ER stress regulates myeloid-derived suppressor cell fate through TRAIL-R-mediated apoptosis

Abstract

Myeloid-derived suppressor cells (MDSCs) dampen the immune response thorough inhibition of T cell activation and proliferation and often are expanded in pathological conditions. Here, we studied the fate of MDSCs in cancer. Unexpectedly, MDSCs had lower viability and a shorter half-life in tumor-bearing mice compared with neutrophils and monocytes. The reduction of MDSC viability was due to increased apoptosis, which was mediated by increased expression of TNF-related apoptosis-induced ligand receptors (TRAIL-Rs) in these cells. Targeting TRAIL-Rs in naive mice did not affect myeloid cell populations, but it dramatically reduced the presence of MDSCs and improved immune responses in tumor-bearing mice. Treatment of myeloid cells with proinflammatory cytokines did not affect TRAIL-R expression; however, induction of ER stress in myeloid cells recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts. The ER stress response was detected in MDSCs isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes, and blockade of ER stress abrogated tumor-associated changes in TRAIL-Rs. Together, these data indicate that MDSC pathophysiology is linked to ER stress, which shortens the lifespan of these cells in the periphery and promotes expansion in BM. Furthermore, TRAIL-Rs can be considered as potential targets for selectively inhibiting MDSCs.

Figure 1. MDSCs have shorter survival than their naive counterparts.

(A–C) BrdU pulse-chase analysis of MDSC turnover (n = 3 mice per time point). (A and B) BrdU⁺ cells in PB monocytes and M-MDSCs (A) and in PB PMNs and PMN-MDSCs (B) during pulse. (C) BrdU⁺ cells in PMNs and PMN-MDSCs in blood and spleen during chase. (D–F) MDSC/IMC ratio in different organs 6 (D) or 20 (E and F) hours after i.v. injection of MDSC/IMC mix (1:1 ratio) into TB recipients. (D and E) 1 representative experiment. Cell count and percentages of boxed regions are indicated. (F) Combined results; each symbol represents 1 mouse (n = 4). (G) Recovery of myeloid cells isolated from BM or spleen of control or EL4 TB mice, after 20 hours of culture in complete media without cytokines (n = 3). 2 × 10⁵ purified cells were plated at the beginning of culture. (H) Naive and EL4 TB mice were injected i.p. with 12% casein 20 and 4 hours prior to harvesting of peritoneal fluid. Ly6G⁺ cells were purified using magnetic beads and cultured overnight in complete media without cytokines. Shown is the proportion of surviving cells (relative to input) in 3 samples per group. (I) Purified IMCs and MDSCs were labeled with 2 different cell trackers (1:1 CMFDA/DDAO). Labeled cells were cultured alone in medium or mixed at a 1:5 ratio with freshly isolated splenocytes or with tumor, lung, or liver cells, then cultured overnight in complete media. The MDSC/IMC ratio was then calculated (n = 3). P values are compared with medium alone. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. MDSCs undergo rapid apoptosis mediated by upregulation of DR5 expression.

(A and B) Apoptosis was analyzed based on percentages of cleaved caspase-3⁺ cells after 3 hours in culture (A) and of annexin V⁺ cells after 6 hours (B). Results represent the average of 4 different experiments. (C) Amount of BCL-2, BCL-XL, cleaved caspase-8, cleaved caspase-9, and cleaved caspase-3 was determined after a 3-hour culture of cells by Western blot. 3 experiments were performed with the same results. (D) Cells were cultured in the presence of the caspase-8 inhibitor Z-IETD-FMK (100 μM) or DMSO (control), and the percentage of apoptotic cells (cleaved caspase-3⁺) was determined after 3 hours. Representative results of 3 different experiments are shown. (E) Receptor expression in BM IMCs or MDSCs, determined using quantitative RT-PCR. Results represent the average of 3 different samples. (F and G) DR5 expression on the surface of PMN-MDSCs (F) or M-MDSCs (G) in spleens of different TB mice or from control PMNs (F) or monocytes (G) isolated from corresponding C57BL/6 or Balb/c mice (3–5 different samples). (H) DR5 expression on the surface of PMN-MDSCs and M-MDSCs from spleen (solid line) or tumor site (dotted line) from EL4 TB mice. Gray filled histogram, isotype control. Results are representative of 4 different experiments. *P < 0.05.

Figure 3. TRAIL-R is a potential target for MDSC removal.

