Abstract
Relatively little is known about factors that initiate immunosuppression in tumors and act at the interface between tumor cells and host cells. In this article, we report novel immunosuppressive properties of the ribosomal protein S19 (RPS19), which is upregulated in human breast and ovarian cancer cells and released from apoptotic tumor cells, whereupon it interacts with the complement C5a receptor 1 expressed on tumor infiltrating myeloid-derived suppressor cells. This interaction promotes tumor growth by facilitating recruitment of these cells to tumors. RPS19 also induces the production of immunosuppressive cytokines, including TGF-β, by myeloid-derived suppressor cells in tumor-draining lymph nodes, leading to T cell responses skewed toward Th2 phenotypes. RPS19 promotes generation of regulatory T cells while reducing infiltration of CD8+ T cells into tumors. Reducing RPS19 in tumor cells or blocking the C5a receptor 1–RPS19 interaction decreases RPS19-mediated immunosuppression, impairs tumor growth, and delays the development of tumors in a transgenic model of breast cancer. This work provides initial preclinical evidence for targeting RPS19 for anticancer therapy enhancing antitumor T cell responses.
Introduction
The vast majority of processes mediated through the complement C5a receptor 1 (C5aR1) are thought to be initiated by the interaction of C5aR1 with its best-studied ligand C5a (1). Ribosomal protein S19 (RPS19) is the only other endogenous ligand of eukaryotic origin that is known to bind to this receptor (2, 3). RPS19 released from apoptotic cells was shown to have chemotactic activity toward leukocytes in vitro through interaction with C5aR1 (4). Interestingly, unlike C5a, RPS19 differentially regulates monocytes versus neutrophils during inflammation (5). It selectively enhances the recruitment of monocytes that give rise to macrophages involved in clearance of dying cells and, simultaneously, halts the migration of neutrophils to sites of inflammation (5). Therefore, RPS19 was postulated to protect tissues from neutrophil-induced damage and to facilitate the clearance of dying cells in inflammation (5). Because cell death resulting from hypoxia is present in virtually all malignancies, even in the early stages of tumor growth (6), and C5aR1 is known to activate and recruit myeloid-derived suppressor cells (MDSCs) to tumors (7, 8), we hypothesize that RPS19 released from dying tumor cells is involved in the initiation of immunosuppression in the tumor microenvironment through its interaction with C5aR1 expressed on MDSCs.
In support of this hypothesis, an early study demonstrated upregulation of RPS19 expression (mRNA) in colon carcinoma compared with normal epithelial colon cells (9). In addition, small quantities of RPS19 were found in feces of patients with colorectal cancer (10), and single-nucleotide polymorphisms in the RPS19 gene were found to be associated with an increased risk for cervical intraepithelial neoplasia and cervical cancer (11). However, the extracellular functions of RPS19 on the whole remain largely unknown. Moreover, knowledge of the involvement of RPS19 in the pathogenesis of diseases is limited to a rare hematologic condition named Diamond-Blackfan anemia, with 25% of patients having mutations in the RPS19 gene (12).
Materials and Methods
Mice, cell lines, and human tissues
Eight- to twelve-week-old FVB/N-Tg(MMTVneu) 202Mul/J, wild-type FVB/NJ, and BALB/c female mice (Jackson Laboratory) were used for this study with the approval of the Institutional Animal Care and Use Committee of the Texas Tech University Health Sciences Center, according to guidelines of the National Institutes of Health. NT-5 tumor cells derived from a spontaneously occurring mammary adenocarcinoma in an FVB/N Her2/neu-transgenic mouse expressing nontransforming rat Her2/neu (13) were injected s.c. (1 × 106) into the rear flanks of mice. 4T1 cells were injected into the mammary fat pad to generate breast tumors, as previously described (14). Tumors were measured with calipers and their volume was calculated as (length × width × depth)/2. Mouse cell lines (RAW 264.7 and 4T1) and human cell lines (benign mammary epithelium: MCF-10A; breast cancer: MCF-7, MDA-MB-231, MDA-MB-453, and BT-20; ovarian adenocarcinoma: SKOV-3; and colorectal cancer: COLO 205) were obtained from the American Type Culture Collection and maintained according to its recommendations; human 1520 melanoma and OCAR-5 ovarian carcinoma cells were obtained from Dr. J. Weidanz (Texas Tech University Health Sciences Center). Cell lines were routinely tested for the absence of mycoplasma contamination (Aldevron). Human cell lines were authenticated by the American Type Culture Collection or by the providing investigator by short tandem repeat profiling and were used within 6 mo after resuscitation. Breast cancer human tissues were procured from the National Disease Research Interchange.
C5aR1 inhibition and C5a detection
For pharmacological blockade of C5aR1, mice were injected s.c. with a selective C5aR1 antagonist (C5aRA), the cyclic peptide Ac-(2, 6)-F[OP(D-Cha)WR]. This compound, acetyl-phenylalanine–[ornithine-proline-(d-cyclohexylalanine)-tryptophan-arginine], originally named 3D53 and also previously licensed as PMX53 (15), does not bind to the second receptor for C5a (C5L2) or to the C3a receptor (16). It was synthesized and characterized as described (17), dissolved in PBS, and administered to mice at a dose of 1 mg/kg body weight every 2–3 d beginning at day 3 or 4 after tumor cell injection (3.3 μmol/kg body weight per week), as previously described (7). Control mice were injected s.c. with PBS as placebo. C5a was detected in mouse plasma by ELISA (MyBiosource), according to the manufacturer’s instructions.
