1α, 25 Dihydroxyvitamin D (1,25(OH)2D) inhibits the T cell suppressive function of myeloid derived suppressor cells (MDSC)

Abstract

Myeloid derived suppressor cells (MDSC) suppress the ability of cytotoxic T cells to attack and clear tumor cells from the body. The active form of vitamin D, 1,25 dihydroxyvitamin D (1,25(OH)₂D), regulates myeloid cell biology and previous research showed that in mouse models 1,25(OH)₂D reduced the tumor level of CD34+ cells, an MDSC precursor, and reduced metastasis. We tested whether MDSC are vitamin D target cells by examining granulocytic- (G-MDSC) and monocytic (M-MDSC) MDSC from tumors, spleen, and bone marrow. Vitamin D receptor (VDR) mRNA levels are low in MDSC from bone marrow and spleen but are 20-fold higher in tumor MDSC. At all sites, M-MDSC have 4-fold higher VDR mRNA expression than G-MDSC. Bone marrow MDSC were induced to differentiate in vitro into tumor MDSC-like cells by treating with IFN-γ, IL-13, and GM-CSF for 48 h. This treatment significantly elevated Arg1 and Nos2 levels, activated the T cell-suppressive function of MDSC, increased VDR expression 50-fold, and made the MDSC responsive to 1,25(OH)₂D treatment. Importantly, 1,25(OH)₂D treatment reduced the T cell suppressive capacity of cytokine-induced total MDSC and M-MDSC by ≥70 % and tumor-derived M-MDSC by 30-50 %. Consistent with this finding, VDR deletion (KO) increased T cell suppressive function of in vitro M-MDSC by 30 % and of tumor-derived M-MDSC by 50 % and G-MDSC by 400 %. VDR KO did not alter Nos2 mRNA levels but significantly increased Arg1 mRNA levels in tumor M-MDSC by 100 %. In contrast, 1,25(OH)₂D treatment reduced nitric oxide production in both in vitro derived M- and G- MDSC. The major finding of this study is that 1,25(OH)₂D signaling through the VDR decreases the immunosuppressive capability of MDSC. Collectively, our data suggest that activation of vitamin D signaling could be used to suppress MDSC function and release a constraint on T-cell mediated clearance of tumor cells.

Keywords: Cancer; Immunology; Myeloid derived suppressor cell; Vitamin D receptor.

Fig. 1.

VDR Expression is Elevated in Tumor MDSC Subtypes. RM-1 cells were injected ip into 12–14 wk old male C57BL/6 mice. After 7 d, MDSC subtypes were isolated from the tumor-containing ip space (Tumor), the spleen (SP), and bone marrow (BM) via flow cytometry-based cell sorting. VDR mRNA levels were analyzed by qPCR. n=3 (SP) to 8 (BM, Tumor) biological replicates comprised of pools from 4 to 5 mice each. Bars with different letter superscripts are significantly different (p < 0.05).

Fig. 2.

In vitro Induction of MDSC Differentiation Stimulates VDR Expression and Makes MDSC Responsive to 1,25(OH)₂D Treatment. Bone marrow MDSC (CD11b⁺Gr1⁺) were isolated from naïve male mice by FACS and then treated with a cocktail of 25 ng/ml IFNγ, 33 ng/ml IL-13, and 10 ug/ml GM-CSF for 48 h. (A) RNA was isolated from pre-culture (Pre) and post-culture (Post) cells then analyzed by qPCR for Arg1, Nos2 and VDR mRNA levels. * p < 0.05 vs pre-culture. (B) Bone Marrow MDSC were cultured with the three cytokines for 24 h and then treated for the last 24 h with cytokines combined with 10 nM 1,25(OH)₂D (1,25D) or ethanol vehicle (EtOH) prior to analysis of RNA by qPCR for two classical vitamin D target genes, VDR and Cyp24a1. * p < 0.05 vs preculture; #p < 0.05 vs EtOH vehicle.

