4V clone was obtained by stably transfecting 4T1 cell line with an shRNA plasmid (Mission shRNA purchased from Sigma-Aldrich) optimized to downregulate GM-CSF expression by RNAi. MBL-2 lymphoma and MCA203, a 3-methylcholanthreneinduced fibrosarcoma, are all derived from C57BL/6 mice (H-2b). C26-GM cell line was derived from C26 colon carcinoma (H-2d) genetically modified to release GM-CSF [24]. These cell lines were grown in DMEM (Invitrogen) or RPMI 1640 (Euroclone), supplemented with 2 mM L-glutamine, 10 mM HEPES, 20 mM 2-ME, 150 U/mL streptomycin, 200 U/mL penicillin, and 10% heat-inactivated FBS (Biochrom).

Eur. J. Immunol. 2010. 40: 22–35

stained for 20 minutes at 41C with the relevant Ab. The Abs used were CD11b PERCPCy5.5, Gr-1 APC, Ly6C FITC, Ly6G PE, CD62L FITC, IL-4Ra PE, CD11c FITC, and I-A/I-E PE from BD Biosciences, F4/80 FITC and CD68 PE from Serotec, Tie2 PE and IL-6R PE from eBioscience, CD31 APC, CD1d Alexa 647, and CD71 biotin from Biolegend. For FACS analysis of bone marrow, mAb conjugated with either biotin, FITC, PE, CyChrome, PerCP, or APC against the following Ag were used: CD8a, CD4, NK1.1, TCRb, TCRd, Mac-1, Gr-1, CD2, CD19, CD11b, CD34, IL-7R, FcgR/CD16/35, and c-Kit/CD117 (eBioscence); CD45R/B220, Sca-1, and APC-Cy7-streptavidin (BD Bioscience). All samples were acquired with a FACScalibur or FACSCanto (Becton Dickinson Immunocytometry Sistems) and analyzed with FlowJo software (Treestar).

Cell sorting In vivo functional tests Total CD11b1 cells from spleen and tumor mass and total Gr-11 cells from spleen were sorted with CD11b-MicroBeads and AntiGr-1-Biotin/Anti-Biotin-MicroBeads respectively, according to the manufacturer’s standard protocol. Gr-1high/Gr-1int subset sorting: 1 108 cells were blocked with 100 mL FcgR blocking reagent/869 mL sorting buffer (PEB: PBS, 2 mM EDTA, 0.5% BSA) for 10 min at 41C; stained with 30.3 mL Anti-Ly6G-Biotin for 10 min at 41C; washed with 10 mL PEB and decanted at 1200 RPM for 6 min; stained with 200 mL Anti-BiotinMicroBeads/800 mL PEB for 15 min at 41C; washed, decanted and resuspended in 1 ml PEB; Gr-1high subset was sorted with 1 LS column. Negative fraction from previous step was decanted and stained with 227 mL Anti-Gr-1-biotin/773 mL PEB for 10 min at 41C; washed and decanted; stained with 100 mL streptavidin-microbeads/900 mL PEB for 15 min at 41C; washed, decanted, and resuspended in 1 mL PEB; Gr-1int subset was sorted with 2 MS columns. Gr-1high/Gr-1int/Gr-1low subset sorting: 1 108 cells were blocked with 100 mL FcgR blocking reagent/869 mL PEB for 10 min at 41C; stained with 31 mL Anti-Ly6G-Biotin for 10 min at 41C; washed and decanted; stained with 200 mL Anti-Biotin-MicroBeads/800 mL PEB for 15 min at 41C; washed, decanted and resuspended in 1 mL PEB; Gr-1high subset was sorted with 1 LS column. Negative fraction from previous step was decanted and stained with 114 mL Anti-Gr-1biotin/386 mL PEB for 10 min at 41C; washed and decanted; stained with 50 mL streptavidin-microbeads/450 mL PEB for 15 min at 41C; washed, decanted and resuspended in 1 mL PEB; Gr-1high subset was sorted with 1 LS column. Negative fraction from previous step was decanted and stained with 50 mL CD11b-MicroBeads/450 mL PEB for 15 min at 41C; washed, decanted and resuspended in 1 mL PEB; Gr1low subset was sorted with 1 LS column. All Ab and columns were from Miltenyi Biotec; PBS, BSA and EDTA from SIGMA Aldrich. Please, refer to Supporting Information Fig. 1 for a more extensive information about the sorting protocols.

