TNF signaling drives myeloid-derived suppressor cell accumulation (original) (raw)
Transplanted tumors are often spontaneously rejected in Tnfr–/– mice. We previously reported that J558L tumors are often spontaneously rejected in mice deficient in both TNFRs (referred to herein as Tnfr–/– mice), for unknown reasons (27). To exclude possible artifacts due to the contamination of microorganisms, J558L cells were passed 3 times in nude mice and then injected into mice. As shown in Figure 1A, beginning at 10 days after tumor cell inoculation, 4 of 7 tumors were spontaneously rejected in Tnfr–/– mice. To exclude the possibility that TNFRs on tumor cells act as antigens causing tumor rejection, we established several Tnfr1–/– and Tnfr–/– tumor cell lines by MCA-induced carcinogenesis in Tnfr1–/– and Tnfr–/– mice. These cells did not express the corresponding TNFRs, and the downstream signaling pathways were defective, as confirmed for FB61 cells by flow cytometry and Western blot (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI64115DS1). Subsequently, the Tnfr1–/– fibrosarcoma FB61 cells were subcutaneously injected into Tnfr–/– and Tnfr+/+ mice (Figure 1B). Whereas 6 of 7 FB61 tumors were spontaneously rejected within around 3 weeks in Tnfr–/– mice, all 5 FB61 tumors in Tnfr+/+ mice grew progressively during the same time period. Tnfr–/– FD99 tumor cells, originating from Tnfr–/– mice, were also tested. As shown in Figure 1C, all 7 FD99 tumors were completely rejected in Tnfr–/– mice, but none of the tumors did so in the Tnfr+/+ counterparts. These results demonstrated that the spontaneous tumor rejection in Tnfr–/– mice was caused not by immunogenicity of TNFR proteins on transplanted tumor cells, but by TNFR expression on host cells.
Transplanted tumors are spontaneously rejected in Tnfr–/– mice. Tnfr+/+ (n = 4–5) and Tnfr–/– (n = 7) mice were subcutaneously injected with (A) 5 × 106 J558L cells, (B) 1 × 106 FB61 cells, or (C) 1 × 106 FD99 cells. Tumor volumes after tumor cell inoculation are shown; each line represents the growth curve of a tumor in a single mouse. Similar results were obtained from 2 other independent experiments.
Tumor rejection is associated with impaired peripheral accumulation of MDSCs. To address the mechanisms responsible for the aberrant tumor growth in Tnfr–/– mice, we first analyzed immune cell populations in peripheral organs, such as tumors and spleens, at days 8–10, when tumors began to regress in Tnfr–/– mice. No clear difference was found for CD11c+ dendritic cells, B220+ B cells, or CD8+ cytotoxic or CD4+ T cells (including CD4+Foxp3+ regulatory T cells and CD4+ Th17 cells) between FB61 tumor–bearing Tnfr–/– and Tnfr+/+ mouse spleens (Supplemental Figure 2A). The density of blood vessels, as well as infiltrated CD4+ and CD8+ T cells in the tumor, were also similar between Tnfr–/– and Tnfr+/+ mice (Supplemental Figure 2B). However, the percentages and absolute numbers of splenic CD11b+Gr1+ myeloid cells in tumor-bearing Tnfr–/– mice were drastically reduced compared with those in their Tnfr+/+ counterparts (Figure 2, A and B). Decreased proportions of CD11b+Gr1+ cells in tumor-bearing Tnfr–/– mice were also observed in the peripheral blood and within the tumor (Figure 2, C and D). CD11b+Gr1+ cells from both Tnfr+/+ and Tnfr–/– tumor-bearing mice, but not from control mice, inhibited the proliferation of CD4+ T cells (Supplemental Figure 3A). Compared with the cells from control animals, they exhibited increased arginase and iNOS activities and produced higher levels of immune-suppressive cytokine IL-10 (Supplemental Figure 3, B–D). Therefore, the CD11b+Gr1+ cells from the tumor-bearing mice were also named as MDSCs (13, 28, 29).