Immunoprecipitation and Western blotting
Binding assay
C5a and RPS19 binding to C5aR1 expressed on murine RAW 264.7 macrophages and MDSCs, isolated using magnetic beads from spleens of NT-5 tumor-bearing mice, was determined by FACS. Approximately 95% purity of isolated cells was achieved (Supplemental Fig. 1
Chemotaxis assay
Supplemental Fig. 2A). For some experiments, cells were preincubated with 10 μM C5aRA.
RPS19 downregulation by short hairpin RNA
Tissue processing, cell isolation, and immunofluorescence
Portions of tumors and tumor-draining lymph nodes (TDLNs) were frozen in OCT at −70°C and sectioned with a cryostat for immunofluorescence or were used for cell isolation for subsequent FACS. The following mAbs were used for immunofluorescence in frozen sections: CD8a (53-6.7; BD Pharmingen), perforin (CB5.4; Abcam), CD11b (M1/70; BD Pharmingen), Gr-1 (RB6-8C5; BD Pharmingen), C3b/iC3b/C3c (2/11; Hycult Biotech) (18 + T cells and MDSC (CD11b+Gr-1+) tumor infiltrates were quantified with Nikon Elements Advanced Research image-analysis software. Cells were counted in entire tissue sections, and mean values per 63× fields were calculated.
FACS and functional assays
+ T cells.
For CD8+ T cell stimulation, cells were incubated with PDSLRDLSVF for 6–8 h with Golgi inhibitors (brefeldin A and monensin; BD Biosciences), and IFN-γ production was assessed with PE–IFN-γ Ab (XMG1.2). For myeloid cell function, cell preparations from TDLNs were stimulated with 1 μg/ml LPS for 8–12 h with Golgi inhibitors and subsequently stained for cytokines.
For Her2/tumor-specific CD8+ T cells, viable lymphocytes were gated based on forward and side scatter, followed by gating on CD3+CD45+ cells and then CD8+Her2+ cells. The frequencies and numbers of Tregs were calculated by gating on CD4+CD25+FOXP3+ cells, whereas MDSCs were identified as CD11b+Gr-1+MHCIIlow. Frequencies of dendritic cells (DCs) were calculated based on MHC class II (MHCII) and CD11c positivity, whereas the expression of CD80 and CD86 was determined as median fluorescence intensity.
To study the impact of C5aR1 signaling in MDSCs on the polarization of T cell responses, groups of tumor-bearing FVB/N Her2/neu-transgenic mice were injected with C5aRA or PBS. On day 28, spleens were harvested for MDSC isolation by magnetic columns (Miltenyi Biotec). Naive CD4+ T cells were negatively sorted (Miltenyi Biotec) from splenocytes of female FVB/N Her2/neu-transgenic mice not bearing tumors. Purity of cells was verified by FACS (>98%). CD4+ T cells and MDSCs were cocultured at 1:5 ratios in a 24-well plate coated with anti-CD3/CD28 Abs. C5aRA was added to some wells (at 10 nM concentration). CD4+ T cell cultures were harvested at day 5, and intracellular staining for IFN-γ, IL-4, and IL-17 was performed as described (14
Statistical analysis
Data were analyzed using an unpaired t test or the nonparametric Mann–Whitney U test, depending on the results of the normality test (Kolmogorov–Smirnov), or one-way ANOVA for more than two mean values. Tumor growth with time was analyzed by two-way ANOVA. Bar graphs indicate mean + SEM. All statistical analyses were done with Graph Pad Prism 6 software.
Results
RPS19 is upregulated in human breast and ovarian cancer cells and interacts with C5aR1 expressed on MDSCs
Ribosomal proteins and ribosomal biogenesis, which are linked to the MDM-p53 pathway, appear to have dual, and sometimes contradictory, functions in cancer development (19). Elevated levels of ribosomal biogenesis and protein translation were linked to formation of tumors in multiple mouse models (20). In contrast, a reduction in ribosomal biogenesis and translational capacity were associated with a high incidence of cancer in humans (21). For example, Diamond-Blackfan anemia, which is associated with mutations in the gene for RPS19 in 25% of patients, increases susceptibility to hematopoietic malignancies (19). Therefore, we tested human tumor cell lines for RPS19 expression; three of four human breast cancer cell lines and both ovarian cancer cell lines overexpress RPS19 compared with normal epithelia from mammary gland (Fig. 1A, 1B). In addition, we found RPS19 on the surface of cells expressing CD11b and CD33, likely representing MDSCs, in tumor sections from patients with ductal breast carcinoma (Fig. 1C, 1D, upper panel). RPS19 on the cell surface colocalized with C5aR1 expressed on these cells, suggesting an interaction of RPS19 with C5aR1 (Fig. 1D, middle and bottom panels). Because complement activation, which leads to generation of C5a, was reported to occur in breast cancer (22), these data suggest that RPS19 interacts with C5aR1, even in the presence of C5a in human tumors. To interrogate a further interrelationship between both ligands in tumor tissue, we stained mouse 4T1 tumors from BALB/c mice, which have C5 and generate C5a in the response to breast tumors, in contrast to FVB/N Her2/neu-transgenic mice (Fig. 1E), for deposition of C3 cleavage fragments, which indicates complement activation and the subsequent generation of C5a in tissue. Importantly, we used anti-C3 Ab, which reacts exclusively with C3 cleavage products but not with intact C3 (18); therefore, this staining indicates areas of tumor where complement activation occurs. We found overlap of C3 fragment deposition with RPS19 accumulation (Fig. 1F, areas outlined by dashed lines, yellow color), likely because RPS19 is released from apoptotic cells (4) that also activate complement (23). These data suggest that both ligands potentially compete for C5aR1 binding. However, in the same tumor areas, we found RPS19 and C3 fragment deposition without apparent overlap (Fig. 1F, areas outlined by dashed lines, green and red color). Moreover, in some portions of the tumor, the large areas of complement deposition were observed at some distance from RPS19 accumulation (Fig. 1F, squares, red color). Despite the presence of complement activation and C5a generation, RPS19 colocalized with C5aR1 on the surface of myeloid (CD11b+) cells in proximity of greater RPS19 accumulation in tumor cells (Fig. 1G, squares, arrows denote myeloid cells, and arrowhead denotes tumor cells). Thus, we concluded that RPS19 interacts with C5aR1, even in the presence of C5a. This conclusion is supported by results of an in vitro competitive binding assay. We found that increasing concentrations of C5a reduced RPS19 binding in a dose-dependent manner; however, even a 125-fold greater concentration of C5a (5 μM) did not entirely eliminate RPS19 binding (Fig. 1H, 1I).
