Immunosuppressive myeloid-derived suppressor cells are increased in splenocytes from cancer patients

Abstract

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells that are increased in the peripheral blood of cancer patients and limit productive immune responses against tumors. Immunosuppressive MDSCs are well characterized in murine splenic tissue and are found at higher frequencies in spleens of tumor-bearing mice. However, no studies have yet analyzed these cells in parallel human spleens. We hypothesized that MDSCs would be increased in the spleens of human cancer patients, similar to tumor-bearing mice. We compared the frequency and function of MDSC subsets in dissociated human spleen from 16 patients with benign pancreatic cysts and 26 patients with a variety of cancers. We found that total MDSCs (Lin^neg CD11b^pos CD33^pos HLA-DR^neg), granulocytic MDSCs (additional markers CD14^neg CD15^pos), and monocytic MDSCs (CD14^pos CD15^neg) were identified in human spleen. The monocytic subset was the most prominent in both spleen and peripheral blood and the granulocytic subset was expanded in the spleen relative to matched peripheral blood samples. Importantly, the frequency of CD15^pos MDSCs in the spleen was increased in patients with cancer compared to patients with benign pancreatic cysts and was associated with a significantly increased risk of death and decreased overall survival. Finally, MDSCs isolated from the spleen suppressed T cell responses, demonstrating for the first time the functional capacity of human splenic MDSCs. These data suggest that the human spleen is a potential source of large quantities of cells with immunosuppressive function for future characterization and in-depth studies of human MDSCs.

Keywords: Cancer; Human spleen; Immunosuppression; MDSCs.

Fig. 1

Myeloid-derived suppressor cell subsets identified in human spleen tissue. a Human splenocytes or PBMC from the same donor were stained with antibodies specific for Lineage markers (CD3, CD19, and CD56), CD11b, and CD33. The frequency of total MDSCs (Lin−, CD11b+, CD33+, HLA-DR−), CD15 + MDSCs (Lin−, CD11b+, CD33+, HLA-DR−, CD15+, CD14−), and CD14 + MDSCs (Lin−, CD11b+, CD33+, HLA-DR−, CD15−, CD14+) was determined by flow cytometry. b The frequency of MDSCs subsets in total PBMC or splenocytes was compared using ANOVA and compared between PBMC and spleen samples using a paired t test (c)

Fig. 2

The frequency of CD15 + MDSCs in human spleen tissue is increased in patients with cancer. a Human splenocytes were stained as in Fig. 1 and the frequency of MDSCs was compared between patients with cancer (see Table 1) and those with benign pancreatic lesions using Student’s t test. b The frequency of MDSCs was compared between different types of cancer by one-way ANOVA. c Three dimensional spleen volumes were calculated using length, width, and thickness measurements recorded after splenectomy and compared between patients with cancer, across cancer types, and across stages of disease

Fig. 3

A low frequency of CD15+ MDSCs correlates with increased overall survival. a Overall survival of cancer patients with a high (≥0.41%) or low (<0.41%) frequency of CD15+ MDSCs was plotted on a Kaplan–Meir survival curve and compared using a log-rank test. Outcomes of each group are shown in the legend (death events/total patients). b Overall survival of cancer patients diagnosed with benign cysts, pancreatic adenocarcinoma, pancreatic neuroendocrine, or other cancers (melanoma, ovarian, and colon cancer) were compared using a log-rank test. Outcomes of each group are shown in the legend (death events/total patients)

Fig. 4

MDSCs isolated from human spleen tissue suppress T cell proliferation and activation. a The purity of CD15+, CD14+ MDSCs, and HLA-DR + control cells was determined after separation by FACS. b H&E stains of sorted CD15+ and CD14+ MDSCs imbedded in paraffin. c CFSE-labeled T cells were stimulated with allogenic dendritic cells in the presence or absence of HLA-DR+ control cells and MDSCs. After 4 days, cells were stained with antibodies specific for CD3, CD8, and CD25 and the frequency of divided (CD25⁺ CFSE^low) CD8 T cells was determined by flow cytometry. The histogram depicts CD3⁺ CD8⁺ cells. d The percentage of divided T cells in the presence of HLA-DR+ control cells and MDSCs was determined as in c and compared using one-way ANOVA (***p < 0.0001), p values shown are adjusted for multiple comparisons across groups. e The percent suppression of proliferation by CD15+ or CD14+ MDSCs was determined for each sample relative to HLA-DR+ cells and compared using a t test (no significant difference) in patient samples with CD14 functional analysis