(A) IMCs and MDSCs (2 × 10⁵ plated) were cultured in complete media overnight in the presence of different concentrations of recombinant OPG. Results represent the average number of cells recovered at the end of culture (n = 3). (B and C) Congenic CD45.1⁺ WT mice were lethally irradiated and reconstituted with WT or Dr5 KO BM cells. (B) 3 weeks after tumor inoculation, mice were sacrificed, and the presence of CD11b⁺Gr1⁺ MDSCs in spleen and liver was analyzed. (C) MDSCs were isolated from spleens and cultured in complete media. After 3 hours, the percentage of cleaved caspase-3⁺ cells was assessed by flow cytometry (results are representative of 5 experiments). (D) Number of cells recovered after overnight culture of 2 × 10⁵ MDSCs (results of 5 different samples). (E and F) Naive CD45.1⁺ mice were lethally irradiated and reconstituted with a 1:1 mix of BM cells from CD45.2⁺ Dr5 KO and CD45.1⁺ WT mice (10⁶ cells). 5 weeks after reconstitution, mice were inoculated s.c. with EL4 cells, and spleens were collected 3 weeks later. The percentage of WT (CD45.1⁺) and Dr5 KO (CD45.2⁺) cells was analyzed in spleens among different cell populations. (E) Typical example of results. (F) Cumulative results of 3 experiments. *P < 0.05; **P < 0.01.

Figure 4. DR5 targeting results in selective MDSC elimination.

(A and B) Splenic PMNs and PMN-MDSCs were cultured overnight in complete media supplemented with 10 ng/ml GM-CSF, in a plate coated with MD5-1 mAb or control IgG (10 μg/ml). After 20 hours, percentages of annexin V⁺ cells (A) and survival (B) were determined. Results are representative of 3 different experiments. (C) Total MDSCs, PMN-MDSCs, and M-MDSCs in EL4 TB spleens, measured by flow cytometry. Treatment with control IgG and MD5-1 mAb (100 μg) was initiated on day 17 after tumor inoculation, when tumor diameter reached 1.5 cm, and repeated on days 20 and 23; mice were sacrificed on day 24. (n = 4). (D) EG7 TB mice were treated with MD5-1 mAb (100 μg) and/or anti-CD8 mAb (200 μg) or left untreated, and tumor growth was determined (n = 4 per group). (E) On day 28 after tumor inoculation, mice were sacrificed; splenic T cells were enriched and stimulated in the presence of control DCs, loaded with OVA or irrelevant protein. IFN-γ secretion was assessed by Elispot after 48 hours of restimulation (n = 3). (F) Naive (freshly isolated) or activated (3 days culture with 100 ng/ml SIINFEKL) OT-I splenocytes were stimulated with control or specific peptide (100 ng/ml SIINFEKL) in the presence of control IgG or MD5-1 mAb (10 μg/ml). IFN-γ secretion was assessed by Elispot after 48 hours of restimulation (n = 3). (G) EL4 TB mice were treated with anti-CTLA4 and/or anti–MD5-1 mAb (100 μg each) or left untreated, and tumor growth was determined (n = 4 per group). *P < 0.05; **P < 0.01.

Figure 5. Short life-span of MDSCs contributes to their expansion.

(A–F) Naive mice received 1 injection of 250 μg anti-Gr1 mAb to deplete PMNs from the periphery, then examined 2 (A–C) and 4 (D–F) days after treatment. (A and D) Gr1 depletion was assessed in blood and spleen; typical results are shown. (B, C, E, and F) Mice were then injected with BrdU i.p. and sacrificed 4 hours later. (B and E) BM IMCs, assessed by flow cytometry (n = 3). (C and F) BrdU incorporation in BM IMCs (CD11b⁺Ly6C⁺ cells gated), assessed by flow cytometry. Shown are 1 representative sample (left) and cumulative results (right; n = 3). (G) 3 weeks after EL4 inoculation, TB WT or Dr5 KO mice were injected i.p. with BrdU for 4 hours, and spleen MDSCs were analyzed for BrdU incorporation (percent BrdU⁺ cells). Shown are 1 representative sample (left) and cumulative results (right; n = 3). (H) Mice were injected i.p. with 250 μg Gr1 or control IgG mAb. The absolute number of monocytes and PMNs was determined in spleens 9 days after injection (n = 3). *P < 0.05; **P < 0.01.

Figure 6. ER stress response drives TRAIL-R upregulation in MDSCs.