RPS19 is overexpressed in breast and ovarian cancer cells and interacts with C5aR1 at the interface between tumor and MDSCs. (A) RPS19 (16 kDa) is overexpressed in breast (MDA-MB-231, 354, and BT-20) and ovarian (OVCAR-5 and SKOV-3) cancer-derived cell lines relative to normal mammary gland epithelium (MCF-10A). (B) Quantification of data from (A) normalized to β-tubulin. *p < 0.0001, one-way ANOVA. (C) H&E staining of the section of tumor from a patient with breast ductal adenocarcinoma. The entire section contains only tumor tissue. Scale bar, 500 μm. (D) Confocal microscopy analysis of human MDSCs (CD11b+ CD33+), RPS19, and C5aR1 in the breast cancer section shown in (C). Arrowheads denote tumor cells, yellow arrows denote MDSCs, and white arrows denote C5aR1 and RPS19 colocalization on MDSCs. Scale bars, 50 μm (top and middle panels), 10 μm (bottom panel). (E) C5a in plasma of 4T1 tumor-bearing BALB/c and NT-5 tumor-bearing FVB/N Her2/neu-transgenic (FVB-Tg) mice. *p = 0.0006, t test. (F) RPS19 and C3 cleavage fragment (C3b/iC3b) deposition in mouse 4T1 tumor. Dashed lines outline areas with abundant RPS19 accumulation. Squares denote prominent C3 fragment deposition that does not colocalize with RPS19. Yellow inside the dashed outlines in the merged image represents RPS19 and C3 fragment colocalization. Scale bar, 200 μm. (G) RPS19, CD11b, and C5aR1 in 4T1 tumors. The squares outline the areas that are magnified in the lower panels. Arrowheads denote tumor cells. Arrows denote CD11b+ myeloid cells expressing C5aR1 that colocalizes with RPS19. Scale bars, 25 μm (lower panels), 50 μm (upper panels). (H) Representative line graphs showing the binding of 40 nM recombinant RPS19 in the presence of increasing concentrations of C5a by FACS. (I) Quantification of data from (H). *p < 0.0001, one-way ANOVA. Data are representative of three independent experiments with n = 3 replicates for (A), (B), (H), and (I), n = 2 for (C) and (D), and n = 5 for (E)–(G).
To confirm that RPS19 interacts with C5aR1 on MDSCs, we used Her2/neu-transgenic mice (on FVB background) that, in contrast to BALB/c mice, do not produce C5 and C5a (Fig. 1E) (https://www.jax.org/strain/002376). To expedite mechanistic studies, these mice were injected with NT-5 cells that do not express C5aR1 (data not shown) and are derived from spontaneous breast carcinoma that develops in these mice at older age (13). Tumor cells were injected prior to the development of spontaneous tumors. The FVB/N Her2/neu mouse exhibits T cell tolerance to rat Her2/neu, which is a self-antigen in this mouse and, thus, is a useful breast cancer model mimicking human cancer and suitable for testing the role of tolerance in antitumor immune responses (24). We chose to inject mice into the rear flank to avoid potential confusion of transplantable tumor with spontaneous breast carcinoma developing in these mice. We found that RPS19 accumulated in tumor cells undergoing apoptosis, as indicated by binding of annexin V to phosphatidylserine on the external surface of tumor cells (Fig. 2A, top panel). Because apoptotic cells activate complement, leading to coating of apoptotic cells with the cleavage products C3b and iC3b of the complement fragment C3 (25), we examined deposition of these fragments on apoptotic (annexin V+) tumor cells and found that these cells were uniformly coated by C3b/iC3b (Fig. 2A, top panel). These complement fragments interact with complement receptor 3, which is composed of CD11b and CD18 (26). Therefore, as anticipated, CD11b-expressing myeloid cells were present in close proximity to apoptotic tumor cells with abundant expression of RPS19 (Fig. 2A, second panel, arrows) and, in some areas, CD11b positivity seemed to be adjacent to C3b/iC3b (Fig. 2A, third panel), pointing to a possible interaction between complement receptor 3 and iC3b in the interphase between tumor and host cells. Importantly, RPS19 staining in tumor cells colocalized with C5aR1 expressed on CD11b+ cells (Fig. 2A, bottom panel, arrows), suggesting an interaction of RPS19 with C5aR1. This interaction is supported by results of IP of RPS19 protein from the tumor whole-cell lysate by C5aR1 Ab (Fig. 2Bi). This IP resulted in the pull-down of only a monomer (16 kDa) of RPS19, likely as a result of the very low concentrations of the dimerized form of RPS19 (32 kDa) in a mouse tumor. We observed only low amounts of RPS19 dimer in the recombinant protein preparation used as a positive control; however, we failed to detect RPS19 dimer in the tumor whole-cell lysate (Fig. 2Bi, input). The lack of RPS19 dimer in tumors is consistent with the study by Yamamoto and colleagues (27) indicating that, although RPS19 dimer is formed in apoptotic cells, it is quickly inactivated by serine proteases released from the same cells. Interestingly, IP of C5aR1 with RPS19 Ab resulted in pull-down of phosphorylated receptor with a reported molecular size of 43 kDa (28) (Fig. 2Bii, iii), whereas IP with C5aR1 Ab caused pull-down primarily of the unphosphorylated form (40 kDa), with very limited contribution from the phosphorylated receptor (Fig. 2Bii, iii). These data suggest that engaging of C5aR1 with interaction with RPS19 leads to receptor internalization/phosphorylation similar to that observed for C5a (28). The dominance of unphosphorylated receptor in IP with C5aR1 Ab is likely associated with the overabundance of C5aR1, which is not engaged in an interaction with a ligand.