In vivo tolerance induced by MDSC subset adoptive transfer (a schematic workflow can be followed in Supporting Information Fig. 2). BALB/c mice received, via i.v. injection, 4 106 immunomagnetically sorted CL4 Thy 1.11/CD81 lymphocytes. Two days later, MDSC fractions were sorted from 9-days-old C26-GM tumorbearing mice and 4 106 cells, the same number as effector CD81 T cells, were injected i.v. within an hour, mice were immunized s.c. with an IFA emulsion containing 100 mg of HA512–520 peptide. Ten days later, mice were euthanized and cells from lymph nodes were tested by means of ELISPOT and FACS analysis. In vivo tolerance of 4T1 and 4V induced MDSC (a schematic workflow can be followed in Supporting Information Fig. 3). Mice were injected s.c. in the flank with 5 105 cells of 4T1 or 4V clone tumor cell lines. After ten days, mice were transferred with 20 106 splenocytes derived from CL4 transgenic mice (bearing HA-specific CD81 T lymphocytes), approximately corresponding to 5 106 HAspecific CD81 cells (determined by cytofluorimetric staining). Two days after lymphocyte transfer, mice were vaccinated s.c. with 100 mg/mouse of IFA-emulsified HA512–520 peptide. Six days after vaccination, animals were euthanized and cells form lymph nodes were tested by means of ELISPOT and FACS analysis.

Chemicals (Calbiochem) was used at 500 mM; Nw-Hydroxy-nor-Larginine (Calbiochem) was used at 2 mM; manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (Sigma-Aldrich) was used at 500 nM; protoporphyrin IX zinc(II) (Sigma-Aldrich) was used at 2.5 mM; Catalase (Sigma-Aldrich) was used at 50 mg/mL. All concentrations have been determinated to minimize effects on control MLPC and MLR in a 5-h 51Cr-release test.

L-NMMA

Flow cytometry analysis

G-CSF and pegylated-GMCSF in vivo treatment

Samples were washed with PBS, after red blood cell lysis when required, and incubated with Fc blocking reagent and then

Mice were injected either i.p. with 5 mg/mouse of human G-CSF (Sanofi Aventis), twice a day for a total of 3 days or i.p.


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with pegylated-GM-CSF (a gift from J. F. Parkinson, Bayer Healthcare Pharmaceuticals, Richmond, CA, USA) for 4 days, at the dose of 5 mg/mouse/day, while control animals only received PBS.

performed using mouse IFN-gELISPOT Kit (BD Pharmingen) following manufacturer’s instructions. Each well contained 5 105 lymph nodes cells. The numbers of spots were evaluated in triplicates by using an automated ELISPOT reader and software.

Cytological preparation CTL assay Freshly isolated MDCS subsets were spun over slides with a Shandon Cytospin: preconditioning 100 mL PBS, 1 min at 800 rpm, 500 mL of 0.1 106 cells/mL single-cell suspension in PBS for 4 min at 800 rpm. Cells were fixed in 201C cold methanol for 20 min. Slides were stained for 2 min in 1:1 May–Gru ¨nwald/phosphate buffer pH 6.8 and for 20 min in 6% Giemsa in phosphate buffer pH6.8.