Peripheral accumulation of CD11b+Gr1+ cells is impaired in Tnfr–/– mice. (A) Tnfr+/+ and Tnfr–/– mice were subcutaneously injected with 1 × 106 FB61 cells or PBS as control. 8–10 days after tumor cell inoculation, single splenocytes were stained for CD11b and Gr1 and assessed by flow cytometry. Left: Gated CD11b+Gr1+ cells. Right: Percent CD11b+Gr1+ cells in tumor-bearing Tnfr+/+ and Tnfr–/– mice and corresponding controls. Bars denote means. **P < 0.01. (B) Absolute number of CD11b+Gr1+ cells in spleens of tumor-bearing mice and corresponding controls. Data are mean ± SEM. *P < 0.05. (C and D) Percent CD11b+Gr1+ cells relative to total cells for (C) peripheral blood and (D) tumor tissues. n = 5 per group. Data are mean ± SEM. *P < 0.05. (E) CD11b+ and Gr1+ cells in tumor and spleen sections of Tnfr+/+ and Tnfr–/– mice were visualized by immunofluorescence staining. Nuclei were counterstained with DAPI. Images are representative of at least 3 mice per group. Original magnification, ×200 (tumor); ×100 (spleen). Scale bars: 300 μm. (F) Spleen cells as above were stained for flow cytometry analysis. Unstained Tnfr+/+ splenocytes were used as a control.
To investigate local distributions of the CD11b+Gr1+ MDSCs in the absence of TNFR, immunofluorescence staining was performed in tumors and spleens. Although the number of CD11b+ and Gr1+ cells was reduced within Tnfr–/– tumor sites, the distribution of cells did not differ in Tnfr+/+ and Tnfr–/– mice (Figure 2E). In spleens, germinal center structures were disturbed in Tnfr–/– mice, but again, the distribution patterns of CD11b+ and Gr1+ cells were similar to those of Tnfr+/+ controls. The activation status of Tnfr–/– compared with Tnfr+/+ MDSCs showed no obvious changes in surface expression of the costimulatory molecules CD80, CD86, and B7-H1; the cytokine receptor IL-4R; the antigenic peptide–presenting molecules H2-Kd and I-Ad; and the apoptosis-related molecules CD95 and CD95L (Figure 2F). These results indicated that TNF signaling contributed to enhanced MDSC accumulation in spleen and tumor, but did not influence the cells’ immunosuppressive function and distribution in organs.
Adoptive transfer of Tnfr+/+ MDSCs restores tumor growth in Tnfr–/– mice in a dose-dependent manner. To confirm that the lack of TNFRs on MDSCs was attributable to impaired Tnfr–/– mouse tumor growth, we purified Tnfr+/+ and Tnfr–/– spleen MDSCs and adoptively transferred them to Tnfr–/– and Tnfr+/+ mice. Subsequently, Tnfr1–/– FB61 fibrosarcoma cells were used to challenge the MDSC recipient mice. In Tnfr–/– recipients of Tnfr–/– MDSCs (referred to herein as Tnfr–/–_→_Tnfr–/– mice), all tumors grew for a few days before the expected regression (Figure 3A). These mice were protected against subsequent FB61 challenges (data not shown), indicative of the establishment of effective antitumor immunity. However, in _Tnfr+/+→_Tnfr–/– mice, all tumors grew progressively and reached a mean volume of 700 ± 100 mm3 within 16 days, which highlighted that TNFR expression on MDSCs was crucial for tumor development. The recovery of tumor growth could be attributed to the attenuated antitumor T cell response, since IFN-γ production and tumor-specific CD8+ T cell proliferation were significantly suppressed in these mice (Supplemental Figure 4). When Tnfr–/–_→_Tnfr+/+ mice were challenged with tumors, all tumors grew, but did so more slowly than those in Tnfr+/+→_Tnfr+/+ mice (Figure 3B). As Tnfr–/– MDSCs — like Tnfr+/+ MDSCs — were immunosuppressive (Supplemental Figure 3), this retarded tumor growth may be due to the quantity, but not the quality, of transferred MDSCs. To confirm this hypothesis, Tnfr–/– mice were adoptively transferred with decreasing dose of Tnfr+/+ CD11b+Gr1+ cells and challenged with FB61 tumor cells. As shown in Figure 3C, a dose of more than 1 × 106_Tnfr+/+ MDSCs per mouse was necessary to make a significant tumor-promoting effect within 16 days of tumor cell injection.
