The Ribosomal Protein S19 Suppresses Antitumor Immune Responses via the Complement C5a Receptor 1

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).

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FIGURE 1.

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.

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FIGURE 2.

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-1^int) versus granulocytic (Gr-1^high) 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⁺MHCII^lowCD11b⁺Gr-1⁺), with higher amounts of RPS19 bound to monocytic (Gr-1^int) MDSCs compared with granulocytic (Gr-1^high) 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.

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FIGURE 3.

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).

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FIGURE 4.

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).

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FIGURE 5.

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).

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FIGURE 6.

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).

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FIGURE 7.

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.