RPS19 binds to C5aR1 expressed on MDSCs and macrophages and triggers chemotaxis of macrophages. (A) RPS19, C3b/iC3b, annexin V, CD11b, and C5aR1 in murine tumors generated in FVB/N Her2/neu-transgenic mice by injecting NT-5 cells. Arrows denote accumulation of CD11b+ cells (second middle) and colocalization of RPS19 with C5aR1 (bottom panel). Scale bars, 5 μm (top, third, and bottom panels), 25 μm (second panel). (Bi and ii) Immunoprecipitation of RPS19–C5aR1 complex from whole-cell lysate (Tum Lys) of NT-5 tumors from Her2/neu-transgenic mouse. Murine RAW 264.7 macrophages (Mac) were used as a positive control for C5aR1 expression, and recombinant RPS19 (rRP S19) was used as control for RPS19. (iii) Quantification of data from (ii). *p = 0.0364 (right panel), *p = 0.003 (left panel), one-way ANOVA. FACS analysis of RPS19 on the surface of monocytic (Gr-1int) versus granulocytic (Gr-1high) MDSCs (C) and C5aR1 expression on monocytic versus granulocytic MDSCs (D). Black lines represent RPS19 and C5aR1 Abs. Gray lines represent isotype controls. (E) Pre- and postsort FACS of spleen MDSCs from NT-5 tumor-bearing mice. Binding of fluorescently labeled RPS19 or C5a, in the absence (solid black lines) or presence (dashed lines) of C5aR1 antagonist (C5aRA), to sorted MDSCs (p < 0.0001 for RPS19, p = 0.0001 for C5a, one-way ANOVA) (F) or murine macrophages (p < 0.0001 for RPS19 and C5a, one-way ANOVA) (G). (H) Chemotaxis assay; y-axis shows the number of murine macrophages (RAW 264.7) in the lower chamber at the end of the experiment. Data are representative of one experiment with n = 5 for (A), three independent experiments for (B)–(G), and four replicates for (H). *p < 0.0001, one-way ANOVA.
To verify that MDSCs are engaged in interactions with RPS19, we mechanically disintegrated NT-5 tumors. Through FACS we found RPS19 on the surface of MDSCs (CD45+MHCIIlowCD11b+Gr-1+), with higher amounts of RPS19 bound to monocytic (Gr-1int) MDSCs compared with granulocytic (Gr-1high) MDSCs (Fig. 2C); this is consistent with reported chemotactic activity of RPS19 toward monocytes/macrophages (4). C5aR1 expression on both MDSC populations was reported previously (7). Consistent with this report, MDSCs from NT-5 tumors were found to express C5aR1 (Fig. 2D). Next, we sorted MDSCs from spleens of NT-5 tumor-bearing mice (Fig. 2E, Supplemental Fig. 1) and incubated them with fluorescently labeled C5aR1 ligands (C5a or RPS19) in the absence or presence of the C5aR1 inhibitor, which binds to C5aR1 but not to C5L2. Preliminary studies established that binding of RPS19 or C5a to C5aR1 is optimal in 40 nM ligand concentrations. We found that C5a and RPS19 bind to sorted MDSCs, and preincubation with C5aR1 inhibitor reduces this binding significantly (Fig. 2F). Similar results were obtained for the murine macrophage cell line (Fig. 2G) that also was used for testing the chemotactic activity of recombinant RPS19 in the Transwell system. We found that the recombinant RPS19 preparation used for this assay contained a small amount of the dimerized form of RPS19 (Supplemental Fig. 2A), which was reported to have chemotactic activity (5). We found that RPS19 was as potent as C5a as a chemoattractant for these cells (Fig. 2H). RPS19 and C5a chemotactic activities were mediated by the interaction of these ligands with C5aR1, because the preincubation of macrophages with the specific C5aR1 inhibitor attenuated C5a- and RPS19-mediated chemotaxis (Fig. 2H). These functional data suggest that the C5aR1 inhibitor used in this study interferes with binding of RPS19 to C5aR1 and blocks the functions of RPS19 that are mediated through its interaction with C5aR1.
