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. 2012 Oct 1;18(19):5212-23.
doi: 10.1158/1078-0432.CCR-12-1108. Epub 2012 Jul 26.

Myeloid cells obtained from the blood but not from the tumor can suppress T-cell proliferation in patients with melanoma

Alena Gros  1 , Simon Turcotte, John R Wunderlich, Mojgan Ahmadzadeh, Mark E Dudley, Steven A Rosenberg
Affiliations

Myeloid cells obtained from the blood but not from the tumor can suppress T-cell proliferation in patients with melanoma

Alena Gros et al. Clin Cancer Res. .

Abstract

Purpose: Myeloid-derived suppressor cells (MDSC) have emerged as an immune-regulatory cell type that is expanded in tumor-bearing mice, but less is known about their immune-suppressive role in patients with cancer.

Experimental design: To study the importance of MDSC in patients with melanoma, we characterized the frequency, phenotype, and suppressive function of blood myeloid-derived cells and tumor-infiltrating myeloid cells in 26 freshly resected melanomas.

Results: Blood and tumor-infiltrating myeloid cells (Lin(-) CD11b(+)) could be phenotypically and morphologically classified into monocytes/macrophages, neutrophils, eosinophils, and immature myeloid cells according to marker expression (CD14(+), CD14(-) CD15(hi), CD14(-) CD15(int), and CD14(-) CD15(-), respectively). In contrast to the expansion of MDSC reported in tumor-bearing mice, we found no differences in the frequency and phenotype of myeloid subsets in the blood of patients with melanoma compared with healthy donors. Myeloid cells represented 12% of the live cells in the melanoma cell suspensions, and were phenotypically diverse with high tumor-to-tumor variability. Interestingly, a positive association was found between the percentage of Tregs and granulocytic cells (Lin(-) CD11b(+) CD14(-)CD15(+)) infiltrating melanoma tumors. However, melanoma-infiltrating myeloid cells displayed impaired suppression of nonspecific T-cell proliferation compared with peripheral blood myeloid cells, in which monocytes and eosinophils were suppressive.

Conclusions: Our findings provide a first characterization of the nature and suppressive function of the melanoma myeloid infiltrate and indicate that the suppressive function of MDSC in patients with melanoma seems far less than that based on murine tumor models.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed by the authors.

Figures

Figure 1.
Figure 1.
Characterization of myeloid cells in peripheral blood of healthy donors and melanoma patients. A, flow cytometry evaluation of expression of Lin (CD3/CD19/CD20/CD56/CD57), CD11b, CD14, and CD15 in whole blood. An example of representative dot plots after excluding aggregates, dead cells, and RBCs is shown. Gates were set based on isotype controls. Numbers represent the percentages from the parental populations gated. Names above FACS plots indicate the population gated that was analyzed. Markers analyzed are indicated in the axis of each FACS plot. The gating strategy used to analyze the samples is illustrated. B, frequency of the different phenotypes of myeloid cells in peripheral blood of healthy donors (HD; n = 10) and patients with melanoma (Mel; n = 20) within all the live cells. Each dot represents one patient. C, frequency of CD14+ HLA-DR− cells within CD14+ cells (left) and frequency of CD14+ HLA-DR− within all live cells (right) in peripheral blood.
Figure 2.
Figure 2.
Morphologic traits of myeloid cells in peripheral blood and tumors of patients with melanoma. Myeloid cells were stained with PI and cell surface antibodies for Lin (CD3/CD56/CD57/CD19/CD20), CD11b, CD14, and CD15, and PI-negative cells (live cells) were sorted according to cell surface expression of Lin, CD11b, CD14, and CD15 and cytospins and Wright-Giemsa stains were conducted. Representative pictures of the populations sorted from patient #2 are shown. Cytospins of myeloid cells sorted from peripheral blood (A) and from melanoma single cells suspension (B).
Figure 3.
Figure 3.
Characterization of myeloid cells infiltrating fresh melanoma tumors. A, flow cytometry evaluation of expression of Lin (CD3/CD19/CD20/CD56/ CD57), CD11b, CD14, and CD15 in melanoma tumor cell digests. An example of representative dot plots after excluding aggregates, dead cells, and RBCs is shown. Gates were set based on isotype controls. B, frequencies of myeloid cells and subpopulations of myeloid cells infiltrating melanoma tumors (n = 26) represented as a percentage of all the live cells in the tumor (after excluding aggregates, dead cells, and RBCs). Each dot represents one melanoma sample. C, differences in the relative frequency of the myeloid subpopulations present in blood and tumors from patients with melanoma (Blood n = 20; Tumor n = 26). D, correlation of tumor-infiltrating myeloid cells and T-regulatory cells. For 11 melanoma tumors, both T-regulatory cell frequency (CD4+ CD25 FOXP3+) and frequency of myeloid cells as well as frequency of CD14+ and CD14- CD15+ within myeloid population were determined and correlation was assessed using a Spearman test.
Figure 4.
Figure 4.
Melanoma-infiltrating myeloid cells are not capable of suppressing T-cell proliferation. Myeloid cells and subpopulations were isolated from blood (A) and fresh melanoma tumor digest (B) of a melanoma patient (P#2) and cocultured together with CFSE-labeled autologous CD3 cells at a 1:1 ratio in presence of anti-CD3/anti-CD28 beads. Four days later, proliferation of T cells was assessed by measuring percentage of T cells that diluted CFSE. C, percentage inhibition of T-cell proliferation exerted by blood and tumor myeloid cells. D, cumulative percentage inhibition of T-cell proliferation exerted by blood (n = 7) and tumor-infiltrating (n = 6) myeloid subsets. Average percentage inhibition of T-cell proliferation in presence of the different myeloid subsets isolated and SEM are represented.
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