Myeloid-derived suppressor cells (MDSCs) are known suppressors of antitumor immunity, affecting amino acid metabolism and T cell function in the tumor microenvironment. However, it is unknown whether MDSCs regulate B cell responses during tumor progression. Using a syngeneic mouse model of lung cancer, we show reduction in percentages and absolute numbers of B cell subsets including pro–, pre–, and mature B cells in the bone marrow (BM) of tumor-bearing mice. The kinetics of this impaired B cell response correlated with the progressive infiltration of MDSCs. We identified that IL-7 and downstream STAT5 signaling that play a critical role in B cell development and differentiation were also impaired during tumor progression. Global impairment of B cell function was indicated by reduced serum IgG levels. Importantly, we show that anti–Gr-1 Ab-mediated depletion of MDSCs not only rescued serum IgG and IL-7 levels but also reduced TGF-β1, a known regulator of stromal IL-7, suggesting MDSC-mediated regulation of B cell responses. Furthermore, blockade of IL-7 resulted in reduced phosphorylation of downstream STAT5 and B cell differentiation in tumor-bearing mice and administration of TGF-β–blocking Ab rescued these IL-7–dependent B cell responses. Adoptive transfer of BM-derived MDSCs from tumor-bearing mice into congenic recipients resulted in significant reductions of B cell subsets in the BM and in circulation. MDSCs also suppressed B cell proliferation in vitro in an arginase-dependent manner that required cell-to-cell contact. Our results indicate that tumor-infiltrating MDSCs may suppress humoral immune responses and promote tumor escape from immune surveillance.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that are drivers of tumor-associated immune suppression (16). Broadly identified as Gr-1+CD11b+ cells in tumor-bearing mice, MDSCs segregate further into granulocytic and monocytic subsets (14). Accumulating evidence suggests that MDSCs modulate T cell responses in the tumor microenvironment (TME) by induction of multiple pathways that regulate oxidative and nitrative stress, such as inducible NO synthase (iNOS), arginase 1(ARG1), and reactive oxygen species, and by the induction of regulatory T (Treg) cells (13, 5, 6). Additionally, recent reports of suppression of B cell responses in experimental autoimmune myasthenia gravis and a murine acquired immunodeficiency model (7, 8) have been attributed to MDSCs. But the potential role of MDSCs in regulation of B cell responses during tumor progression is currently unknown.

B cells can either positively or negatively regulate immune responses (9). B cells positively regulate cellular immune responses by producing Abs (10), by serving as APCs (11), by secreting cytokines and chemokines, and by providing costimulatory signals to T cells (12, 13). Tumor-reactive B cells play a pivotal role in generating potent and long-term T cell responses against cancer (13, 14). A recently identified subset of regulatory B (Breg) cells is also known to promote tumor growth (1518).

IL-7, a cytokine which plays a pivotal role in B cell lineage commitment, regulation of B cell survival, proliferation, and maturation (19, 20), is primarily produced by nonhematopoietic cells, including fibroblastic stromal cells in the bone marrow (BM) and in the TME (21). Stromal IL-7 can be regulated by TGF-β (22), one of the key immunoregulatory cytokines produced by MDSCs (3). IL-7/IL-7R axis regulates early B cell development by activation of downstream signal transducer and activator of transcription 5 (STAT5) (23). Additionally, suppressor of cytokine signaling 1 (SOCS1) inhibits IL-7 responses in developing B lineage cells (24). A significant contribution of IL-7 and STAT5 signaling in B cell responses has not been described during tumor progression.

In the current study, we show that B cell differentiation and function are impaired during tumor progression. We provide evidence that MDSCs directly suppress B cell responses by inhibiting IL-7 and downstream STAT5 signaling that are essential for B cell differentiation. Anti–Gr-1 Ab-mediated depletion of MDSCs reduced TGF-β1 levels and partially rescued serum IgG, IL-7, phosphorylation of STAT5, and B cell differentiation in tumor-bearing mice. These data show that MDSCs directly inhibit B cell responses to tumors and suggest that targeted deletion of MDSCs could have a beneficial effect by enhancing B cell responses in cancer.

Female C57BL/6 mice and C57BL/6 congenic CD45.1+ mice at 6–8 wk of age were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were kept in pathogen-free conditions and handled in accordance with the Guidelines for Animal Experiments at the University of Alabama at Birmingham. The murine Lewis lung carcinoma (LLC) cell line was purchased from the American Type Culture Collection (Manassas, VA). LLC cells were cultured in DMEM supplemented with 10% FBS, 1 mmol/l sodium pyruvate, 2 mmol/l l-glutamine, 10 μg/ml penicillin-streptomycin, and 0.1 mmol/l nonessential amino acids (Life Technologies, Waltham, MA). Then, 106 LLC cells in 100 μl PBS were injected either i.v. via tail-vein injection or via an intracardiac (i.c.) route (25). BM and spleens were collected for analyses on day 16 or at other specified time points after injection of LLC cells.

BM from tibias and femurs as well as spleens were harvested, as previously described (25). RBCs were removed by ACK lysis buffer. Fc receptors were blocked with 3% BSA in PBS containing 2.4G2 Ab (anti-mouse CD16/CD32; BD Pharmingen) followed by staining with relevant Abs. FITC-conjugated anti-IgM (II/41), anti-CD21 (eBio4E3), PE-conjugated anti-CD43 (eBioR2/60), anti-CD23 (B3B4), anti–Gr-1 (RB6-8C5), anti-CD25 (PC61.5), anti–IL-10 (JES5-16E3), allophycocyanin-conjugated anti-CD93 (AA4.1), anti-CD24 (M1/69), anti-CD45.2 (104), anti-F4/80 (BM8), anti-CD5 (53-7.3), eFluor 450–conjugated anti-IgM (II/41), anti-CD1d (1B1), PerCP-eFluor 710–conjugated anti-IgD (11-26C), PerCP-Cyanine (Cy) 5.5–conjugated anti-Ly6C (HK1.4), anti-Foxp3 (FJK-16s), PE-eFluor 610–conjugated anti-CD45.1 (A20), PE-Cy7–conjugated anti-CD19 (eBio1D3), anti-CD4 (GK1.5), and allophycocyanin-eFluor 780–conjugated anti-CD45R (B220; RA3-6B2) Abs were purchased from Life Technologies (Grand Island, NY). FITC-conjugated anti-Ly6G (1A8), anti–Annexin V, and allophycocyanin-Cy7–conjugated CD11b Abs were purchased from BD Biosciences (San Jose, CA). Data were collected with LSR-II flow cytometer (BD) and analyzed with FlowJo software (version 8.5.2; Tree Star, Ashland, OR).

Anti–Gr-1 or IgG2b control Ab was administered i.p. into tumor-bearing mice, as previously described (25, 26). Mice transplanted with LLC cells i.v. were given i.p. injections of anti–Gr-1 Ab (250 μg/100 μl/mouse; Bio X Cell, West Lebanon, NH) or Ig G2b (IgG2b) control (Bio X Cell) on days 4, 7, and 11. BM, serum, and spleen tissues were collected for analyses on day 16 after injection of LLC cells.

Mice transplanted with LLC i.c. were given i.p. injections of anti–TGF-β Ab (clone 1D11.16.8, 300 μg/100 μl/mouse; Bio X Cell) on days 4 and 7. BM, serum, and spleen tissues were collected for analyses at day 11 after injection of LLC cells.

Mice transplanted with LLC i.c. were given i.p. injections of anti–IL-7 Ab (clone M25, 300 μg/100 μl/mouse; Bio X cell) or mouse IgG2b isotype control (Clone MPC-11, 300 μg/100 μl/mouse; Bio X Cell) on days 4 and 7. BM, serum, spleen, and lung tissues were collected for analyses at day 11 after injection of LLC cells.