RPS19 downregulation in tumor cells correlates with slower tumor growth and enhanced antitumor immunity
To investigate a role for RPS19 in tumor growth in vivo, we used a doxycycline-inducible shRNA approach to downregulate RPS19 in NT-5 cells (Supplemental Fig. 2B, 2C). We injected FVB/N Her2/neu-transgenic mice with NT-5 cells transduced with an empty lentiviral vector (control) or inducible lentiviral RPS19 shRNA prior to the development of spontaneous mammary tumors. Neither the transgenic mice nor NT-5 cells produce C5 and C5a. Consequently, RPS19 is the only known ligand to interact with C5aR1 in this experimental setting. The doxycycline-induced RPS19 shRNA expression led to the downregulation of RPS19 protein in the majority (but not all) of NT-5 tumors (Fig. 3A, representative examples are shown) in vivo and reduced tumor growth (Fig. 3B). In contrast, doxycycline did not impact tumor growth in control mice that were injected with NT-5 cells transduced with empty lentivirus (data not shown). To avoid prolonged exposure of mice to doxycycline, the experiments were terminated when statistically significant differences in tumor growth were first observed.
The downregulation of RPS19 in NT-5 tumor cells leads to reduced tumor growth and improved immunity in FVB/N Her2/neu-transgenic mice. (A) RPS19 in whole-cell lysates of tumors composed of NT-5 cells transduced with an empty lentiviral vector (Ctrl) or inducible lentiviral RPS19 shRNA (i-shRNA) from FVB/N Her2/neu vehicle (PBS)-treated transgenic mice (No Dox) or mice treated with doxycycline (Dox) by Western blot. (B) Volumes of tumors generated by injecting NT-5 cells, transduced with inducible lentiviral RPS19 shRNA, into Her2/neu-transgenic mice treated with vehicle (▪, i-shRNA-No dox [RPS19+]) or doxycycline (□, shRNA-Dox [RPS19−]). p = 0.0201, two-way ANOVA. (C–L) Immunity in mice bearing RPS19+ tumors versus RPS19− tumors by FACS. (C) CD8+ T cells in spleens. *p = 0.0081, t test. (D) IFN-γ–producing CD4+ T cells in spleens. *p = 0.0499, t test. (E) Representative plots for data shown in (D). (F) IFN-γ–producing CD8+ T cells in spleens, *p = 0.04, t test. (G) Representative plots for data shown in (F). (H) DCs in tumors. (I) CD80 on DCs. *p = 0.0373, t test. (J) CD86 on DCs. *p = 0.0402, t test. MHCII on DCs (K) and CD45− tumor cells (L). *p = 0.0193, t test. Legend in (B) applies to all panels. Data are representative of two independent experiments with n = 3–5. MFI, median fluorescence intensity.
Mice expressing lower amounts of RPS19 in tumor cells also have higher numbers of CD8+ T cells (Fig. 3C) and IFN-γ–producing CD4+ (Fig. 3D, 3E) and CD8+ (Fig. 3F, 3G) T cells in their spleens, suggesting enhanced Th1 and cytotoxic T cell responses. Importantly, for IFN-γ intracellular staining in CD8+ T cells, splenocytes were stimulated ex vivo with peptide corresponding to the Her2/neu immunodominant epitope (29). Therefore, the evaluated CD8+ T cell responses are tumor specific. We found no difference in the number of tumor-infiltrating CD4+ or CD8+ T cells (data not shown); however, DCs (CD45+CD11c+MHCII+) show a trend to be more frequent (not statistically significant) in tumors with RPS19 downregulation (Fig. 3H), and they have better Ag-presenting functions, as demonstrated by the increased expression of CD80 (Fig. 3I) and CD86 (Fig. 3J). MHCII expression showed a trend toward higher expression on DCs from tumors with RPS19 downregulation (Fig. 3K). Interestingly, tumor cells identified by the lack of CD45 expressed more MHCII in the context of reduced RPS19 expression (Fig. 3L), rendering them more susceptible to immune recognition and destruction (30).
Pharmacological blockade of the interaction of RPS19 with C5aR1 delays the development of spontaneous breast tumors, reduces tumor growth, and enhances antitumor immunity
Although we did not observe an impact of a moderate downregulation of RPS19 on NT-5 cell growth in vitro, as demonstrated by identical morphology and growth rate of the cultured cells treated with doxycycline (induced RPS19 shRNA) versus nontreated cells (Supplemental Fig. 2D), there is concern that the downregulation of RPS19, which is involved in ribosomal biogenesis, may affect tumor cell proliferation and/or survival in vivo. Thus, it may affect tumor growth regardless of antitumor immunity. Therefore, we next used a pharmacological approach to block RPS19 interaction with C5aR1. We found that the C5aR1 inhibitor used in our study efficiently blocks the functions of RPS19 that are mediated through the C5aR1 receptor (Fig. 2H); therefore, we used this inhibitor in a FVB/N Her2/neu-transgenic or FVB wild-type mice. Because of the lack of C5 and C5a in these mice, C5aR1 inhibitor blocks the interaction of C5aR1 with the only remaining known C5aR1 ligand, RPS19.
Because hypoxia and associated tumor cell death occur early during tumor development and we found that RPS19 is released from apoptotic tumor cells, we hypothesize that RPS19 may be one of the factors involved in the initiation of tumor-associated immunosuppression, which is pivotal for the escape of tumors from immune surveillance (31). To test this hypothesis, we treated FVB/N Her2/neu-transgenic mice with C5aR1 inhibitor prior to the development of spontaneous tumors. Focal mammary tumors first appear in these mice at 4 mo, with a median incidence at 205 d (https://www.jax.org/strain/002376). Therefore, we used randomized 6-mo-old female mice without palpable breast tumors for these studies. C5aR1 antagonism delayed the development and reduced the growth of spontaneous tumors in these mice (Fig. 4A).
