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Journal of Clinical Oncology, Vol 25, No 18 (June 20), 2007: pp. 2546-2553 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.08.5829 Identification of a New Subset of Myeloid Suppressor Cells in Peripheral Blood of Melanoma Patients With Modulation by a Granulocyte-Macrophage Colony-Stimulation Factor–Based Antitumor Vaccine
From the Unit of Immunotherapy of Human Tumors and Unit of Medical Statistics and Biometry, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico, Istituto Nazionale Tumori, Milan, Italy Address reprint requests to Licia Rivoltini, MD, Unit of Immunotherapy of Human Tumors, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133, Milan, Italy; e-mail: licia.rivoltini{at}istitutotumori.mi.it
Purpose: Phenotypic and functional features of myeloid suppressor cells (MSC), which are known to serve as critical regulators of antitumor T-cell responses in tumor-bearing mice, are still poorly defined in human cancers. Here, we analyzed myeloid subsets with suppressive activity present in peripheral blood of metastatic melanoma patients and evaluated their modulation by a granulocyte-macrophage colony-stimulating factor (GM-CSF) –based antitumor vaccine. Patients and Methods: Stage IV metastatic melanoma patients (n = 16) vaccinated with autologous tumor-derived heat shock protein peptide complex gp96 (HSPPC-96) and low-dose GM-CSF provided pre- and post-treatment whole blood specimens. Peripheral-blood mononuclear cells (PBMCs) were analyzed by flow cytometry, separated into cellular subsets, and used for in vitro proliferation assays. PBMCs from stage-matched metastatic melanoma patients (n = 12) treated with non–GM-CSF-based vaccines (ie, HSPPC-96 alone or interferon alfa/melanoma–derived peptides) or sex- and age-matched healthy donors (n = 16) were also analyzed for comparison. Results: The lack of or low HLA-DR expression was found to identify a CD14+ cell subset highly suppressive of lymphocyte functions. CD14+HLA-DR–/lo cells were significantly expanded in all metastatic melanoma patients, whereas they were undetectable in healthy donors. Suppressive activity was mediated by transforming growth factor beta (TGF-ß), whereas no involvement of the arginase and inducible nitric oxide synthase pathways could be detected. CD14+HLA-DR–/lo cells, as well as spontaneous ex vivo release and plasma levels of TGF-ß, were augmented after administration of the HSPPC-96/GM-CSF vaccine. No enhancement of the CD14+-mediated suppressive activity was found in patients receiving non–GM-CSF-based vaccines. Conclusion: CD14+HLA-DR–/lo cells exerting TGF-ß–mediated immune suppression represent a new subset of MSC potentially expandable by the administration of GM-CSF–based vaccines in metastatic melanoma patients.
Melanoma is considered the prototype of immunogenic tumors in humans.1 However, a role of the immune system in controlling disease may be claimed only in the initial phases, when the presence of T cells deeply infiltrating tumor lesions favorably impacts prognosis and reduces recurrence risk.2,3 With disease progression, immune responses start displaying functional defects and may even turn into mere indicators of tumor burden.4 In mouse models, these deficiencies are, at least in part, attributed to the accumulation of early differentiated myeloid cells, characterized by the coexpression of CD11b and Gr-1, known as myeloid suppressor cells (MSC).5 This highly plastic population affects T-cell functions through different molecular pathways, mostly involving arginase metabolism products,6 inducible nitric oxide synthase (iNOS),7 reactive oxygen species,8,9 and/or production of soluble inhibitory factors such as transforming growth factor beta (TGF-ß), interleukin (IL) -10, prostaglandin E2, and nitric oxide.10-14 Expansion of MSC should stem from tumor ability to secrete myeloid-influencing factors (ie, colony-stimulating factor-1, IL-10, IL-6, vascular endothelial growth factor, and granulocyte-macrophage colony-stimulating factor [GM-CSF]).15 In particular, GM-CSF recruits MSC into lymphoid secondary organs and suppresses antigen-specific T cells when produced by gene-modified cancer cells or administrated exogenously in tumor-bearing mice.16,17 This evidence raises the possibility that GM-CSF, a cytokine broadly used in cancer patients for its properties on bone marrow mobilization and immune functions, may promote the expansion of myeloid suppressive components, with negative consequences on tumor antigen–specific immune responses.18 Nevertheless, GM-CSF represents one of the adjuvant therapies most commonly used in combination with cancer vaccines for recruiting antigen-presenting cells (APC) at the injection site.19 Indeed, in a recent vaccination study with autologous melanoma cell–derived heat shock protein peptide complex gp96 (HSPPC-96; Oncophage; Antigenics Inc, Lexington, MA), a promising approach both in terms of immunologic and clinical efficacy,20 we injected low-dose GM-CSF to promote dendritic cell accrual and to synergize with HSPPC-96 stimulatory activity on APC.21 However, the inclusion of GM-CSF in the schedule was associated with a lower immunologic potency (with only 29% of patients developing enhanced CD8-mediated antitumor T-cell responses compared with 49% of patients treated with HSPPC-96 alone) and no major antitumor effect.20,21 Although the two treatment schedules were not simultaneously explored in a randomized trial and despite the use of low doses of GM-CSF, these data prompted us to investigate whether the addition of this cytokine could have caused MSC expansion and/or activation, with detrimental consequences on antitumor immune responses. In contrast to murine models, the human counterparts of MSC are still poorly defined, although recent studies in head and neck, breast, lung, and renal carcinomas have reported a potential role of CD34+,22-24 lineagenegHLA-DRneg,25 and CD11b+CD15+26 cells. In the present study, we investigated the phenotypic/functional features of MSC in melanoma patients and identified a new subset of CD14+ monocytes exerting TGF-ß–mediated immunosuppressive activity, which seemed significantly boosted by an antitumor vaccine including GM-CSF.