MDSCs from BM or tumor tissues from CD45.2+ mice challenged with LLCs were purified by cell sorting on FACSAria cell sorter (BD Biosciences). A total of 5 × 105 sorted CD11b+Gr-1+ MDSCs were injected into tibias of CD45.1+ recipients. Peripheral blood, spleen, and BM samples were collected for analysis on day 7 after MDSC transfer.

Splenocytes from naive mice were labeled with CFSE (Molecular Probes, Eugene, OR) and cultured for 72 h with 20 μg/ml LPS (Sigma-Aldrich, St. Louis, MO) and 10 ng/ml IL-4 (PeproTech, Rocky Hills, NJ). The preactivated splenocytes were then cocultured for 48 h with MDSCs purified from the BM of tumor-bearing mice at a ratio of 1:1 in the absence or presence of 20 μM arginase inhibitor nor-NOHA (Cayman Chemical, Ann Arbor, MI), 500 nM iNOS inhibitor 1400W (Cayman Chemical), or 1 mM IDO inhibitor 1-D-MT (Sigma-Aldrich). The percentages of CD19+CFSElow cells (proliferating cells) were determined by FACS analysis. In some experiments, splenocytes were depleted of T cells by using Dynabeads FlowComp Mouse Pan T kit (Life Technologies). The same coculture assay was performed with the T cell–depleted splenocytes, as described above.

In some experiments, splenocytes from naive mice were stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 h. To determine whether the inhibitory effect of MDSCs on B cells is T cell–dependent or not, B220+CD19+ cells were sorted and labeled with CFSE. The sorted B cells were cocultured with MDSCs purified from the BM of tumor-bearing mice or immature myeloid cells from naive mice in the absence or presence of nor-NOHA, 1400W for 72 h. To determine whether suppression of B cells by MDSCs is T cell–dependent, T cells from the spleens of tumor-bearing mice were isolated with Dynabeads FlowComp Mouse Pan T kit (Life Technologies). The purified T cells were then added to the B cells and MDSCs in the cocultures described above. In blocking experiments, anti–TGF-β Ab (clone 1D11.16.8; Bio X Cell) or isotype control mouse IgG1(clone MOPC21; Bio X Cell) were added to the B cells and MDSCs coculture assays. To determine whether MDSCs suppress B cell proliferation through direct cell-to-cell contact, MDSC–B cell coculture experiments were established in the presence or absence of transwell inserts (Corning, NY).

Treg cells were purified from tumor-bearing Foxp3-DTR-GFP mice (kindly provided by Dr. T. Randall at the University of Alabama at Birmingham). Splenocytes were stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 h. B220+CD19+ cells were sorted and labeled with CFSE. These preactivated B cells were cocultured with CD4+GFP+ Treg cells or CD4+GFP T cells purified from spleens of tumor-bearing mice. The percentages of CD19+CFSElow cells (proliferating cells) were determined by FACS analysis.

The mouse sera were harvested from naive and tumor-bearing mice. The supernatants were collected from the in vitro B cell inhibitory assays, as described above. The levels of IgG and IgM were determined by ELISA kits from Life Technologies in sera and B cell culture supernatants following the instructions of the manufacturer. The levels of IL-7 were measured by a mouse IL-7 ELISA kit from R&D Systems (Minneapolis, MN). The levels of IL-2 in sera were quantified by an ELISA kit from Abcam (Cambridge, MA). The levels of TGF-β1 in sera or supernatants were measured using a commercial ELISA kit purchased from Life Technologies.

Sorted CD19+B220+ cells from BM were collected in radioimmunoprecipitation assay lysis buffer. A total of 15–20 μg of protein was used for the immunoblotting, unless otherwise indicated. Immunoblotting for total STAT5, phospho-STAT5, SOCS1, phospho–Bruton’s tyrosine kinase (Btk) (Try223), or Btk (Cell Signaling, Danvers, MA) was performed, as described previously (25). β-Actin was purchased from Sigma-Aldrich and used for the loading control.

One-way ANOVA with Tukey’s multiple comparisons test was used for multiple groups. Unpaired t test was used for the statistical analyses between two groups by using GraphPad Prism 5. A p value < 0.05 was considered statistically significant.

We used an established syngeneic orthotopic mouse model of lung cancer to evaluate mechanisms by which MDSCs affect B cell responses during tumor progression (26). Increased infiltration of MDSCs has been associated with tumor progression in murine models of lung cancer (25, 26). The percentages and total numbers of BM-MDSCs were increased in tumor-bearing mice on day 16 after i.v. implantation of LLC cells and on day 11 after i.c. transplantation, compared with naive tumor-free mice (Fig. 1A). Infiltration of both granulocytic (CD11b+Ly6G+Ly6Clow) and monocytic (CD11b+Ly6GLy6Chigh) MDSC subsets were significantly increased (Supplemental Fig. 1A, 1B) in BM and in the spleens of tumor-bearing mice (Fig. 1B, Supplemental Fig. 1C, 1D).

FIGURE 1.

Increased MDSCs in the BM and spleens of tumor-bearing mice. (A) FACS plots, percentages, and cell numbers of Gr-1+CD11b+ MDSCs in the BM from naive mice and tumor-bearing mice on day 16 post–LLC i.v. injection and on day 11 post–LLC i.c. injection (n = 8 mice per group). (B) FACS plots, percentages, and cell numbers of Gr-1+CD11b+ MDSCs in the spleens from naive mice and tumor-bearing mice on day 16 post–LLC i.v. injection and on day 11 post–LLC i.c. injection (n = 8 mice per group). ***p < 0.001.

FIGURE 1.

Increased MDSCs in the BM and spleens of tumor-bearing mice. (A) FACS plots, percentages, and cell numbers of Gr-1+CD11b+ MDSCs in the BM from naive mice and tumor-bearing mice on day 16 post–LLC i.v. injection and on day 11 post–LLC i.c. injection (n = 8 mice per group). (B) FACS plots, percentages, and cell numbers of Gr-1+CD11b+ MDSCs in the spleens from naive mice and tumor-bearing mice on day 16 post–LLC i.v. injection and on day 11 post–LLC i.c. injection (n = 8 mice per group). ***p < 0.001.

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We then determined if B cell populations/numbers and/or their differentiation were impaired with increasing infiltration of MDSCs by enumerating B cell subsets in the BM and spleens of tumor-bearing mice (27, 28). The percentages of total B220+ cells, B220+IgDIgMCD24intCD43+ pro–B cells and B220+IgD+IgM+ mature B cells were decreased (Fig. 2A, i.v. model), whereas the absolute numbers of B220+IgDIgMCD24hiCD43 pre–B cells and B220+IgDIgM+ immature B cells, in addition to the above subsets of B cells, were significantly decreased in the BM of tumor-bearing mice (Fig. 2B, i.v. model). To determine if modulation of B cell responses occurs with increasing tumor burden and correlates with increasing infiltration of tumor-promoting MDSCs, we investigated the dynamics of this response. Time course studies revealed that this reduction in the numbers of B220+ cells, pro–B cells and pre–B cells in the BM occurred as early as 5 d post–tumor challenge (Supplemental Fig. 2A, 2B), whereas immature and mature B cells in the BM were reduced at day 11 (Supplemental Fig. 2C, 2D). Additionally, the dynamics of MDSC infiltration in BM coincided with the decline of B cell populations in the BM (Supplemental Fig. 3A, 3B). In the spleens of tumor-bearing mice, both the percentages and numbers of B220+CD93+ immature B cells were increased, whereas B220+CD93CD21intCD23+ follicular B cells were decreased (Fig. 2C, 2D, i.v. challenged). Time course studies showed that the modulation of immature and follicular B cells was observed as early as 5 d after tumor challenge (Supplemental Fig. 3C–F). In the lungs of tumor-bearing mice, both the percentages and numbers of B cell populations were reduced in a time-dependent manner (Supplemental Fig. 3G, 3H). Similar results were obtained in an i.c. challenged tumor model (data not shown). These data suggest that impaired B cell differentiation was associated with MDSC infiltration and tumor progression.