Adoptive transfer of Tnfr+/+ CD11b+Gr1+ cells restores tumor growth in Tnfr–/– mice in a dose-dependent manner. (A and B) Purified Tnfr+/+ or Tnfr–/– CD11b+Gr1+ cells (5 × 106) were intravenously injected into Tnfr–/– (A; n = 5–6) or Tnfr+/+ (B; n = 3–4) mice. Untreated mice served as controls. 12 hours later, mice were subcutaneously injected with 1 × 106 FB61 cells. Shown are tumor volumes after tumor cell inoculation. Lines represent growth curves of a tumor in a single mouse; bars denote mean tumor size of each group at day 16. *P < 0.05; **P < 0.01. (C) Tnfr–/– mice were adoptively transferred without or with 2 × 105, 1 × 106, or 5 × 106 purified Tnfr+/+ CD11b+Gr1+ cells by intravenous injection; 12 hours later, they were injected subcutaneously with 1 × 106 FB61 cells. Tumor volumes are presented as mean ± SEM. n = 5 or 7 per group. *P < 0.05; **P < 0.01.
To further study the fate of MDSCs after adoptive transfer, CD11b+Gr1+ cells were isolated from eGFP-transgenic mice bearing MCA205 (also an MCA-induced fibrosarcoma cell line in C57BL/6 mice) tumors. Similar to the experiments above, cells were adoptively transferred, and MCA205 tumor cells were inoculated into syngeneic C57BL/6 recipient mice. Although in peripheral blood, eGFP+ cells were hardly detectable at day 3 after tumor cell inoculation (Supplemental Figure 5, A and B), a group of these eGFP+ cells could be traced in spleen until day 7 and in tumors until day 10, which indicates that adoptively transferred MDSCs did participate in tumor development. Furthermore, eGFP+ MDSCs were detected in the vicinity of T cells in the outer rim of tumors (Supplemental Figure 5C), indicative of effective suppression of local T cell immunity by donor MDSCs. Together, these results strongly suggest that impaired peripheral accumulation of MDSCs accounts for impaired tumor growth in Tnfr–/– mice. TNFR expression on MDSCs was necessary and sufficient for the MDSC-mediated tumor-promoting effect.
Enhanced apoptosis is responsible for reduced MDSC numbers in Tnfr–/– mice. It is well known that TNF elicits different cellular responses, such as cell proliferation or apoptosis (30). Because the reduced numbers of MDSCs in Tnfr–/– mice could be attributable to decreased proliferation or increased apoptosis, we used BrdU to determine the proliferation of MDSCs. BrdU incorporation in bone marrow MDSCs showed no differences between FB61 tumor–bearing Tnfr–/– and Tnfr+/+ mice (Figure 4A), which indicates that MDSC proliferation was not influenced. Similar results were obtained in spleen MDSCs (data not shown). However, in the same time period, when CD11b+Gr1+ cells were stained with anti–annexin V antibody to detect apoptosis (Figure 4B), a drastic increase of annexin V+ CD11b+Gr1+ cells was found in Tnfr–/– mice compared with that in controls (24% ± 5% vs. 4% ± 3%; Figure 4C). These results suggest that the impaired accumulation of MDSCs in Tnfr–/– mice during tumor growth was not due to the decreased proliferation, but the increased apoptosis of these cells in the absence of TNFR.