Patients Sixteen pretreated patients with stage IV metastatic melanoma (according to the American Joint Committee on Cancer) were studied before and after administration of a vaccine including HSPPC-96 (Oncophage; Antigenics Inc), GM-CSF (Leukomax; Novartis-Farma, Varese, Italy), and interferon alfa (IFN-  ; IFN--2b; Schering-Plough, Milan, Italy). Patient characteristics are detailed elsewhere.21 The treatment schedule included four weekly plus four biweekly vaccinations with HSPPC-96, GM-CSF (75 µg administered subcutaneously [SC] at the vaccine site at days –1, 0, and +1 with respect to HSPPC-96), and IFN- (3 MU administered SC 4 and 6 days after HSPPC-96). Twelve stage-matched melanoma patients vaccinated with either HSPPC-96 alone (four weekly plus four biweekly injections, n = 6)20 or Melan-A/MART-126-35/27L and gp100209-217/210M peptides (four biweekly plus four monthly SC administrations, n = 6) and IFN- (3 MU SC on days –1, 0, and +1 with respect to peptides)27 and 16 age- and sex-matched healthy donors were also evaluated. Blood samples were obtained from patients before and after the first vaccination cycle. Peripheral-blood mononuclear cells (PBMCs) were isolated by Ficoll gradient (Ficoll-Paque; Amersham Biosciences, Uppsala, Sweden). These studies were approved by the internal independent ethics committee, and informed consent was obtained from all patients.
Flow Cytometry
Cell Subset Separation and Proliferation Assays
Enzyme-Linked Immunosorbent Assay
Statistical Analysis
Phenotype of Peripheral Myeloid Cell Subsets in Melanoma Patients Compared With Healthy Donors and Modulation by HSPPC-96/GM-CSF Vaccine For identifying MSC in melanoma patients, we first assessed whether the candidate phenotypes previously described in other cancers22-26 could be enriched in these patients or modulated in vivo by an antimelanoma vaccine including low-dose GM-CSF. Patients' PBMCs, obtained before and after treatment with HSPPC-96, GM-CSF, and IFN- (hereafter referred to as HSPPC-96/GM-CSF vaccine), were analyzed for the expression of different myeloid markers and compared with PBMCs from healthy donors. The frequency of MSC candidates (ie, CD34+,22-24 lineagenegHLA-DRneg,25 or CD15+26 cells) was found to be rather low (< 1%) in melanoma patients and overlapping with the frequency detected in healthy donors (Fig 1A). Additionally, no significant change in these frequencies was observed after HSPPC-96/GM-CSF vaccination. In contrast, in postvaccine samples, we noticed a consistent increase of monocytes staining positively for CD14+ and CD11b+, which were coexpressed in the totality of the gated cells (as exemplified in Fig 1B, center panel). As shown in Figure 1B (left panel), the mean difference in the percentage of CD14+CD11b+ cells after versus before vaccine was 7.33% (95% CI of the difference [CID], 1.93% to 3.72%), and the effect size (ES; mean difference scaled over the standard deviation in the control group) was 1.2 (P = .001). These modifications were associated with a significant reduction of CD68 expression in CD14+ cells, which already expressed this marker at a lower level in PBMCs of prevaccine melanoma patients compared with healthy donors.