Western blot and gene expression analysis Total RNA was extracted by TRIzol (Invitrogen) from sorted cells according to the manufacturer’s instructions with minor modifications. The quality and quantity of RNA sample were determined by Agilent RNA 6000 Nano Chip (Agilent Technologies). cDNA from purified total RNA was obtained by M-MLV reverse transcriptase (Invitrogen) following manufacturer protocol, and 20 ng of template cDNA were used in TaqMans Real Time PCR (2 min. at 501C, 10 min 951C, 15 s at 951C, 1 min at 601C, for 45 cycles) performed on ABI prism 7900HT (Applied Biosystems) using the following assays-on-demand (Applera): glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as endogenous reference; Arginase 1 liver (Arg1), Ariginase 2 (Arg2), nitric oxide synthase 1, neuronal (Nos1), nitric oxide synthase 2, inducible, macrophage (Nos2), nitric oxide synthase 3, endothelial cell (Nos3), cytochrome b-245, alpha polypeptide (Cyba), cytochrome b-245, beta polypeptide (Cybb), as target gene. Threshold Cycle (Ct) was manually determined and the expression fold change was calculated as described by Applied Biosystem’s User Bulletin ]2. Arginase 1 Western blots were performed with rabbit polyclonal Ab Arginase 1 (H-52): sc-20150 from Santa Cruz Biotecnology; nitric oxide syntase 2 and actin Western blots were performed as described previously [4].

To generate in vitro CTL we performed two different cultures: MLR and MLPC. In MLR, splenocytes (6 105) from BALB/c mice were incubated with 6 105g-irradiated C57BL/6 splenocytes. In some experiments, MLR were also performed using C57BL/6 splenocytes as responders andg-irradiated BALB/c as allogenic stimulators. MLPC were set up using 6 105 BALB/c splenocytes together with 1% HA-specific CD81 T lymphocytes, derived from the spleen of CL4 transgenic mice (bearing a HA512-520 peptidespecific TCR in CD81 T cells), pulsed with 1 mg/mL HA512-520 peptide for 5 days. Alternatively, MLPC were set up with C57BL/ 6, instead of BALB/c, splenocytes together with 2% gp100 specific CD81 T lymphocytes, derived from the spleen of pmel-1 mice (bearing a hgp10025–33 peptide-specific TCR in CD81 T cells), pulsed with 1 mg/mL gp100 peptide for 5 days.

Statistical analysis Student’s t-test was used to compare parametric groups. p-values are all two sides. In figures, asterisks were used as follows: pr0.05; pr0.01; pr0.001.

Acknowledgements: The authors thank Serena Zilio for technical support and Pierantonio Gallo for the assistance with graphics. This work was supported by grants from the Italian Ministry of Health, Fondazione Cassa di Risparmio di Padova e Rovigo, Italian Association for Cancer Research (AIRC), Progetto Locale SUN 2008, Istituto Superiore Sanita` -Alleanza Contro il Cancro (project no. ACC8). Conflict of interest: Markus Geilich and Gregor Winkels are employees of Miltenyi Biotec GmbH, Germany.

ELISA Briefly, 1 106 tumor cells were plated for 48 h in 24-well plates and supernatants were assayed by ELISA for mouse GM-CSF (Endogen), IL-6 (eBioscience), and G-CSF (Biosource) release according to manufacturer’s instructions.

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Abbreviations: CMP: common myeloid progenitor GMP: granulocyte/ macrophage progenitor IFA: incomplete freund adjuvant L-NMMA: NGmonomethyl-L-arginine L.U.30: lytic unit 30 MDSC: myeloid derived suppressor cell MEP: megacaryocyte/erytroid progenitor MnTBAP: manganese (III) tetrakis (4-benzoic acid) porphyrin chloride MO: mononuclear nor-NOHA: Nw-hydroxy-nor-L-arginine PMN: polymorphonuclear shRNA: short hairpin RNA TDSF: tumor derived soluble factors Znpp: protoporphyrin IX zinc(II)

Supporting Information for this article is available at www.wiley-vch.de/contents/jc_2040/2009/39903_s.pdf

Received: 14/8/2009 Revised: 7/10/2009 Accepted: 9/11/2009

Full correspondence: Dr. Vincenzo Bronte, Istituto Oncologico Veneto (IOV), via Gattamelata, 64, 35128 Padua, Italy Fax: 139-049-8072854 e-mail: enzo.bronte@unipd.it


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