Enhanced caspase-8 activity is responsible for the impaired accumulation of CD11b+Gr1+ cells in Tnfr–/– mice. (A) Tnfr+/+ and Tnfr–/– mice were intraperitoneally injected with 1 mg BrdU 3, 5, and 8 days after FB61 inoculation. After an additional 24 hours, CD11b+Gr1+ cells in total bone marrow cells were gated (left), and BrdU incorporation was determined (right). Numbers indicate percent BrdU+ cells in gated cells. n = 5 per group. (B) Spleen cells were prepared 8–10 days after FB61 cell inoculation and stained for CD11b, Gr1, and annexin V. Numbers denote percent annexin V+ cells in gated CD11b+Gr1+ cells. (C) Proportion of annexin V+CD11b+Gr1+ cells, expressed as percent of total CD11b+Gr1+ cells (mean ± SEM). n = 3 per group. **P < 0.01. (D) Splenic CD11b+Gr1+ cells from tumor-bearing Tnfr+/+ and Tnfr–/– mice were lysed, and caspase-8 activities were determined by colorimetric activity assay (mean ± SEM). n = 3–5 per group. **P < 0.01. (E) Total cell lysates were subjected to cleaved caspase-8–specific Western blot. β-actin served as internal control. (F) MDSCs were in vitro induced from Tnfr+/+ and Tnfr–/– bone marrow cells. 6 hours later, z-VAD, z-IETD, or DMSO (as control) was added to the medium. Data (mean ± SEM) denote percent CD11b+Gr1+ cells in total living cells in the culture. *P < 0.05.
To further investigate why TNF signaling is crucial for protecting MDSCs from apoptosis, activities of apoptosis-related caspases were determined in splenic MDSCs. More than a 3-fold increase of caspase-8 activity was found in purified Tnfr–/– versus Tnfr+/+ CD11b+Gr1+ cells (1.28 ± 0.18 vs. 0.32 ± 0.06; Figure 4D), but no increase of caspase-3 or caspase-9 activity was observed (data not shown). Western blot analysis confirmed this observation, since the levels of cleaved caspase-8 also increased in purified Tnfr–/– relative to Tnfr+/+ MDSCs (Figure 4E).
To confirm that increased MDSC apoptosis in the absence of TNFR signaling is caused by enhanced caspase-8 activity, MDSCs were induced from bone marrow cells of Tnfr–/– and Tnfr+/+ mice. Within 5 days of in vitro culture, significantly fewer CD11b+Gr1+ cells were induced in the absence of the TNFRs (25% ± 2% vs. 42% ± 3%; P < 0.01; Figure 4F). Addition of either the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F (z-VAD; 41% ± 2%) or the caspase-8–specific inhibitor Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (z-IETD; 44% ± 2%) restored accumulation of CD11b+Gr1+ cells, to the level of Tnfr+/+ cells (Figure 4F). These results demonstrated that lack of TNFR signaling led to enhanced caspase-8 activation and therefore accelerated MDSC apoptosis. Thus, TNFR signaling is necessary for maintaining antiapoptotic activities in MDSCs.
NF-κB–mediated c-FLIP expression is downregulated in Tnfr–/– MDSCs. c-FLIP is a natural inhibitor of caspase-8. To investigate whether c-FLIP is involved in influencing MDSC apoptosis, its expression was determined at the transcriptional level in purified splenic CD11b+Gr1+ cells. Corresponding to the enzymatic activities (Figure 4, D and E), significantly decreased c-FLIP — rather than Bcl-xL, another antiapoptotic protein that serves as a caspase-9 inhibitor — was found in Tnfr–/– MDSCs (Figure 5A). Similarly, Western blot confirmed decreased intracellular c-FLIP and increased cleaved caspase-8 in Tnfr–/– MDSCs, whereas Bcl-xL level did not markedly change (Figure 5B).
NF-κB–mediated c-FLIP expression is downregulated in Tnfr–/– MDSCs. (A) CD11b+Gr1+ cells were freshly isolated from Tnfr+/+ and Tnfr–/– tumor-bearing mice. Amounts of c-FLIP or Bcl-xL mRNA were determined by real-time RT-PCR and are shown relative to β-actin mRNA (mean ± SEM). *P < 0.05. (B) Total cell lysates were extracted from purified Tnfr+/+ and Tnfr–/– MDSCs. Levels of c-FLIP, cleaved caspase-8, and Bcl-xL were determined by Western blot. β-actin served as internal control. (C) Purified MDSCs were stimulated with 20 ng/ml TNF for the indicated times. Levels of p–NF-κB p65, p-IκBα, and p-IKKα/β, as well as c-FLIP and cleaved caspase-8, were determined from total cell lysates by Western blot. β-actin served as internal control. Representative images are shown for 3 independent experiments.