Among the three components of the vaccine, this effect was apparently linked to GM-CSF because no comparable alterations were detected in stage-matched melanoma patients receiving either HSPPC-96 alone (data not shown) or IFN- /melanoma–derived peptides.27
CD14+ Monocytes From Melanoma Patients Exert Immunosuppressive Activity That Is Increased by HSPPC-96/GM-CSF Vaccine
The boost of lymphocyte proliferation mediated by CD14+ depletion was also detected in six of 12 additional stage-matched melanoma patients, but no further amplification after treatment with either HSPPC-96 alone or IFN- /melanoma–derived peptides was observed (Fig 2B). Furthermore, CD14+ suppressive monocytes were a specific feature of melanoma patients because CD14+ depletion did not result in any proliferative increase in PBMCs from healthy donors (Fig 2C).
The immunosuppressive activity of CD14+ monocyte subsets affected T-cell effector functions as well. Indeed, CD3+ T cells produced higher levels of IFN-
Suppressive Activity of CD14+ Monocytes Involves TGF-ß
Products of the arginase metabolism have also been considered as responsible for MSC activity in tumor-bearing mice.28 Still, we could not detect any arginase expression or activity in PBMCs from melanoma patients or healthy donors, thereby excluding a role of arginase and iNOS metabolism products (online-only Appendix Fig A1).
CD14+HLA-DR–/lo Subset Is Responsible for the TGF-ß–Mediated Suppressive Activity of Melanoma Patients' Monocytes
The present study shows that MSC may be represented in melanoma patients by a subset of circulating monocytes, identified as CD14+HLA-DR–/lo TGF-ß–producing cells. These cells, which likely correspond to monocytes in the early stage of maturation, are significantly and reproducibly increased in melanoma patients compared with healthy donors and are further expanded after administration of an antitumor vaccine that includes GM-CSF. Conversely, melanoma patients do not present any of the myeloid alterations described in patients affected by other cancer histotypes, which include expansion of lineage-negative cells, myeloid CD34 precursors, and immature cells expressing both monocyte (ie, CD11b) and granulocyte (ie, CD15) markers.22-26
The CD14+HLA-DR–/lo monocyte subset of melanoma patients displays a clear suppressive activity on T cells. Indeed, its depletion results in enhanced proliferation and improved effector functions (in terms of perforin and IFN- To the best of our knowledge, immunosuppressive activity has not been previously described as a feature of peripheral monocytes, with the only exception reported by Jaffe et al,29 showing an accumulation of suppressive monocytes in the spleen of colon carcinoma–bearing mice compared with healthy animals. The CD14+HLA-DR–/lo monocyte subset exerts its suppressive activity by release of TGF-ß, as shown by the ability of these cells to spontaneously secrete bioactive TGF-ß ex vivo, a feature undetectable in the CD14+HLA-DR+ counterpart, and by functional experiments with neutralizing mAb. Higher amounts of TGF-ß were also found in sera from melanoma patients compared with healthy donors, with a further amplification after treatment with an HSPPC-96/GM-CSF–based vaccine. In contrast, we could not detect any contribution of arginase and iNOS metabolism products, which have been considered thus far as a functional hallmark of MSC suppressive activity.6,30 However, the key role played by TGF-ß in our system is in agreement with data obtained in several animal models showing that MSC may use a pathway involving TGF-ß for negatively regulating antitumor T-cell immunity.10-11,31 Supporting the link between MSC activity and TGF-ß is our recent observation that melanoma cells can directly induce the in vitro generation of CD14+HLA-DR–/lo cells with TGF-ß–mediated suppressive activity through the release of vesicular organelles.13 The evidence that the lack of HLA-DR expression in myeloid cells may identify an immunosuppressive cell subset is in line with the data reported by Almand et al,25 who described murine MSC as immature myeloid cells expressing low or undetectable levels of major histocompatibility complex class II antigens. In this regard, the reduced levels of HLA-DR expression may not be simply markers of suppressive monocytes, but they may also be directly involved in the ability of these cells to affect lymphocyte activity. Indeed, it has been demonstrated that continuous contact of the T-cell receptor with selected major histocompatibility complex class II molecules is required for T-cell survival.32-34 Our data show that CD14+HLA-DR–/lo TGF-ß–producing cells may represent a new component of the MSC population in melanoma patients. In this regard, it is worth mentioning that the presence of suppressive monocytes, a high percentage of CD14+HLA-DR–/lo cells, and a significant increase in TGF-ß sera levels were associated with a poor immunologic response to the HSPPC-96/GM-CSF vaccine (Fig 2A and data not shown). It should be also pointed out that myeloid cells with similar phenotypic and functional properties have been described in ascitic fluid of ovarian carcinoma patients,14 suggesting that this immunosuppressive cell subset may not be a peculiar feature of melanoma patients.