Fig. 3.

Vitamin D Signaling Inhibits the Suppression of T cell Proliferation by in vitro Induced MDSC. Bone marrow from naïve mice was used to isolate total MDSC (CD11b⁺Gr1⁺) or M-MDSC (CD11b⁺Ly6C^hiLy6G^lo) and these cells were then treated with a cocktail of 25 ng/ml IFNγ, 33 ng/ml IL-13, and 10 ug/ml GM-CSF for 48 h. After 24 h of cytokine treatment, the cells were treated with 10 nM 1,25(OH)₂D or ethanol vehicle (EtOH) in the presence of cytokines for an additional 24 h. Afterwards, total MDSC (A) or M-MDSC (B) were isolated then co-cultured with activated OT-I splenocytes for 18 h. EdU was added for the last 1.5 h to assess T cell proliferation. Two different MDSC:OT-I ratios were used to assess the ability of MDSC to suppress T cell proliferation. (C) M-MDSC were isolated from bone marrow of naïve WT and VDR knockout (KO) Mice. Cells were co-cultured with OT-I splenocytes for 48 h. 24 h before harvest, ovalbumin peptide was added to activate splenocytes (SIINFKL to 1 ug/ml) and EdU was added for the last 1.5 h to assess T-cell proliferation. Differences between groups were determined on ArcSIN-transformed data by t-test (p < 0.05), n = 4 biological replicates of cells pooled from n = 3–5 mice. * p < 0.05 vs EtOH control at the same MDSC:OT-1 ratio.

Fig. 4.

VDR Deletion (VDR KO) Enhances the Ability of Freshly Isolated tumor MDSC to Suppress T Cell Proliferation. RM-1 tumor cells were injected ip into wild-type (WT) and VDR KO mice and after 7 d, tumor MDSC were isolated from the ip space for use in an 18 h T cell proliferation suppression test using pre-activated OT-1 splenocytes. EdU was added for the last 1.5 h to assess T cell proliferation. (A) total tumor MDSC, (B) tumor M-MDSC, and (C) tumor G-MDSC were studied at three MDSC:OT-I cell ratios to capture the maximal suppressive effect on T cell proliferation (n = 3 independent biological replicates from pools of 3–5 mice) * p < 0.05 vs WT at a specific ratio.

Fig. 5.

Effects of VDR Deletion (KO) on Arg1 and Nos2 mRNA levels from Freshly Isolated Tumor MDSC. RM-1 tumor cells were injected ip into wild type (WT) and VDR KO mice. After 7 d, tumor MDSC subtypes were isolated from the ip space for RNA isolation and PCR analysis. (A) Arg1 mRNA and (B) Nos2 mRNA levels were assessed by qPCR (n = 4–6 biological replicates from pools of 3–5 mice). * p < 0.05 G vs M; # p < 0.05 WT vs VDR KO for a subtype within a tissue.

Fig. 6.

1,25(OH)₂D Treatment Reduces the Ability of Freshly Isolated Tumor M-MDSC to Suppress T Cell Proliferation. M-MDSC were isolated from the ip space of mice with RM-1 ip tumors by FACS. Cells were incubated with activated OT-I splenocytes for 18 h in a short-term T cell suppression test. EdU was added for the last 1.5 h to assess T cell proliferation. Different MDSC:OT-I ratios were used to capture the maximal suppressive effect on T cell proliferation. During this time, cells were co-treated with 10 nM 1,25(OH)₂D or ethanol (EtOH) vehicle. (A) Percent suppression of T cell proliferation (n = 4 biological replicates of pooled samples from 3 to 5 mice per replicate). * p < 0.05 vs EtOH group; (B) A separate set of tumor G- or M-MDSC were cultured in the presence of absence of 1,25(OH)₂D for 24 h at which point nitrite production was assessed with the Griess assay. (n = 3 biological replicates from pools of 3–5 mice). Paired t-tests were conducted on log10 transformed data. P values provided for comparisons.