FIGURE 2.

Impairment of B cell subsets in the BM and spleens of tumor-bearing mice. Percentages of pro– and mature B cells were reduced, whereas absolute numbers of pro–, pre–, immature, and mature B cells decreased in BM of tumor-bearing mice after i.v. challenge with LLC tumor cells. (A) The percentages of total B220+, pro–, pre–, immature, and mature B cells were determined by FACS analysis of cells harvested from the BM of naive and tumor-bearing mice on day 16 post–LLC i.v. challenge (n = 8 mice per group). (B) Absolute numbers of total B220+, pro–, pre–, immature, and mature B cells in the BM were calculated. Percentages and absolute numbers of follicular B cells were reduced, whereas immature B cells were increased in the spleens of tumor-bearing mice on day 16 post–LLC i.v. challenge with LLC tumor cells (n = 8 mice per group). (C) The percentages of total B, immature B, marginal zone B, and follicular B cells were determined by FACS analysis of cells harvested from the spleens of naive and tumor-bearing mice on day 16 post–LLC i.v. challenge (n = 8 mice per group). (D) Absolute numbers of total B, immature B, marginal zone B, and follicular B cells in the spleens are presented (n = 8 mice per group). **p < 0.01, ***p < 0.001.

FIGURE 2.

Impairment of B cell subsets in the BM and spleens of tumor-bearing mice. Percentages of pro– and mature B cells were reduced, whereas absolute numbers of pro–, pre–, immature, and mature B cells decreased in BM of tumor-bearing mice after i.v. challenge with LLC tumor cells. (A) The percentages of total B220+, pro–, pre–, immature, and mature B cells were determined by FACS analysis of cells harvested from the BM of naive and tumor-bearing mice on day 16 post–LLC i.v. challenge (n = 8 mice per group). (B) Absolute numbers of total B220+, pro–, pre–, immature, and mature B cells in the BM were calculated. Percentages and absolute numbers of follicular B cells were reduced, whereas immature B cells were increased in the spleens of tumor-bearing mice on day 16 post–LLC i.v. challenge with LLC tumor cells (n = 8 mice per group). (C) The percentages of total B, immature B, marginal zone B, and follicular B cells were determined by FACS analysis of cells harvested from the spleens of naive and tumor-bearing mice on day 16 post–LLC i.v. challenge (n = 8 mice per group). (D) Absolute numbers of total B, immature B, marginal zone B, and follicular B cells in the spleens are presented (n = 8 mice per group). **p < 0.01, ***p < 0.001.

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To determine whether B cell function is also impaired during tumor progression, we evaluated serum IgM and IgG levels in tumor-bearing mice. Serum IgG was decreased in tumor-bearing mice (Fig. 3A, 3B) as early as 5 d after tumor challenge (Fig. 3C) and correlated with the decline in B cell numbers (Fig. 3D, 3E); however, no difference was observed in IgM levels between tumor-bearing mice and tumor-free naive mice (Fig. 3A, 3B).

FIGURE 3.

Reduced IgG and IL-7 levels in the serum of tumor-bearing mice. (A) IgG and IgM levels were detected in the serum of tumor-bearing mice on day 16 post–LLC i.v. challenge (n = 8 mice per group). (B) IgG and IgM levels were detected in the serum of tumor-bearing mice on day 11 post–LLC i.c. challenge (n = 8 mice per group). (C) Time course of serum IgG at the indicated time points post–LLC i.v. injection (n = 4 mice per group). (D) Pearson correlation analysis of IgG levels with the percentages of total B cells in the BM of tumor-bearing mice (n = 8). (E) Pearson correlation analysis of IgG levels with the absolute numbers of B cells in the BM of tumor-bearing mice (n = 8). (F) IL-7 was reduced in the serum of tumor-bearing mice on 16 post–LLC i.v. challenge or on day 11 post–LLC i.c. challenge (n = 6 mice per group). (G) Time course of serum IL-7 at the indicated time points after LLC i.v. challenge (n = 4 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Reduced IgG and IL-7 levels in the serum of tumor-bearing mice. (A) IgG and IgM levels were detected in the serum of tumor-bearing mice on day 16 post–LLC i.v. challenge (n = 8 mice per group). (B) IgG and IgM levels were detected in the serum of tumor-bearing mice on day 11 post–LLC i.c. challenge (n = 8 mice per group). (C) Time course of serum IgG at the indicated time points post–LLC i.v. injection (n = 4 mice per group). (D) Pearson correlation analysis of IgG levels with the percentages of total B cells in the BM of tumor-bearing mice (n = 8). (E) Pearson correlation analysis of IgG levels with the absolute numbers of B cells in the BM of tumor-bearing mice (n = 8). (F) IL-7 was reduced in the serum of tumor-bearing mice on 16 post–LLC i.v. challenge or on day 11 post–LLC i.c. challenge (n = 6 mice per group). (G) Time course of serum IL-7 at the indicated time points after LLC i.v. challenge (n = 4 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

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To further understand the regulatory mechanisms that may affect B cell differentiation and maturation during lung cancer progression, we focused on IL-7 signaling. B cell precursors are associated with stroma in the BM through expression of c-kit, which interacts with stem cell factor (29). IL-7 plays a key role in initiating proliferation and differentiation of pro–B cells to pre–B cells (30, 31). We investigated whether modulation of IL-7 contributed to the impaired B cell differentiation observed in tumor-bearing mice. As shown in Fig. 3F, IL-7 was significantly reduced in tumor-bearing mice, with reduction observed as early as 5 d after tumor challenge (Fig. 3G).

STAT5 activation plays a crucial role in directing IL-7–dependent B cell differentiation (23, 32). STAT5 signaling was impaired with reduced level of STAT5 phosphorylation in the BM of tumor-bearing mice on day 16 after LLC i.v. (Fig. 4A, 4B) or on day 11 after LLC i.c. challenge (Fig. 4C). Importantly, phosphorylation of STAT5 was reduced as early as day 5 (Fig. 4D), consistent with the decline of IL-7 and B cell numbers and infiltration of MDSCs and remained reduced in the BM of tumor-bearing mice on day 11 (Supplemental Figs. 2, 3).

FIGURE 4.

Impairment of STAT5 signaling in the BM of tumor-bearing mice. (A and B) Impairment of STAT5 signaling in the BM of tumor-bearing mice. BM lysates were collected from tumor-bearing mice on day 16 post–LLC i.v. challenge. Western blotting was performed with p-STAT5, STAT5, and SOCS1 Abs. The relative expression of p-STAT5 or SOSC1 was normalized with STAT5 or β-actin, respectively. (C) Phosphorylation of STAT5 was reduced in the BM of tumor-bearing mice on day 11 post–LLC i.c. challenge. The relative expression of p-STAT5 was normalized with STAT5 or β-actin, respectively. (D) Time course of impairment of STAT5 signaling in the BM of tumor-bearing mice at the indicated time points post–LLC i.v. challenge. The relative expression of p-STAT5 or SOSC1 was normalized with STAT5 or β-actin, respectively. Data are presented as mean ± SEM of triplicates. (E) Time course of impairment of STAT5 signaling in the sorted CD19+B220+ cells from the BM of naive or tumor-bearing mice at the indicated time points post–LLC i.v. challenge. The relative expression of p-STAT5 or SOSC1 was normalized with STAT5 or β-actin, respectively. **p < 0.01, ***p < 0.001.

