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. 2010 Aug;123(1):39-49.
doi: 10.1007/s10549-009-0622-8. Epub 2009 Nov 8.

GM-CSF is one of the main breast tumor-derived soluble factors involved in the differentiation of CD11b-Gr1- bone marrow progenitor cells into myeloid-derived suppressor cells

Johanna K Morales  1 , Maciej Kmieciak, Keith L Knutson, Harry D Bear, Masoud H Manjili
Affiliations

GM-CSF is one of the main breast tumor-derived soluble factors involved in the differentiation of CD11b-Gr1- bone marrow progenitor cells into myeloid-derived suppressor cells

Johanna K Morales et al. Breast Cancer Res Treat. 2010 Aug.

Abstract

Recent reports have shown the involvement of tumor burden as well as GM-CSF in supporting myeloid-derived suppressor cells (MDSC). However, it is not known what progenitor cells may differentiate into MDSC in the presence of GM-CSF, and whether FVBN202 transgenic mouse model of spontaneous breast carcinoma may exhibit distinct subset distribution of CD11b+Gr1+ cells. In addition, it is not known why CD11b+Gr1+ cells derived from tumor-free and tumor-bearing animals exhibit different functions. In this study, we determined that GM-CSF was one of the tumor-derived soluble factors that induced differentiation of CD11b-Gr1- progenitor cells from within monocytic/granulocytic bone marrow cells into CD11b+Gr1+ cells. We also showed that CD11b+Gr1+ cells in FVBN202 mice consisted of CD11b+Ly6G-Ly6C+ suppressive and CD11b+Ly6G+Ly6C+ non-suppressive subsets. Previously reported variations between tumor-free and tumor-bearing animals in the function of their CD11b+Gr1+ cells were found to be due to the variations in the proportion of these two subsets. Therefore, increasing ratios of CD11b+Gr1+ cells derived from tumor-free animals revealed their suppressive activity on T cells, in vitro. Importantly, GM-CSF supported the generation of CD11b+Ly6G-Ly6C+ suppressor subsets that inhibited proliferation as well as anti-tumor function of neu-specific T cells. These findings suggest revisiting the use of GM-CSF for the expansion of dendritic cells, ex vivo, for cell-based immunotherapy or as an adjuvant for vaccines for patients with cancer in whom MDSC play a major role in the suppression of anti-tumor immune responses.