On the basis of the evidences reported here, the clinical use of GM-CSF could be considered potentially detrimental in cancer patients.18 Indeed, we observed that low-dose GM-CSF, which was administered to recruit dendritic cells at the vaccine site, was associated with the expansion of suppressive CD14+HLA-DR–/lo cells, which seemed to affect vaccine ability to stimulate tumor antigen–specific immune responses. In addition, the spontaneous ex vivo secretion of TGF-ß by PBMCs and the sera levels of TGF-ß were reproducibly increased after GM-CSF–based vaccine. In contrast, none of these boosting effects was observed when other vaccine schedules, including HSPPC-96 alone20 and IFN-
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment: N/A Leadership: N/A Consultant: Giorgio Parmiani, Antigenics Inc Stock: N/A Honoraria: Giorgio Parmiani, Antigenics Inc Research Funds: N/A Testimony: N/A Other: N/A
Conception and design: Paola Filipazzi, Roberta Valenti, Giorgio Parmiani, Licia Rivoltini Financial support: Giorgio Parmiani, Licia Rivoltini Administrative support: Giorgio Parmiani, Licia Rivoltini Provision of study materials or patients: Lorenzo Pilla, Licia Rivoltini Collection and assembly of data: Paola Filipazzi, Roberta Valenti, Veronica Huber, Paola Canese, Manuela Iero, Licia Rivoltini Data analysis and interpretation: Paola Filipazzi, Roberta Valenti, Veronica Huber, Lorenzo Pilla, Chiara Castelli, Licia Rivoltini Manuscript writing: Paola Filipazzi, Veronica Huber, Licia Rivoltini Final approval of manuscript: Giorgio Parmiani, Licia Rivoltini Other: Luigi Mariani [Statistical analysis]
We thank R. Patuzzo, MD, E. Pennacchioli, MD, A. Maurichi, MD, and M. Santinami, MD, (Melanoma-Sarcoma Unit, Istituto Nazionale Tumori of Milan) for patient accrual; R. Milani, MD (Division of Hematology), for hematologic analyses; and A. Cova, MS, G. Sovena, MS, and P. Squarcina, MS, (Immunotherapy Unit of Human Tumors) for technical help.
Supported by grants from the Italian Association for Cancer Research, Program No. 518234 from European Community, and Grant No. 530/F-A17 the Italy-USA Project. R.V. is a scholarship holder of the Italian Foundation for Cancer Research (Milan, Italy). Presented in part at the 20th Annual Meeting of the International Society of Biological Therapy, November 10-13, 2005, Alexandria, VA, and the Keystone Symposium on Advances in the Understanding and Treatment of Melanoma, January 18-23, 2006, Santa Fe, NM. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Bronte V, Zanovello P: Regulation of immune responses by L-arginine metabolism. Nat Rev Immunol 5:641-654, 2005[CrossRef][Medline] 7. Mazzoni A, Bronte V, Visintin A, et al: Myeloid suppressor lines inhibit T cell responses by an NO-dependent mechanism. J Immunol 168:689-695, 2002 8. Kusmartsev S, Gabrilovich DI: Inhibition of myeloid cell differentiation in cancer: The role of reactive oxygen species. J Leukoc Biol 74:186-196, 2003 9. Kusmartsev S, Nefedova Y, Yoder D, et al: Antigen-specific inhibition of CD8+ T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species. J Immunol 172:989-999, 2004 10. Ghiringhelli F, Puig PE, Roux S, et al: Tumor cells convert immature myeloid dendritic cells into TGF-beta-secreting cells inducing CD4+CD25+ regulatory T cell proliferation. J Exp Med 202:919-929, 2005 11. Terabe M, Matsui S, Park JM, et al: Transforming growth factor-beta production and myeloid cells are an effector mechanism through which CD1d-restricted T cells block cytotoxic T lymphocyte-mediated tumor immunosurveillance: Abrogation prevents tumor recurrence. J Exp Med 198:1741-1752, 2003 12. Thomas DA, Massague J: TGF-beta directly targets cytotoxic T cell functions during tumor evasion of immune surveillance. Cancer Cell 8:369-380, 2005[CrossRef][Medline] 13. Valenti R, Huber V, Filipazzi P, et al: Human tumor-released microvesicles promote the differentiation of myeloid cells with TGF-beta-mediated suppressive activity on T lymphocytes. Cancer Res 66:9290-9298, 2006 14. Loercher AE, Nash