FIGURE 4.

Impairment of STAT5 signaling in the BM of tumor-bearing mice. (A and B) Impairment of STAT5 signaling in the BM of tumor-bearing mice. BM lysates were collected from tumor-bearing mice on day 16 post–LLC i.v. challenge. Western blotting was performed with p-STAT5, STAT5, and SOCS1 Abs. The relative expression of p-STAT5 or SOSC1 was normalized with STAT5 or β-actin, respectively. (C) Phosphorylation of STAT5 was reduced in the BM of tumor-bearing mice on day 11 post–LLC i.c. challenge. The relative expression of p-STAT5 was normalized with STAT5 or β-actin, respectively. (D) Time course of impairment of STAT5 signaling in the BM of tumor-bearing mice at the indicated time points post–LLC i.v. challenge. The relative expression of p-STAT5 or SOSC1 was normalized with STAT5 or β-actin, respectively. Data are presented as mean ± SEM of triplicates. (E) Time course of impairment of STAT5 signaling in the sorted CD19+B220+ cells from the BM of naive or tumor-bearing mice at the indicated time points post–LLC i.v. challenge. The relative expression of p-STAT5 or SOSC1 was normalized with STAT5 or β-actin, respectively. **p < 0.01, ***p < 0.001.

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SOCS1 is an important transcriptional regulator of cytokine signaling (24, 33). We investigated whether SOCS1 was upregulated during tumor progression and associated with the infiltration of tumor promoting MDSCs along with the inhibition of STAT5 activation. SOCS1 expression was increased in the BM tumor-bearing mice (Fig. 4A, 4B) as early as day 5 after tumor challenge (Fig. 4D).

Next, we sorted CD19+B220+ cells from BM of tumor-bearing mice. Phosphorylation of STAT5 was reduced whereas SOCS1 was elevated in a time-dependent manner (Fig. 4E). The results indicate that STAT5 signaling was impaired in B cells of BM from tumor-bearing mice.

Taken together, these results suggest that IL-7–mediated activation of downstream STAT5 signaling is disrupted during tumor progression, which contributes to the impairment of B cell development.

Because the dynamics of MDSC infiltration during tumor progression was associated with suppression of B cell differentiation and function, we investigated whether MDSCs would directly inhibit the B cell responses during tumor progression. TGF-β, an MDSC-associated cytokine that promotes MDSC-mediated Treg induction, has been shown to negatively regulate stromal levels of IL-7 (22). Therefore, we first investigated whether levels of immunoregulatory TGF-β were altered during tumor progression. We observed a significant increase in serum TGF-β1 during tumor progression (Fig. 5A, 5B).

FIGURE 5.

Anti–Gr-1 treatment partially rescued serum TGF-β1, IgG, and IL-7 levels as well as B cell subsets and STAT5 signaling in tumor-bearing mice. (A) Serum TGF-β1 levels were elevated in tumor-bearing mice, and TGF-β1 levels were reduced after MDSC depletion by anti–Gr-1 treatment (n = 5 mice per group). (B) Time course of serum TGF-β1 at the indicated time points after LLC i.v. injection (n = 4 mice per group). (C) Serum IgG levels were elevated after anti–Gr-1 treatment (n = 5 mice per group). (D) Serum IL-7 levels were increased after anti–Gr-1 treatment (n = 5 mice per group). (E) Percentages and absolute numbers of total B220+, pro–, pre–, immature, and mature B cells in the BM of tumor-bearing mice treated with anti–Gr-1 Ab (n = 5 mice per group). (F) The percentages and absolute numbers of immature B cells were decreased, whereas the percentages and absolute numbers of follicular B cells were increased after anti–Gr-1 treatment (n = 5 mice per group). (G) Phospho-STAT5 was elevated, whereas SOCS1 was reduced after anti–Gr-1 treatment in tumor-bearing mice. Densitometry data were quantified with ImageJ software. The relative expression of p-STAT5 or SOSC1 was normalized with STAT5 or β-actin, respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Anti–Gr-1 treatment partially rescued serum TGF-β1, IgG, and IL-7 levels as well as B cell subsets and STAT5 signaling in tumor-bearing mice. (A) Serum TGF-β1 levels were elevated in tumor-bearing mice, and TGF-β1 levels were reduced after MDSC depletion by anti–Gr-1 treatment (n = 5 mice per group). (B) Time course of serum TGF-β1 at the indicated time points after LLC i.v. injection (n = 4 mice per group). (C) Serum IgG levels were elevated after anti–Gr-1 treatment (n = 5 mice per group). (D) Serum IL-7 levels were increased after anti–Gr-1 treatment (n = 5 mice per group). (E) Percentages and absolute numbers of total B220+, pro–, pre–, immature, and mature B cells in the BM of tumor-bearing mice treated with anti–Gr-1 Ab (n = 5 mice per group). (F) The percentages and absolute numbers of immature B cells were decreased, whereas the percentages and absolute numbers of follicular B cells were increased after anti–Gr-1 treatment (n = 5 mice per group). (G) Phospho-STAT5 was elevated, whereas SOCS1 was reduced after anti–Gr-1 treatment in tumor-bearing mice. Densitometry data were quantified with ImageJ software. The relative expression of p-STAT5 or SOSC1 was normalized with STAT5 or β-actin, respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

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We then investigated whether MDSC depletion would reduce TGF-β1 levels, which would then potentially rescue IL-7, which would then support B cell development, maturation, and function. As shown in Fig. 5A, anti–Gr-1 Ab-mediated MDSC depletion reduced circulating TGF-β1 and partially rescued serum IL-7 and IgG levels in vivo (Fig. 5C, 5D).

Next, we tested whether dysregulation of B cell differentiation in the BM and spleens of tumor-bearing mice was reversed following MDSC depletion. The percentages of total B cells, pro–B cells, and mature B cells in the BM of tumor-bearing mice were significantly increased in the anti–Gr-1 Ab–treated group compared with the controls (Fig. 5E). Moreover, the absolute numbers of total B cells, pro–B cells, pre–B cells, immature B cells, and mature B cells in the BM of tumor-bearing mice were also elevated after administration of anti–Gr-1 Ab (Fig. 5E). In the spleen, both the percentages and absolute numbers of immature B cells were reduced, whereas both the percentages and absolute numbers of follicular B cells were increased by anti–Gr-1 treatment (Fig. 5F).

We then investigated whether rescue of levels of IL-7 following MDSC depletion would restore activation of STAT5. Phospho-STAT5 was increased in the BM of tumor-bearing mice after anti–Gr-1 treatment (Fig. 5G) compared with the control groups. In contrast, SOCS1 expression was reduced by MDSC depletion (Fig. 5G). Taken together, our studies show a direct effect of MDSCs on B cell differentiation and function mediated potentially by TGF-β–dependent regulation of IL-7 and its downstream effects on STAT5 signaling.

We further tested whether TGF-β depletion would rescue IL-7 signaling and B cell differentiation in tumor-bearing mice. As shown in Fig. 6A, anti–TGF-β depletion partially recued serum IL-7 level in vivo. Phospho-STAT5 was increased in the BM of tumor-bearing mice after anti–TGF-β treatment (Fig. 6B, 6C) compared with the control groups. In contrast, SOCS1 expression was reduced by TGF-β depletion (Fig. 6B, 6C). Both the percentages and absolute numbers of total B cells, pro–B cells, pre–B cells, and mature B cells in the BM of tumor-bearing mice were significantly increased in the anti–TGF-β Ab–treated group compared with the controls (Fig. 6D, 6E). In the spleen, both the percentages and absolute numbers of immature B cells were reduced, whereas both the percentages and absolute numbers of follicular B cells were increased by anti–TGF-β treatment (Fig. 6F, 6G). These results show that depletion of MDSC-associated cytokine TGF-β partially rescued serum IL-7 level and its downstream effects on STAT5 signaling.