(A and B) Hematopoietic progenitor cells were cultured for 5 days in the presence of 20 ng/ml GM-CSF. (A) DR5 expression on the surface of PMNs stimulated for 24 hours in the presence of IL-1β, TNF-α, IL-6, IFN-γ, or tunicamycin (TUN) or left unstimulated (NS). Results of 3 experiments are shown. (B) DR5 expression on the surface of PMNs and monocytes stimulated with tunicamycin (dotted line) or left unstimulated (solid line) for 24 hours. Gray filled histogram, isotype control. Shown is 1 representative experiment of 4. (C and D) Purified PMNs from BM were treated overnight with thapsigargin (THG) or left untreated. DR5 expression (C) and cell viability (D) were assessed by flow cytometry (n = 3). (E and F) Electron microscopy analysis of cell morphology. PMNs and PMN-MDSCs (E) and monocytes and M-MDSCs (F) were isolated from spleen of control and EL4 TB mice. Shown are representative microphotographs (arrows denote ER dilation) and the proportion of cells with each ER dilation score (see Methods). Scale bars: 1 μm. (G) Chop, Xbp1, Atf4, and Bip expression in splenic PMNs and PMN-MDSCs, determined using quantitative RT-PCR. Results are from 3 different samples. (H and I) Spliced XBP1 and CHOP in PMNs and PMN-MDSCs from EL4 TB mice (H) or KPC TB mice (spleen cells only; I), determined by Western blot. Results are representative of 4 different samples. *P < 0.05; **P < 0.01.

Figure 7. Human MDSC survival and TRAIL-R expression.

(A) Human PMN-MDSCs and PMNs were isolated from blood of NSCLC patients and cultured overnight in complete media. The percentage of cells was determined by flow cytometry at the beginning and end of culture, and the total number of surviving PMN-MDSCs and PMNs was calculated (n = 3). (B and C) Human PMN-MDSCs and PMNs were cultured in complete media supplemented with 10 ng/ml GM-CSF, in the presence of agonistic anti-DR5 mAb (200 ng/ml) (B) or recombinant TRAIL (100 ng/ml) (C), and the percentage of surviving cells was determined (n = 3). (D) DR4, DR5, DCR1, and DCR2 expression on the surface of PMN-MDSCs and PMNs from the blood of healthy donors (HD) or NSCLC patients. (E) DCR1 and DCR2 expression in PMNs (solid line) and PMN-MDSCs (dotted line). Gray filled histogram, isotype control. Results show a typical example of 6 different patient samples. (F) DR5, DCR1, and DCR2 expression on the surface of blood or tumor PMN-MDSCs from 5 HNC patients and PMNs from healthy donors. *P < 0.05; **P < 0.01.

Figure 8. ER stress response modulates TRAIL-R expression in human MDSCs.

(A) Amounts of spliced XBP1 and CHOP in PMNs and PMN-MDSCs purified from healthy donor or NSCLC patient blood, determined by Western blot. Results are representative of 5 different samples. (B) Human PMNs from healthy donors were cultured in complete media supplemented with 10 ng/ml GM-CSF for 24 hours in the presence of 1 μM thapsigargin. DCR1 and DCR2 expression was assessed by flow cytometry. Shown are representative flow histograms and percentage of MFI decrease (n = 3). (C and D) Healthy donor PMNs were cultured with GM-CSF for 48 hours in the presence of 20% TCM (PCI30 cells). (C) Chop, Xbp1, Atf4, and Bip expression, determined by quantitative RT-PCR. Results represent the average of 4 different samples (D) PMNs were preincubated with 500 μg/ml TUDCA (Calbiochem) for 3 hours prior to the addition of TCM. After culture, cells were harvested, and DCR1 and DCR2 expression was analyzed. Data are mean ± SD of 4 different samples. *P < 0.05.

Figure 9. Mechanism of different regulation of DR5 in human and mouse PMNs.

(A) SK-MEL melanoma tumor cells and MDA231 breast cancer cells were treated overnight with thapsigargin (dotted line) or left untreated (solid line), and DR5 expression was assessed by flow cytometry. Gray filled histogram, isotype control. Typical result is shown. (B) Mouse PMNs were purified from BM of naive mice and cultured for 24 hours in the presence of thapsigargin in combination with VPA. DR5 expression was assessed by flow cytometry (n = 3). (C) Effect of VPA and THG combination on DR5 expression in PMNs. DR5 expression was assessed by flow cytometry. Gray filled histogram, isotype control; black solid line, untreated; gray solid line, VPA alone; dotted line, VPA plus THG. Shown is 1 representative staining of 3 different experiments. (D) ChIP of DR5 promoter with acetylated histone H3 or acetylated histone H4 antibodies in healthy donor PMNs and the SK-MEL melanoma cell line cultured overnight with or without VPA. Results are expressed as DNA enrichment normalized to corresponding input value. (E) Potential role of ER stress and TRAIL-Rs in the fate of MDSCs.