Pharmacological disruption of the RPS19–C5aR1 interaction delays the development and growth of tumors and enhances antitumor immunity. (A) Volume of spontaneous mammary tumors in FVB/N Her2/neu-transgenic mice treated with C5aR1 antagonist (C5aRA) or vehicle (PBS). Red and green arrows indicate the onset of clinically detectable tumors in vehicle- or C5aRA-treated mice, respectively. *p = 0.0373, two-way ANOVA. (B) Volumes of NT-5 tumors in FVB wild-type mice that were treated with C5aRA or vehicle once palpable tumors were present. *p = 0.0063, two-way ANOVA. (C) Volumes of NT-5 tumors in transgenic FVB/N Her2/neu mice treated as in (B). *p = 0.064, two-way ANOVA. (D) CD8+ T cells in NT-5 tumors of FVB wild-type mice treated as in (B), by FACS. *p = 0.0511, confidence interval −0.9229 to 0.0029. (E) Tumor CD8+Perforin+ T cells from mice shown in (B). (F) Quantification of data shown in (E). *p = 0.0419. (G) CD8+Perforin+ T cells from mice shown in (C). (H) Quantification of data shown in (G). *p = 0.0125. (I) Her2-specific CD8+ T cells from NT-5 tumors of FVB wild-type mice treated as in (B). *p = 0.0014. (J) FACS plots for data shown in (I). (K) Her2-specific CD8+ T cells from NT-5 tumors from FVB/N Her2/neu-transgenic mice treated as in (B). (L) FACS plots for data shown in (K). (M) IFN-γ–producing CD8+ T cells from NT-5 tumors from FVB wild-type mice treated as in (B). *p = 0.037. (N) FACS plots for data shown in (M). (O) IFN-γ–producing CD8+ T cells from NT-5 tumors of Her2/neu-transgenic mice treated as in (B). *p = 0.0006. (P) FACS plots for data shown in (O). Scale bars, 50 μm. Data are representative of at least three independent experiments with n = 4–10. All p values in (D)–(P) were calculated using a t test.
To expedite the next studies, FVB wild-type mice were injected s.c. with syngeneic NT-5 cells, expressing normal levels of RPS19 and not expressing C5aR1 (data not shown) and treated with C5aR1 antagonist when flank tumors were palpable. C5aR1 antagonism reduced tumor growth (Fig. 4B). To determine the impact of C5aR1 blockade on tumor growth in mice that are tolerant to tumor-associated Ags (Her2/neu), NT-5 cells, which also overexpress Her2/neu, were injected s.c. to the rear flanks of FVB/N Her2/neu-transgenic mice prior to the development of spontaneous breast tumors. Similar to FVB wild-type mice, C5aR1 antagonism reduced the growth of syngeneic tumors in transgenic mice (Fig. 4C), although the rate of tumor growth was more rapid in transgenic mice as a result of poorer immunosurveillance.
The reduced tumor growth in wild-type and transgenic mice treated with C5aR1 inhibitor was associated with the increased infiltration of tumors by cytotoxic CD8+ T cells (Fig. 4D, FACS data from wild-type mice) that expressed high level of perforin, indicating their tumoricidal capacity (Fig. 4E–H). Importantly, tumor (Her2/neu)-specific T cells were increased in tumors from wild-type and transgenic mice treated with C5aR1 antagonist (Fig. 4I–L). In addition, C5aR1 inhibitor improved CD8+ T cell functions, as indicated by the increased production of IFN-γ by these cells stimulated with peptide corresponding to the Her2/neu immunodominant epitope (Fig. 4M–P).
Tumor-promoting RPS19 regulates tumor-associated immunosuppression
Because the C5aR1/C5a axis is thought to regulate immunosuppression in the tumor microenvironment (7), we hypothesized that RPS19 has similar immunosuppressive functions through its interaction with C5aR1. Therefore, we examined immunosuppressive cells using a similar experimental setting as for Fig. 4, in which C5/C5a are absent. C5aR1 inhibition led to the reduction of Tregs in mouse tumors (Fig. 5A) and spleens (Fig. 5B).
Pharmacological disruption of the RPS19–C5aR1 interaction reduces Tregs and MDSCs in tumors and periphery. (A) Tregs in tumors of FVB/N Her2/neu-transgenic NT-5 tumor–bearing mice treated with PBS or C5aR1 antagonist (C5aRA). *p = 0.0052. (B) Tregs in spleen of FVB/N Her2/neu-transgenic NT-5 tumor–bearing mice treated with PBS or C5aR1 antagonist (C5aRA). *p < 0.0001. (C) MDSCs in blood of NT-5 tumor–bearing FVB/N Her2/neu mice treated as in (A). *p = 0.04. (F) MDSCs in blood of NT-5 tumor–bearing FVB wild-type mice treated as in (A). *p = 0.0361. MDSCs (CD11b+GR-1+) in tumors of FVB/N Her2/neu-transgenic mice (D) and FVB wild-type mice (G) treated as in (A). (E) Quantification of data shown in (D). *p = 0.0025. (H) Quantification of data shown in (G). *p = 0.04. Scale bars, 50 μm. Data are representative of at least three independent experiments with n = 4–10. All p values were calculated using a t test.
Treg generation is thought to be controlled by MDSCs (32), and C5aR1 was demonstrated to facilitate the recruitment of these cells to tumors (7). In addition, our studies demonstrated the interaction of RPS19 with C5aR1 expressed on human and murine MDSCs (Figs. 1, 2). Therefore, we examined MDSCs by FACS and immunofluorescence. Using FACS, we found a reduction in the absolute number of MDSCs in peripheral blood of FVB/N Her2/neu-transgenic mice (Fig. 5C) and FVB wild-type mice (Fig. 5F) bearing tumors after treating with C5aR1 inhibitor. FACS failed to demonstrate statistically significant differences in tumor MDSCs (data not shown); however, immunofluorescence indicated a reduction in tumor-infiltrating MDSCs in FVB/N Her2/neu-transgenic mice (Fig. 5D, 5E) and FVB wild-type mice (Fig. 5G, 5H) that were treated with C5aR1 antagonist. This discrepancy between FACS and immunofluorescence studies may be related to difficulties in obtaining MDSCs embedded in tumor stroma as a single-cell suspension.