FIGURE 6.

Anti–TGF-β treatment partially rescued serum IL-7 levels and STAT5 signaling as well as B cell subsets in tumor-bearing mice. (A) Serum IL-7 levels were increased after anti–TGF-β treatment (n = 3 mice per group). (B) Phospho-STAT5 was elevated, whereas SOCS1 was reduced after anti–TGF-β treatment in tumor-bearing mice (n = 3 mice per group). (C) Densitometry data were quantified with ImageJ software. The relative expression of p-STAT5 or SOSC1 was normalized with STAT5 or β-actin, respectively. (D and E) Percentages and absolute numbers of total B220+, pro–, pre–, immature, and mature B cells in the BM of tumor-bearing mice treated with anti–TGF-β Ab (n = 3 mice per group). (F and G) The percentages and absolute numbers of immature B cells were decreased, whereas the percentages and absolute numbers of follicular B cells were increased in the spleens after anti–TGF-β treatment (n = 3 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6.

Anti–TGF-β treatment partially rescued serum IL-7 levels and STAT5 signaling as well as B cell subsets in tumor-bearing mice. (A) Serum IL-7 levels were increased after anti–TGF-β treatment (n = 3 mice per group). (B) Phospho-STAT5 was elevated, whereas SOCS1 was reduced after anti–TGF-β treatment in tumor-bearing mice (n = 3 mice per group). (C) Densitometry data were quantified with ImageJ software. The relative expression of p-STAT5 or SOSC1 was normalized with STAT5 or β-actin, respectively. (D and E) Percentages and absolute numbers of total B220+, pro–, pre–, immature, and mature B cells in the BM of tumor-bearing mice treated with anti–TGF-β Ab (n = 3 mice per group). (F and G) The percentages and absolute numbers of immature B cells were decreased, whereas the percentages and absolute numbers of follicular B cells were increased in the spleens after anti–TGF-β treatment (n = 3 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

We next determined whether IL-7 blockade further impaired B cell differentiation in tumor-bearing mice. Serum levels of IL-7 were markedly reduced in the tumor-bearing mice by anti–IL-7 treatment (Fig. 7A). Phospho-STAT5 was further reduced in purified B cells from BM of tumor-bearing mice after anti–IL-7 treatment (Fig. 7B, 7C). The percentages and total numbers of MDSCs were increased in the BM and lungs of tumor-bearing mice by anti–IL-7 treatment (Fig. 7D, 7F). These results indicate IL-7 blockade further impaired STAT5 signaling in tumor-bearing mice.

FIGURE 7.

Serum IL-7 levels and STAT5 signaling were reduced after IL-7 blockade. (A) Serum IL-7 levels were decreased after anti–IL-7 treatment. (B and C) Phospho-STAT5 was reduced after anti–IL-7 treatment in tumor-bearing mice. The percentages and cell numbers of Gr-1+CD11b+ MDSCs were elevated in the BM (D) and lungs (F) after anti–IL-7 treatment in tumor-bearing mice. No changes of MDSCs were observed in the spleens (E) of tumor-bearing mice after anti–IL-7 treatment (n = 5 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7.

Serum IL-7 levels and STAT5 signaling were reduced after IL-7 blockade. (A) Serum IL-7 levels were decreased after anti–IL-7 treatment. (B and C) Phospho-STAT5 was reduced after anti–IL-7 treatment in tumor-bearing mice. The percentages and cell numbers of Gr-1+CD11b+ MDSCs were elevated in the BM (D) and lungs (F) after anti–IL-7 treatment in tumor-bearing mice. No changes of MDSCs were observed in the spleens (E) of tumor-bearing mice after anti–IL-7 treatment (n = 5 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

As shown in Fig. 8A and 8B, both the percentages and absolute numbers of total B cells, pro–B cells, pre–B cells, immature B cells, and mature B cells in the BM of tumor-bearing mice were significantly reduced by neutralizing IL-7. In the spleen, both the percentages and absolute numbers of immature B cells were increased, whereas both the percentages and absolute numbers of follicular B cells were reduced by anti–IL-7 treatment (Fig. 8C, 8D). Additionally, both the percentages and absolute numbers of B cell populations in the lung were reduced with IL-7 blockade (Fig. 8E, 8F). Taken together, these results indicated that IL-7 blockade further impaired B cell differentiation in tumor-bearing mice.

FIGURE 8.

Anti–IL-7 treatment further reduced B cell subsets in the BM, spleens, and lungs of tumor-bearing mice. (A and B) The percentages and absolute numbers of total B220+, pro–, pre–, immature, and mature B cells in the BM of tumor-bearing mice treated with anti–IL-7 Ab (n = 5 mice per group). (C and D) The follicular B cells were reduced in the spleens of tumor-bearing mice after anti–IL-7 treatment (n = 5 mice per group). (E and F) B cell subsets were decreased in the lung of tumor-bearing mice after anti–IL-7 treatment (n = 5 mice per group). *p < 0.05, **p < 0.01.

FIGURE 8.

Anti–IL-7 treatment further reduced B cell subsets in the BM, spleens, and lungs of tumor-bearing mice. (A and B) The percentages and absolute numbers of total B220+, pro–, pre–, immature, and mature B cells in the BM of tumor-bearing mice treated with anti–IL-7 Ab (n = 5 mice per group). (C and D) The follicular B cells were reduced in the spleens of tumor-bearing mice after anti–IL-7 treatment (n = 5 mice per group). (E and F) B cell subsets were decreased in the lung of tumor-bearing mice after anti–IL-7 treatment (n = 5 mice per group). *p < 0.05, **p < 0.01.

Close modal

MDSCs are known to suppress both CD4+ and CD8+ T cell responses in the TME (1, 3). Our studies also show suppression of tumor-specific T cells in the TME coinciding with MDSC infiltration, which was restored following MDSC depletion (25, 26). In parallel with MDSCs, Treg cells also contribute to the immunosuppression in tumor-bearing hosts (34). MDSCs are known to induce Treg cells in the TME (35). Because TGF-β1 levels were modulated upon MDSC depletion, we investigated whether this had an impact on Treg frequencies in the TME. As shown in Fig. 9A and 9B, percentages of CD4+CD25highFoxp3+ Treg cells were elevated in both BM and spleen of tumor-bearing mice. Treg infiltration in the BM was suppressed upon MDSC depletion by anti–Gr-1 treatment (Fig. 9A), whereas Treg infiltration in the spleens remained unaffected (Fig. 9B).

FIGURE 9.