c-FLIP expression can be regulated by NF-κB pathway activation (26). To address whether the NF-κB pathway is involved here, recombinant mouse TNF was added to cultures of Tnfr+/+ or Tnfr–/– MDSCs, and phosphorylation of NF-κB p65, IκBα, and IKKα/β over time was studied. In Tnfr+/+ MDSCs, the protein level of p-IKKα/β steadily increased over 30 minutes, whereas p–NF-κB p65 and p-IκBα peaked at 15 minutes (Figure 5C). This was accompanied by an increase of c-FLIP and inhibited cleavage of caspase-8 within 48 hours. However, in MDSCs from Tnfr–/– mice, steady low levels of p–NF-κB p65 and absent p-IKKα/β and p-IκBα confirmed the lack of functional receptors of TNF. This was accompanied by reduced c-FLIP between 12 and 36 hours and a simultaneous increase in cleaved caspase-8. These data indicate that TNF induces c-FLIP upregulation through the NF-κB pathway in MDSCs.
TNFR-2 alone is crucial for the accumulation of MDSCs. TNF binds to 2 receptors, the ubiquitously expressed TNFR-1 and the hematopoietic cell–restricted TNFR-2. Flow cytometry analysis showed that both TNFR-1 and TNFR-2 were expressed on MDSCs (Supplemental Figure 6). We next analyzed whether both receptors are equally important for peripheral accumulation of MDSCs during tumor development. FB61 fibrosarcoma cells were subcutaneously injected into Tnfr+/+, Tnfr1–/–, Tnfr2–/–, and Tnfr–/– mice, and tumor growth as well as peripheral accumulation of MDSCs in spleen were observed. Again, tumors grew progressively in Tnfr+/+ mice (Figure 6A). In contrast, in Tnfr–/– and Tnfr2–/– mice, but not Tnfr1–/– mice, tumor growth was significantly retarded from day 14 after tumor cell inoculation. Furthermore, 5 of 10 FB61 tumors were spontaneously rejected in Tnfr2–/– mice, similar to Tnfr–/– mice (data not shown). In keeping with the retarded tumor growth, peripheral accumulation of CD11b+Gr1+ cells in Tnfr–/– and Tnfr2–/– spleens was significantly impaired, by nearly half (Figure 6B), but the reduced CD11b+ or Gr1+ cells still located around the germinal centers (Figure 6C).
TNFR-2 signaling maintains survival of CD11b+Gr1+ cells. (A) Tnfr+/+, Tnfr1–/–, Tnfr2–/–, and Tnfr–/– mice were subcutaneously injected with 1 × 106 FB61 cells. Tumor volumes were measured after tumor cell inoculation (mean ± SEM). n = 5–7 per group. *P < 0.05. (B) Spleen cells were prepared 8–10 days after tumor cell inoculation, stained for CD11b and Gr1, and analyzed by flow cytometry. Data (mean ± SEM) represent percent CD11b+Gr1+ cells of total spleen cells. *P < 0.05. (C) Accumulation of CD11b+ and Gr1+ cells in the spleen of tumor-bearing mice, determined by immunofluorescence staining (see Methods). Dotted outlines denote germinal centers. Images are representative of at least 3 mice. Original magnification, ×100 (CD11b); ×200 (Gr1). Scale bars: 300 μm. (D) Total cell lysates were prepared from isolated Tnfr1–/– and Tnfr2–/– CD11b+Gr1+ cells after stimulation with 20 ng/ml TNF for the indicated times. Levels of TRAF2, p–NF-κB p65, p-IκBα, and p-IKKα/β, c-FLIP, and cleaved caspase-8 were determined by Western blot. See Figure 5C for expression levels of respective molecules in Tnfr+/+ mice (controls). β-actin was used as an internal control. Representative images are shown for 3 independent experiments.
To further confirm the importance of TNFR-2 for MDSC accumulation, CD11b+Gr1+ cells from tumor-bearing Tnfr1–/– and Tnfr2–/– mice were stimulated in vitro with TNF. The molecules involved in the activation of NF-κB and apoptosis were determined. As shown in Figure 6D, in Tnfr1–/– MDSCs, levels of TNFR-associated factor 2 (TRAF2) and p–NF-κB p65 increased and peaked at 10 minutes. Levels of p-IκBα and p-IKKα/β in Tnfr1–/– MDSCs exhibited increases similar to those in Tnfr+/+ cells (Figure 5C), although the pattern was altered slightly. However, in Tnfr2–/– MDSCs, there was no substantial upregulation of TRAF2 or p–NF-κB p65, and hardly any p-IκBα or p-IKKα/β was detected. This indicates that TNFR-2 is sufficient for activation of the NF-κB signaling pathway in MDSCs.