The expansion of Tregs is controlled by several cytokines, such as TGF-β and IL-10, produced by cells of myeloid origin (33). In addition, the cross-talk between macrophages and MDSCs producing IL-6 and IL-10, which enhance the immunosuppressive properties of MDSCs and reduce IL-12 production in macrophages, plays an important role in this process (32). Furthermore, TGF-β is considered a major immunosuppressive cytokine that inhibits effector T cell responses, with the exception of Th17 cells, which are induced by TGF-β plus IL-6 (34). Therefore, we examined the production of TGF-β1, IL-6, IL-10, and IL-12, individually or in combination in MDSCs (CD11b+Gr-1+). We focused on TDLNs, because they were predicted as preferred sites for inducible Treg generation (35, 36), and it was suggested that the acquisition of Treg lineage and phenotypes requires antigenic stimulation of naive T cells in the presence of TGF-β (37, 38). In addition, we found large quantities of extracellular RPS19 colocalized with MDSCs (CD11b+Gr-1+) in TDLNs (Fig. 6A). We found that, in tumor-bearing PBS-injected control mice, the majority of MDSCs, expressing at least one of the measured cytokines (3% of total MDSCs), produced only TGF-β1, with lower fractions of cells producing only IL-6 or IL-12 (Fig. 6B). The fractions of multifunctional cells producing more than one of the measured cytokines were low. However, we observed distinct subpopulations of cells that coproduced IL-6 and TGF-β1, IL-12 and TGF-β1, or IL-6, IL-12, and TGF-β1. Cells producing IL-10 alone or coproducing IL-6 and IL-12 were rare (Fig. 6B). Fractions of cells producing all four cytokines (IL-6+ IL-12+ IL-12+ TGF-β1+) were negligible. The well-established immunosuppressive properties of TGF-β, together with the high proportions of cells producing this cytokine alone or in combination with other cytokines, suggest a key role for TGF-β in augmenting the immunosuppressive mechanisms in TDLNs. In contrast to the tumor microenvironment, the role of IL-10 in TDLNs was less significant based on these data. C5aR1 blockade reduced the fractions and frequencies of TGF-β1–producing MDCS, including single TGF-β1 producers and cells that coproduced TGF-β1 with IL-12 or IL-6 (Fig. 6B, 6C), although these differences did not reach statistical significance in this experimental setting. However, the significant impact of C5aR1 signaling on TGF-β1 production was confirmed by the reduction of TGF-β1 levels in plasma of tumor-bearing mice treated with C5aR1 inhibitor (Fig. 6D). In contrast, targeting C5aR1 modestly increased the fractions and frequencies of cells that produced IL-6 (Fig. 6B, 6C). Although IL-6 in the tumor microenvironment is reported to impart immunosuppression by regulating IL-10 production in MDSCs, other studies found that IL-6 can counteract TGF-β1 inhibition of CD3 cell activation (39). In addition, IL-6 is a pleiotropic cytokine that favors priming, generation, and survival of Ag-specific CD8+ T cells (40, 41). Thus, we hypothesize that an increase in IL-6 production promotes induction and survival of tumor-specific T cells upon C5aR1 blockade. Furthermore, with a simultaneous decrease in TGF-β production, IL-6 can inhibit the induction of Tregs and promote other T cell lineages (42).
The RPS19–C5aR1 interaction controls the cytokine milieu in TDLNs. (A) H&E staining of TDLNs (dashed lines surround cells with neutrophil-like MDSC morphology) and immunofluorescent detection of RPS19 in TDLNs in the proximity of MDSCs (CD11b+Gr-1+). Scale bars, 100 μm (H&E), 50 μm (immunofluorescence). (B) Fractions of MDSCs that produced TGF-β1, IL-6, IL-10, and/or IL-12 alone or in various combinations after ex vivo stimulation with LPS obtained from TDLNs of control (PBS) and C5aR1 antagonist (C5aRA) NT-5 tumor–bearing FVB/N Her2/neu-transgenic mice. (C) Frequencies of MDSCs producing various cytokines as in (B). Legend in (B) also applies to (C). (D) TGF-β1 levels in plasma of mice as in (B). Each point represents an individual mouse. Horizontal lines represent mean + SEM. Data are representative of one experiment with n = 5. *p < 0.0001, t test.
Inhibiting C5aR1/RPS19 signaling favors Th1 and Th17
To confirm that C5aR1 signaling in MDSCs can impact the polarization of T cell responses, naive CD4+ T cells not expressing C5aR1 (43) were isolated from the spleens of tumor-free FVB/N Her2/neu-transgenic mice and stimulated with CD3/CD28 in the presence of MDSCs from control (PBS-injected) or C5aR1 inhibitor–treated tumor-bearing mice, with or without addition of C5aR1 inhibitor to the culture. We found that C5aR1 blockade increased the frequencies of CD4+ T cells producing IFN-γ (Fig. 7A) and IL-17 (Fig. 7C), indicating Th1 and Th17 polarization. In contrast, C5aR1 antagonism reduced CD4+ T cells producing IL-4 (Fig. 7B), which suggests a reduction in Th2 responses. These changes in polarization of T cells correspond to alterations in the TGF-β/IL-6 ratio resulting from C5aR1 inhibition and to established functions of these cytokines in regulating adaptive antitumor immune responses (42).