Treg cells and apoptosis of B cell subsets in the BM and spleens of tumor-bearing mice. (A) Percentages of Treg cells in total cells and CD4+ T cells in the BM (n = 4 mice per group). (B) Percentages of Treg cells in total cells and CD4+ T cells in the spleens (n = 4 mice per group). (C) Serum IL-2 levels were not altered in tumor-bearing mice (n = 4 mice per group). (D) Serum IL-2 levels were not changed after anti–Gr-1 treatment (n = 4 mice per group). (E) Percentages of Annexin V+ B220+ cells, Annexin V+ pro–B cells, Annexin V+ pre–B cells, Annexin V+ immature B cells, and Annexin V+ mature B cells in the BM (n = 4 mice per group). (F) Percentages of Annexin V+ B220+ cells, Annexin V+ immature B cells, Annexin V+ marginal zone B cells, and Annexin V+ follicular B cells in the spleens (n = 4 mice per group). Naive: naive tumor-free mice; IgG2b: tumor-bearing mice treated with IgG2b control Ab; αGr-1: tumor-bearing mice treated with anti–Gr-1 Ab. (G) Splenocytes from naive mice were stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 h. B220+CD19+ cells were sorted and labeled with CFSE. The sorted B cells were cocultured with CD4+GFP+ Treg cells or CD4+GFP T cells purified from spleens of tumor-bearing Foxp3-DTR-GFP mice. The percentages of CD19+CFSElow cells were determined by FACS analysis. (H) Percentages and cell numbers of CD19+CD1d+CD5+IL-10+ Breg cells in the spleens of naive and tumor-bearing mice on day 16 post–LLC i.v. injection (n = 4 mice per group). (I) Percentages and cell numbers of CD19+CD1d+CD5+IL-10+ Breg cells in the lungs of naive and tumor-bearing mice on day 16 post–LLC i.v. injection (n = 4 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 9.

Treg cells and apoptosis of B cell subsets in the BM and spleens of tumor-bearing mice. (A) Percentages of Treg cells in total cells and CD4+ T cells in the BM (n = 4 mice per group). (B) Percentages of Treg cells in total cells and CD4+ T cells in the spleens (n = 4 mice per group). (C) Serum IL-2 levels were not altered in tumor-bearing mice (n = 4 mice per group). (D) Serum IL-2 levels were not changed after anti–Gr-1 treatment (n = 4 mice per group). (E) Percentages of Annexin V+ B220+ cells, Annexin V+ pro–B cells, Annexin V+ pre–B cells, Annexin V+ immature B cells, and Annexin V+ mature B cells in the BM (n = 4 mice per group). (F) Percentages of Annexin V+ B220+ cells, Annexin V+ immature B cells, Annexin V+ marginal zone B cells, and Annexin V+ follicular B cells in the spleens (n = 4 mice per group). Naive: naive tumor-free mice; IgG2b: tumor-bearing mice treated with IgG2b control Ab; αGr-1: tumor-bearing mice treated with anti–Gr-1 Ab. (G) Splenocytes from naive mice were stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 h. B220+CD19+ cells were sorted and labeled with CFSE. The sorted B cells were cocultured with CD4+GFP+ Treg cells or CD4+GFP T cells purified from spleens of tumor-bearing Foxp3-DTR-GFP mice. The percentages of CD19+CFSElow cells were determined by FACS analysis. (H) Percentages and cell numbers of CD19+CD1d+CD5+IL-10+ Breg cells in the spleens of naive and tumor-bearing mice on day 16 post–LLC i.v. injection (n = 4 mice per group). (I) Percentages and cell numbers of CD19+CD1d+CD5+IL-10+ Breg cells in the lungs of naive and tumor-bearing mice on day 16 post–LLC i.v. injection (n = 4 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Recent studies have demonstrated that IL-2 is involved in the differentiation and proliferation of B cells (3639). In addition to IL-7, IL-2 can also regulate STAT5 signaling (40). We hypothesized that increased infiltration of Treg cells in TME may lead to increased consumption of IL-2, which may then indirectly contribute to suppression of B cell differentiation in tumor-bearing hosts. As shown in Fig. 9C, serum IL-2 levels did not change in tumor-bearing mice. Additionally, IL-2 was not altered by anti–Gr-1 treatment (Fig. 9D), suggesting an IL-2 independent mechanism for the impaired B cell responses.

To determine whether Treg cells directly affect the proliferation of B cells, preactivated B cells were cocultured with Treg cells purified from tumor-bearing Foxp3-DTR-GFP mice. Proliferation of B cells was not inhibited in the coculture of Treg and B cells (Fig. 9G), indicating that impaired B cell responses are independent of Treg cells.

Breg cells are characterized as a CD19+CD1d+CD5+IL-10+ subpopulation that plays important roles in promoting tumor growth (12, 1517). We next investigated whether Breg cells were increased in tumor-bearing mice. As shown in Fig. 9H and 9I, the percentages and absolute numbers of CD19+CD1d+CD5+IL-10+ Breg cells were elevated in the spleens and lungs of tumor-bearing mice. These results suggested that, although B cell differentiation was impaired in tumor-bearing mice, differentiation of immunosuppressive Breg cells were increased.

We then determined whether B cell apoptosis accounted for the reduction in B cell subsets in tumor-bearing mice. As shown in Fig. 9E, the percentage of Annexin V+ B220+IgD+IgM+ mature B cells in the BM was significantly increased in tumor-bearing mice, which was rescued by anti–Gr-1 treatment. However, no significant differences were found in the percentages of pro–B cells, pre–B cells, and immature B cells in the BM as well as splenic B cell subsets (Fig. 9E, 9F). These data suggest a role for MDSC-mediated mechanisms in potentially modulating the apoptosis of mature B cells in the BM.

To further investigate the potential effects of MDSCs on B cell differentiation in vivo, we adoptively transferred MDSCs derived from the BM or tumors of CD45.2+ tumor-bearing mice by intratibial injection into congenic CD45.1+ mice. As shown in Supplemental Fig. 4A, CD45.2+CD11b+ cells were detected in the peripheral blood of the CD45.1+ recipient mice 48 h after transfer. Both the percentages and absolute numbers of total B220+ cells, B220+IgDIgM+ immature B cells, and B220+IgD+IgM+ mature B cells were reduced in circulation of congenic CD45.1+ mice on day 7 after MDSC transfers (Fig. 10A). Additionally, both the percentages and cell numbers of pre–B cells and immature B cells were reduced in the BM of congenic CD45.1+ recipients of BM-MDSCs from CD45.2+ tumor-bearing mice (Fig. 10B). The percentages and absolute numbers of total B220+ cells and B220+CD93+ immature B cells were also reduced in the recipient spleens, whereas only the numbers of B220+CD93CD21intCD23+ follicular B cells were reduced in these animals (Supplemental Fig. 4B). There was a trend toward reduction in serum IgG in the recipients of MDSC transfer, which did not reach statistical significance (Supplemental Fig. 4C).

FIGURE 10.

Adoptive intratibial transfer of MDSCs reduced circulating B cells as well as pre–B and immature B cells in the BM. (A) Percentages and absolute numbers of total B, immature B, and mature B cells were reduced in peripheral blood of congenic CD45.1+ mice on day 7 after intratibial injection of BM-MDSCs and tumor-MDSCs from CD45.2+ tumor-bearing mice (n = 4 mice per group). (B) Percentages and absolute numbers of pre–B and immature B cells were reduced in the BM of congenic CD45.1+ mice on day 7 after intratibial injection of BM-MDSCs from tumor-bearing mice (n = 4 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 10.

Adoptive intratibial transfer of MDSCs reduced circulating B cells as well as pre–B and immature B cells in the BM. (A) Percentages and absolute numbers of total B, immature B, and mature B cells were reduced in peripheral blood of congenic CD45.1+ mice on day 7 after intratibial injection of BM-MDSCs and tumor-MDSCs from CD45.2+ tumor-bearing mice (n = 4 mice per group). (B) Percentages and absolute numbers of pre–B and immature B cells were reduced in the BM of congenic CD45.1+ mice on day 7 after intratibial injection of BM-MDSCs from tumor-bearing mice (n = 4 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

We then investigated if MDSCs suppress B cell proliferation and IgG production in vitro. Preactivated CFSE-labeled splenocytes from naive mice were cocultured for 48 h with BM-MDSCs purified from tumor-bearing mice. The percentages of CD19+CFSElow cells were reduced in these cocultures, which were partially rescued by the addition of nor-NOHA or 1400W (Fig. 11A), inhibitors of arginase and iNOS, the regulatory pathways of MDSCs. IgG production in culture supernatants was decreased by BM-MDSCs, and this inhibition could also be partially reversed by nor-NOHA or 1400W, but not 1-D-MT (Fig. 11B), an inhibitor of indoleamine 2,3-dioxygenase that regulates tryptophan metabolism. These results indicate that MDSCs suppress B cell proliferation and impair IgG production by B cells in an arginase- and iNOS-dependent manner but not by an IDO-dependent mechanism.