Correspondingly, an increase of c-FLIP and a decrease of the cleaved form of caspase-8 in Tnfr1–/– CD11b+Gr1+ cells was observed during the first 36–48 hours of TNF stimulation. On the contrary, lack of TNFR-2 signaling showed a proapoptotic pattern, with reduced c-FLIP and increased caspase-8 over time. Interestingly, TNFR-2 deficiency in MDSCs did not affect their T cell–suppressive function, and there was also no significant difference in arginase or iNOS activity between Tnfr2–/– MDSCs and their Tnfr+/+ counterparts (Supplemental Figure 7). These results demonstrated that TNFR-2 signaling was necessary and also sufficient for the protection of MDSCs from apoptosis.
Neutralization of endogenous TNF impairs transplanted tumor growth and accumulation of MDSCs. Both TNF and lymphotoxin can bind to TNFR-1 and TNFR-2 in vivo. To investigate whether endogenous TNF is necessary for MDSC accumulation during tumor growth, mice were treated with the TNF-neutralizing mAb V1q or its isotype control, then subcutaneously injected with FB61 cells. Neutralization of endogenous TNF led to a significant delay of tumor growth (Figure 7A). Whereas all FB61 tumors grew out in the control mice, 4 of 6 FB61 tumors were spontaneously rejected in the V1q-treated group. Similar antitumor effects were observed in J558L plasmacytoma and TSA mammary adenocarcinoma after V1q treatment (data not shown). Accordingly, splenic MDSC accumulation was also reduced after neutralization of endogenous TNF, especially at a later time point (Figure 7B). For example, at day 15 after tumor cell injection, whereas CD11b+Gr1+ cells accounted for 32% ± 6% of total spleen leukocytes in control mice, they accounted only for 8% ± 5% in the TNF-neutralized mice (Figure 7B). Similarly, intratumoral accumulation of MDSCs also decreased after TNF neutralization (1.25% ± 0.4% vs. 3.9% ± 0.7% in isotype controls at days 8–10; Figure 7C).
Neutralization of endogenous TNF impairs transplanted tumor growth and peripheral accumulation of CD11b+Gr1+ cells. (A) Tnfr+/+ mice were intraperitoneally injected with the TNF-neutralizing mAb V1q (n = 6) or isotype control mAb (n = 4) 2 days prior to subcutaneous injection of 1 × 106 FB61 cells. mAb injection was repeated 3 and 8 days after tumor cell inoculation. Each line represents the growth curve of a tumor in a single mouse; bars denote mean tumor size of each group at day 15. *P < 0.05. (B and C) Spleen or tumor cells were isolated after tumor cell inoculation and stained for flow cytometry. Shown are percent CD11b+Gr1+ cells in (B) total spleen cells and (C) total tumor cells at days 8–10 (mean ± SEM). n = 3–5 per group. *P < 0.05. (D) Bone marrow cells isolated from Tnfr+/+ mice were induced for MDSC generation in vitro (see Methods). V1q, z-IETD, or DMSO (as control) was added to the culture after 6 hours. Cells were collected 5 days later and stained for CD11b and Gr1. Data (mean ± SEM) represent percent CD11b+Gr1+ cells within total living cells in the culture. *P < 0.05.
The in vitro MDSC induction was also performed to confirm the role of TNF in MDSC accumulation. After a 5-day culture, CD11b+Gr1+ cell frequency increased from 13% ± 2% to 42% ± 6% (Figure 7D). Addition of V1q abrogated this increase to 22% ± 12%. However, addition of z-IETD drastically reverted the effect of TNF neutralization on MDSC induction (38% ± 12%). Taken together, these observations indicate that both host-derived TNF and expression of TNFR-2 on MDSCs are required for MDSC accumulation during transplanted tumor growth.