Inhibition of C5aR1 signaling in MDSCs favors Th1 and Th17 responses. CD4+ T cells were stimulated with CD3/CD28 and cocultured in the absence (C5aRA in vitro −) or presence (C5aRA in vitro +) of C5aR1 antagonist with MDSCs from tumor-bearing mice injected with PBS (C5aRA in vivo −) or C5aR1 antagonist (C5aRA in vivo +). Percentage of cells producing IFN-γ (*p = 0.0203) (A), IL-4 (*p = 0.0174) (B), or IL-17 (*p = 0.0296) (C). Each point represents an individual mouse. Horizontal lines represent mean + SEM. Data are representative of one experiment with n = 5. All p values were calculated using one-way ANOVA.
Discussion
The complement system, and, in particular, C5aR1, were repeatedly demonstrated to promote tumor growth and immunosuppression in primary tumors (7, 44) and at metastatic sites (14, 45). C5aR1-mediated tumor-promoting and immunosuppressive functions were attributed to the well-characterized C5aR1 ligand C5a (7, 8), which is generated through the proteolytic cleavage of C5 by the C5 convertases and perhaps other serine proteases (46). Therefore, complement-mediated immunosuppression and tumor-promoting functions have been unequivocally associated with activation of the complement cascade and generation of complement effector proteins, such as C3a and C5a (47). In fact, several studies demonstrated complement activation leading to generation of C5a in human (47, 48) and experimental mouse (7) malignancies. In addition, deficiency in the complement protein C3, which is a pivotal junction upstream of C5a for multiple arms of the complement activation cascade and is required for complement functions, is associated with reduced tumor growth (7, 44). Therefore, it appears that complement activation and complement effectors promote tumor development and play important roles in suppressing antitumor immunity.
This study identifies a novel additional control mechanism linked to complement-mediated regulation of tumor growth and antitumor immunity, because we discovered that C5aR1 is also actively engaged in immunosuppression through its interaction with RPS19, even in the absence of C5 and C5a. Therefore, it appears that complement activation is not mandatory for complement-mediated immunosuppression. Interrupting or altering the RPS19–C5aR1 interaction through downregulation of RPS19 in tumor cells or pharmacological blockade of C5aR1 by C5aRA reduced this immunosuppression and led to the generation of tumor-specific T cell responses and slower tumor growth, pointing to the likely importance of a direct RPS19–C5aR1 interaction for immunosuppression. Furthermore, the delay in the development of clinically overt mammary tumors in C5-deficient FVB/N Her2/neu-transgenic mice treated with C5aRA suggests the participation of a C5aR1–RPS19 axis in the initiation of immunosuppression during the early stages of tumor development when tumors start to escape immune surveillance (31). RPS19 is an abundant intracellular protein that is expressed by virtually all cells in the body, and its extracellular functions, including interaction with C5aR1, are activated upon its release from dying cells (5). Therefore, it is possible that the immunosuppressive actions of RPS19 occur not only in tumors but also in other cells undergoing apoptosis. This notion is supported by the accumulation of RPS19 in TDLNs, where circulating immunosuppressive cells, including MDSCs, are undergoing apoptosis (Fig. 6A). Because inflammatory cells also undergo rapid apoptosis, it is conceivable that a C5aR1–RPS19 interaction contributes to the tumor-promoting properties of chronic inflammatory conditions (49).
Although the pharmacological blockade of C5aR1 in mice on an FVB background (lacking C5/C5a) may theoretically block the interaction of C5aR1 with other, unidentified ligands in addition to blocking C5aR1–RPS19 interactions, the concordance between data from these studies and from those using RPS19 shRNA supports important contributions of RPS19 to the suppression of antitumor immunity. The less robust phenotype observed in the studies using shRNA approaches likely results from moderate downregulation of RPS19 in only a portion of the mice in the study. However, significant downregulation of RPS19 does not appear to be feasible, without impacting cell survival and proliferation, given the pivotal role of RPS19 for protein translation.
We report original findings on a new player, RPS19, that interacts with the complement system in the tumor microenvironment and suppresses antitumor immunity. These findings further highlight the importance of the complement receptor C5aR1 in cancer and the need to better understand its interactions with endogenous ligands that can regulate immunosuppression and tumorigenicity. The current study demonstrates the high redundancy of mechanisms involved in tumor-associated immunosuppression and underscores the need to target multiple immunosuppressive mechanisms for successful cancer immunotherapy.
Disclosures
The authors have no financial conflicts of interest.
Footnotes
This work was supported by grants from the Australian National Health and Medical Research Council (456060 and 1027369) and Australian Research Council (DP150104609, DP160104442, and CE140100011) (to D.P.F.), Cancer Prevention and Research Institute of Texas Grant RP 120168 and Department of Defense Tuberous Sclerosis Research Program Grant TS 140010 (to M.K.), and National Institutes of Health Grant 1R01CA190209 (to M.M.M.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- AF
- Alexa Fluor
- C5aR1
- C5a receptor 1
- C5aRA
- C5aR1 antagonist
- DC
- dendritic cell
- IP
- immunoprecipitation
- MDSC
- myeloid-derived suppressor cell
- MHCII
- MHC class II
- RPS19
- ribosomal protein S19
- shRNA
- short hairpin RNA
- TDLN
- tumor-draining lymph node
- Treg
- regulatory T cell.
- Received December 7, 2016.
- Accepted January 27, 2017.
- Copyright © 2017 by The American Association of Immunologists, Inc.