FIGURE 11.

Suppression of B cell proliferation and function by MDSCs from tumor-bearing mice. (A) Splenocytes from naive mice were labeled with CFSE and cultured with LPS (20 μg/ml) and IL-4 (10 ng/ml). Seventy-two hours later, the preactivated splenocytes were cocultured with MDSCs purified from the BM of tumor-bearing mice in the absence or presence of arginase inhibitor nor-NOHA, iNOS inhibitor 1400W, or IDO inhibitor 1-D-MT for 48 h. The percentages of CD19+CFSElow cells were determined by FACS analysis. (B) IgG detection from the supernatant collected from the coculture. (C and D) Splenocytes depleted of T cells were labeled with CFSE and cultured with LPS and IL-4. The same experiments were performed as described in (A) and (B). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 11.

Suppression of B cell proliferation and function by MDSCs from tumor-bearing mice. (A) Splenocytes from naive mice were labeled with CFSE and cultured with LPS (20 μg/ml) and IL-4 (10 ng/ml). Seventy-two hours later, the preactivated splenocytes were cocultured with MDSCs purified from the BM of tumor-bearing mice in the absence or presence of arginase inhibitor nor-NOHA, iNOS inhibitor 1400W, or IDO inhibitor 1-D-MT for 48 h. The percentages of CD19+CFSElow cells were determined by FACS analysis. (B) IgG detection from the supernatant collected from the coculture. (C and D) Splenocytes depleted of T cells were labeled with CFSE and cultured with LPS and IL-4. The same experiments were performed as described in (A) and (B). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

To address whether the suppression of B cells by MDSCs is T cell–dependent, we examined the responses of B cells to MDSCs cocultured with CFSE-labeled splenocytes depleted of T cells. Both the percentages of CD19+CFSElow cells and IgG production were reduced in the cocultures, which were partially recovered by the addition of arginase inhibitor, but not iNOS inhibitor or IDO inhibitor (Fig. 11C, 11D). These results suggest that the suppression of B cells by MDSCs through the iNOS pathway is T cell–dependent.

Next, we further determined if the suppression of B cells by MDSCs is T cell–dependent. CD19+B220+ B cells were sorted from LPS- and IL-4–stimulated splenocytes and then cocultured with BM-MDSCs from tumor-bearing mice in the presence of nor-NOHA or 1400W. The percentages of CD19+CFSElow cells were reduced in the BM-MDSC cocultures, which were partially rescued by treatment with arginase inhibitor, but not iNOS inhibitor (Fig. 12A). We then examined the effects of T cell addition to the coculture of B cells with BM-MDSCs. The percentages of CD19+CFSElow cells were further reduced in the coculture with the addition of T cells purified from the spleens of tumor-bearing mice, which were partially rescued by iNOS inhibitor (Fig. 12C). Both the T cell depletion (Fig. 11C, 11D) and the T cell addition (Fig. 12C) experiments indicate that the suppression of B cells by MDSCs through iNOS pathways is T cell–dependent. Cocultures in transwells showed that suppression of B cell proliferation by BM-MDSCs was dependent on cell–cell contact (Fig. 12B).

FIGURE 12.

Suppression of B cell proliferation by MDSCs through iNOS is dependent on T cells. (A) Splenocytes from naive mice were stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 h. B220+CD19+ cells were sorted and labeled with CFSE. The sorted B cells were cocultured with MDSCs purified from BM of tumor-bearing mice in the absence or presence of arginase inhibitor nor-NOHA or iNOS inhibitor 1400W for 72 h. The percentages of CD19+CFSElow cells were determined by FACS analysis. (B) Purified B220+CD19+ cells were cocultured with MDSCs from tumor-bearing mice in the absence or presence of a transwell system for 72 h. The percentages of CD19+CFSElow cells were determined by FACS analysis. (C) T cells purified from spleens of tumor-bearing mice were added to the cocultures of B cells and MDSCs described in (A) in the absence or presence of arginase inhibitor nor-NOHA or iNOS inhibitor 1400W. The percentages of CD19+CFSElow cells were determined by FACS analysis. (D) TGF-β1 levels were elevated in the supernatants from the cocultures of B cells and MDSCs. (E) TGF-β1 was reduced in the cocultures of B cells and MDSCs in the presence of TGF-β neutralizing Ab. (F) The proliferation of B cells was increased in the cocultures after TGF-β blockade. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 12.

Suppression of B cell proliferation by MDSCs through iNOS is dependent on T cells. (A) Splenocytes from naive mice were stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 h. B220+CD19+ cells were sorted and labeled with CFSE. The sorted B cells were cocultured with MDSCs purified from BM of tumor-bearing mice in the absence or presence of arginase inhibitor nor-NOHA or iNOS inhibitor 1400W for 72 h. The percentages of CD19+CFSElow cells were determined by FACS analysis. (B) Purified B220+CD19+ cells were cocultured with MDSCs from tumor-bearing mice in the absence or presence of a transwell system for 72 h. The percentages of CD19+CFSElow cells were determined by FACS analysis. (C) T cells purified from spleens of tumor-bearing mice were added to the cocultures of B cells and MDSCs described in (A) in the absence or presence of arginase inhibitor nor-NOHA or iNOS inhibitor 1400W. The percentages of CD19+CFSElow cells were determined by FACS analysis. (D) TGF-β1 levels were elevated in the supernatants from the cocultures of B cells and MDSCs. (E) TGF-β1 was reduced in the cocultures of B cells and MDSCs in the presence of TGF-β neutralizing Ab. (F) The proliferation of B cells was increased in the cocultures after TGF-β blockade. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

TGF-β1 levels were elevated in the coculture of B cells and BM-MDSCs (Fig. 12D). The proliferation of B cells was also increased in the cocultures with anti–TGF-β Ab (Fig. 12F). These results suggest that MDSCs suppressed B cell proliferation in an arginase- and TGF-β–dependent as well as in a cell-to-cell contact-dependent manner.

B cells play a significant role in antitumor immune responses (4144). In this report, we show that B cell differentiation and humoral immunity were impaired in vivo during tumor progression. We present evidence that immunosuppressive MDSCs directly impact B cell differentiation by TGF-β–mediated modulation of IL-7 and downstream STAT5 signaling that are both essential for B cell differentiation and function. Our observation that B cell responses are impaired during tumor progression is consistent with studies by Richards et al. (45) who have demonstrated that tumor growth decreases NK and B cells as well as common lymphoid progenitors. They observed a significant decrease in the percentage and absolute number of total B220+ cells in the BM of EL4 tumor-bearing mice (45). In humans, B cell dysfunction is correlated with loss of CD27+ B cells in patients with advanced melanomas and other solid tumors (46); however, the underlying mechanisms involved in the impairment of B cell subsets remain largely unknown.