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Figures

Fig. 1
Fig. 1
MMC-derived soluble factors cause the accumulation of CD11b+Gr1+ cells in FVB mice. FVB mice (3 per group) were injected intradermally with MMC-conditioned medium (CM) or with control medium (RPMI) for 3 consecutive days and killed on the fourth day. a The total percentage of CD11b+Gr1+ cells in the spleens, bone marrow, and blood of medium-treated (RPMI) or CM-treated (CM) mice as measure by flow cytometry. b Multiplex array analysis of MMC-derived supernatant after a 24-h culture shows substantial levels of GM-CSF, VEGF, and MCP-1. Data are averages of duplicate samples
Fig 2
Fig 2
MMC-derived soluble factors and GM-CSF can generate CD11b+Gr1+ cells from the CD11b-Gr1-progenitor cells, and can maintain existing CD11b+Gr1+ cells. Bone marrow cells from naïve FVBN202 mice were stained and sorted into two populations: CD11b+Gr1+ and CD11b-Gr1-cells. a Flow cytometry plots of the purity of CD11b+Gr1+ and CD11b-Gr1- populations after sorting. Data are representative of three experiments. b Representative flow cytometry plots showing the percentage of CD11b+Gr1+ cells 6 days after culture of sorted CD11b-Gr1-cells (left) and averages of 3–4 experiments (right). c Representative flow cytometry plots showing the percentage of CD11b+Gr1+ cells remaining on day 6 after culture of sorted CD11b+Gr1+ cells (left) and averaged data from 3–4 experiments (right). (d) Representative flow cytometry staining for CD11b and Gr1 from naïve FVBN202 BM cells on day 0 (left) and after 6 days of culture with 100 ng/ml of GM-CSF. Data are representative of 3–5 separate experiments. e Representative data from duplicate experiments showing BrdU incorporation in gated CD4+ and CD8+ T cells derived from FVB mice and cultured for 72 h in the absence (top panel) or presence of CD11b+Gr1+ cells (bottom panel) generated from culture of unfractionated bone marrow cells (d) with GM-CSF
Fig. 3
Fig. 3
MMC-derived soluble factors and GM-CSF can protect newly derived and existing CD11b+Gr1+ cells from apoptosis. Sorted populations of CD11b+Gr1+ or CD11b-Gr1-cells were cultured for 6 days with medium, MMC, or GM-CSF and stained on day 6 for CD11b, Gr1, and Annexin V. a Representative flow cytometry plots of duplicate experiments showing the expression of Annexin V on sorted CD11b+Gr1+ cells (left) or sorted CD11b-Gr1- cells (right) just after sorting. b Representative flow cytometry plots of CD11b+Gr1+ cells after 6 days of culture of sorted CD11b-Gr1- cells (boxed region) that are Annexin V positive. Bar graph shows the average percentage of CD11b+Gr1+AnnexinV+ cells from 3–4 experiments. c Representative flow cytometry plots of CD11b+Gr1+ after 6 days of culture of sorted CD11b+Gr1+ cells (boxed region) that are Annexin V positive. Bar graph depicts the averages of 3–4 experiments
Fig. 4
Fig. 4
The CD11b+Ly6G−Ly6C+ MDSC cause inhibition of T cell proliferation. Bone marrow (BM) cells (a) or splenocytes (b) were isolated from tumor-free (TF) or tumor-bearing (TB) FVBN202 mice (n = 3 per group) and stained for CD11b, Ly6G, and Ly6C and analyzed by flow cytometry. Results are the percentage total cells. (c–d) Sorted CD11b+Ly6G−Ly6C+ and CD11b+Ly6G+Ly6C+ cells from tumor-bearing FVBN202 spleens were cultured at a 1:2 ratio with BrdU-pulsed splenocytes from FVB mice with CD3/CD28 stimulation and stained on day 3 for BrdU incorporation and CD4 and CD8 expression. Representative BrdU plots are gated either on CD4+ (c) or CD8+ (d) lymphocytes. Bar graphs are the averages of triplicate experiments, with the percent suppression of wells containing MDSC derived by: (percent BrdU+ cells in experimental wells/percent BrdU+ cells in splenocyte alone wells) × 100. Each control well was set at 100% and each experimental well was divided by its own corresponding control well and these numbers averaged and presented ±SEM
Fig. 5
Fig. 5
MMC-derived supernatant and GM-CSF cause the generation of suppressive CD11b+Ly6G−Ly6C+ MDSC from CD11b−Gr1− precursor cells. a Sorted CD11b+Gr1+ cells from bone marrow (BM) of tumor-free FVBN202 mice (n = 3) were analyzed on day 0 for the expression of Ly6G and Ly6C by flow cytometry. CD11b−Gr1− cells sorted from these mice and cultured with GM-CSF were analyzed on day 6 for the expression of Ly6G and Ly6C by flow cytometry. b Representative data from duplicate experiments showing BrdU incorporation in gated CD4+ and CD8+ T cells 72 h after culture in the absence (top panel) or presence of cells (bottom panel) that were generated from a 6-day culture of CD11b−Gr1− progenitor cells with GM-CSF. c Gated neu positive MMC were analyzed for Annexin V and PI after a 48 h culture with neu-specific T cells. d Representative data from duplicate experiments showing BrdU incorporation in gated CD4+ and CD8+ T cells 72 h after culture in the absence (top panel) or presence of CD11b+Gr1+ cells derived from tumor-free mice and used at 1:1 (middle panel) or 1:2 (bottom panel) rations
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