MA, Kavanagh JJ, et al: Identification of an IL-10-producing HLA-DR-negative monocyte subset in the malignant ascites of patients with ovarian carcinoma that inhibits cytokine protein expression and proliferation of autologous T cells. J Immunol 163:6251-6260, 1999 15. Serafini P, Borrello I, Bronte V: Myeloid suppressor cells in cancer: Recruitment, phenotype, properties, and mechanisms of immune suppression. Semin Cancer Biol 16:53-65, 2006[CrossRef][Medline] 16. Bronte V, Chappell DB, Apolloni E, et al: Unopposed production of granulocyte-macrophage colony-stimulating factor by tumors inhibits CD8+ T cell responses by dysregulating antigen-presenting cell maturation. J Immunol 162:5728-5737, 1999 17. Serafini P, Carbley R, Noonan KA, et al: High-dose granulocyte-macrophage colony-stimulating factor-producing vaccines impair the immune response through the recruitment of myeloid suppressor cells. Cancer Res 64:6337-6343, 2004 18. Parmiani G, Castelli C, Pilla L, et al: Opposite immune functions of GM-CSF administered as vaccine adjuvant in cancer patients. Ann Oncol 18:226-232, 2007 19. Pan PY, Li Y, Li Q, et al: In situ recruitment of antigen-presenting cells by intratumoral GM-CSF gene delivery. Cancer Immunol Immunother 53:17-25, 2004[CrossRef][Medline] 20. Belli F, Testori A, Rivoltini L, et al: Vaccination of metastatic melanoma patients with autologous tumor-derived heat shock protein gp96-peptide complexes: Clinical and immunologic findings. J Clin Oncol 20:4169-4180, 2002 21. Pilla L, Patuzzo R, Rivoltini L, et al: A phase II trial of vaccination with autologous, tumor-derived heat-shock protein peptide complexes Gp96, in combination with GM-CSF and interferon-alpha in metastatic melanoma patients. Cancer Immunol Immunother 55:958-968, 2006[CrossRef][Medline] 22. Pak AS, Wright MA, Matthews JP, et al: Mechanisms of immune suppression in patients with head and neck cancer: Presence of CD34(+) cells which suppress immune functions within cancers that secrete granulocyte-macrophage colony-stimulating factor. Clin Cancer Res 1:95-103, 1995[Abstract] 23. Young MR, Lathers DM: Myeloid progenitor cells mediate immune suppression in patients with head and neck cancers. 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Rodriguez PC, Quiceno DG, Zabaleta J, et al: Arginase I production in the tumor microenvironment by mature myeloid cells inhibits T-cell receptor expression and antigen-specific T-cell responses. Cancer Res 64:5839-5849, 2004 29. Jaffe ML, Arai H, Nabel GJ: Mechanisms of tumor-induced immunosuppression: Evidence for contact-dependent T cell suppression by monocytes. Mol Med 2:692-701, 1996[Medline] 30. Kusmartsev S, Gabrilovich DI: Role of immature myeloid cells in mechanisms of immune evasion in cancer. Cancer Immunol Immunother 55:237-245, 2006[CrossRef][Medline] 31. Young MR, Wright MA, Matthews JP, et al: Suppression of T cell proliferation by tumor-induced granulocyte-macrophage progenitor cells producing transforming growth factor-beta and nitric oxide. J Immunol 156:1916-1922, 1996[Abstract] 32. Brocker T: Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells. J Exp Med 186:1223-1232, 1997 33. Kirberg J, Berns A, von Boehmer H: Peripheral T cell survival requires continual ligation of the T cell receptor to major histocompatibility complex-encoded molecules. J Exp Med 186:1269-1275, 1997 34. Garcia S, DiSanto J, Stockinger B: Following the development of a CD4 T cell response in vivo: From activation to memory formation. Immunity 11:163-171, 1999[CrossRef][Medline] Submitted August 9, 2006; accepted March 12, 2007. Related Correspondence
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
detection, cells were stimulated with phobol-12-myristate-13-acetate (50 ng/mL) plus ionomycin (1 µg/mL), both of which were supplied by Sigma-Aldrich (Deisenhofen, Germany). Cells were then treated with Brefeldin-A (10 µg/mL; Sigma-Aldrich), stained with PerCP-CD3 mAb, permeabilized using BD-Cytofix/Cytoperm Kit (BD Biosciences), and labeled with PE-perforin and APC-IFN-