Mice deficient in IL-7 or IL-7R display a severe block of early pro–B cell development (47, 48). IL-7 was significantly reduced in our tumor-bearing mice, along with reduced IgG and impairment of B cell differentiation. Total cell numbers of pro–, pre–, immature and mature B cell subsets were reduced in tumor-bearing mice, suggesting that B cell subsets were broadly suppressed in BM in tumor-bearing mice, which might partially be mediated by the reduction of IL-7. In this regard, our observation that alterations in TGF-β levels correlating with tumor progression and the progressive decline in IL-7 levels suggests a role for immunoregulatory TGF-β in modulating IL-7. As TGF-β has been shown to regulate stromal IL-7 (22) and as MDSCs are significant contributors of TGF-β, the rescue of serum IL-7 levels following MDSC depletion suggests that MDSC-associated TGF-β–mediated mechanisms may account for the reduced IL-7 and downstream signaling and the impaired B cell responses. Additional evidence from our studies that treatment with TGF-β–blocking Ab rescued IL-7 and downstream STAT5 activation and B cell differentiation strongly support TGF-β–mediated regulation of IL-7 and STAT5 and their impact on B cell differentiation and function. Targeting the recovery of IL-7 and B cell differentiation and function may provide new approaches for boosting humoral immunity against cancer.

MDSCs and Treg cells are major components of the immunosuppressive TME. Several studies have shown that MDSCs support Treg cell development through TGF-β and IL-10 (49), (ARG1) (50), or through CD40/CD40L interactions (51). These results suggest that the cross-talks between MDSCs and Treg cells are fine-tuned and dependent on the context of different tumor models. In our study, the elevation of Treg cells in the BM could be reduced by anti–Gr-1–mediated MDSC depletion, indicating that MDSCs contribute to the expansion of Treg cells in the BM of tumor-bearing mice. The reduction in TGF-β following the depletion of MDSCs thus not only regulated IL-7 levels but also contributed to the reduction of Treg cells.

Although MDSCs have been shown to regulate B cell functions in different disease models, including experimental autoimmune myasthenia gravis (7), murine retrovirus-induced AIDS (8), and autoimmune arthritis (52), none of these studies have elucidated a direct mechanism involving TGF-β– and IL-7–mediated signaling. IL-7/IL-7R signaling stimulates the JAK-STAT transcription factor pathway that leads to STAT5 phosphorylation (53). Our study identified that IL-7 and downstream STAT5 signaling are impaired in tumor-bearing mice, which contributes to the disruption of B cell differentiation. Anti–Gr-1 treatment could partially rescue the phosphorylation of STAT5, suggesting that MDSCs impact signaling pathways that regulate B cell differentiation. Moreover, anti–TGF-β depletion also partially restored IL-7 and downstream STAT5 signaling as well as B cell differentiation, indicating that MDSC-associated TGF-β may negatively influence B cell differentiation. IL-7 blockade further impaired B cell differentiation in tumor-bearing mice. MDSCs were increased in the BM and lung of tumor-bearing mice following anti–IL-7 Ab treatment. This finding is consistent with the report that radiofrequency thermal ablation combined with the administration of intralesional IL-7 and IL-15 reduced MDSCs in breast tumor models (54). Additionally, the contribution of apoptosis to the observed reduction in B cell differentiation may be mediated by other soluble mediators produced by MDSCs, such as reactive oxygen and nitrogen species. Thus, MDSCs are central regulators of B cell differentiation and function.

It is unclear whether these suppressive responses of B cells mediated by MDSCs are T cell–dependent. Our in vitro coculture study in the presence of T cells suggests that MDSCs suppress B cell proliferation and IgG production by B cells in an arginase- and iNOS-dependent manner but not in an IDO-dependent manner. When splenocytes depleted of T cells were added to the coculture, MDSCs suppressed B cell proliferation and IgG production by B cells in an arginase-dependent manner. Furthermore, when T cells were added to the coculture of B cells with MDSCs, MDSCs suppressed B cell proliferation in an iNOS-dependent manner, indicating that the suppression of B cells by MDSCs through the iNOS pathway is T cell–dependent.

In the absence of T cells in the coculture, MDSCs suppress B cell proliferation in an arginase- and TGF-β–dependent manner. It is possible that the induction of arginase expression and enzymatic activity in MDSCs may use all available l-arginine (l-Arg) substrates and may override the availability of l-Arg for iNOS, which shares this substrate with arginase. It has also been reported that arginine deficiency affects early B cell maturation and lymphoid organ development in transgenic mice overexpressing arginase in their enterocytes (55). It is intriguing to speculate whether l-Arg deficiency induced by the increased activity of arginase in MDSCs might affect B cell differentiation and maturation in vivo in tumor-bearing mice. Durante et al. (56) reported that TGF-β1 stimulates l-Arg transport and metabolism in vascular smooth muscle cells. It has also been shown that TGF-β stimulates arginase activity in macrophages (57, 58). These evidence may account for the TGF-β– and arginase-dependent regulation of B cell proliferation. These combined results suggest that tumor-associated MDSCs may potentially regulate humoral immune responses in this murine lung cancer model.

Btk is a Tec family kinase with a well-defined role in BCR signaling (59, 60). Mutations in Btk result in X-linked agammaglobulinemia in humans. In the mouse, mutations in Btk cause xid, characterized by blockage in B cell development at multiple stages and impaired function of mature B cells (61). We investigated whether Btk signaling is involved in the impairment of B cell responses. There was no difference in the level of Btk phosphorylation in the purified B220+CD19+ BM-derived B cells between tumor-bearing mice and naive mice (data not shown), suggesting the involvement of Btk-independent signaling pathways in the impairment of B cell subsets in the BM of tumor-bearing mice.

In summary, we found that B cell differentiation and function are impaired during tumor progression and that much of this is attributable to direct effects of MDSCs. They were correlated and seemingly mediated via a reduction of IL-7–mediated STAT5 signaling and a dysfunctional B cell response. MDSC depletions and adoptive transfers show a direct role of these cells and their regulatory mechanisms in suppressing not only B cell differentiation but also their function. Additionally, we uncover TGF-β, a MDSC-associated cytokine, as a central suppressive regulator of IL-7 signaling in B cells in the TME. Furthermore, we demonstrate that the suppressive effects of MDSCs require cell-to-cell contact. Taken together, our findings suggest that tumor-infiltrating MDSCs may potentially regulate B cell response during tumor progression. Further efforts to investigate the potential mechanisms of B cell dysregulation during tumor progression may provide new insights into the immune incompetence in cancer, thereby suggesting novel approaches for targeted immunotherapies.

We thank Marion Spell at the University of Alabama at Birmingham Flow Cytometry Core Facilities for technical assistance in acquiring sorted cell samples. We thank Dr. Chad Steele at the University of Alabama at Birmingham for use of the laboratory’s 96-well plate reader.

This work was supported by American Cancer Society Institutional Research Grant Award IRG-60-001-53-IRG and National Institutes of Health Grant 1R01HL128502-01A1 to J.S.D., National Institutes of Health Grant R01-AI-083705 and the Lupus Research Institute Novel Research Award to H.-C.H., National Institutes of Health Grant R01 AI14782 to J.F.K., and National Institutes of Health P30 AR048311 and National Institutes of Health P30 AI27667 for service and support provided by the University of Alabama at Birmingham Comprehensive Flow Cytometry Core.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BM

bone marrow

Breg

regulatory B

Btk

Bruton’s tyrosine kinase

Cy

cyanine

i.c.

intracardiac, intracardially

iNOS

inducible NO synthase

LLC

Lewis lung carcinoma

MDSC

myeloid-derived suppressor cell

SOCS1

suppressor of cytokine signaling 1

STAT5

signal transducer and activator of transcription 5

TME

tumor microenvironment

Treg

regulatory T.

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The authors have no financial conflicts of interest.

Supplementary data