B cell regulation of the anti-tumor response and role in carcinogenesis

B regulatory cells suppress cellular immune responses and promote tumor growth in vivo through diverse mechanisms

1. I)

   Bregs promote tumor growth and metastasis through generation of Treg cells and suppression of effector T cell and NK cell responses

Several murine tumors including the MC38 colorectal cancer, EL4 thymoma, and EMT-6 mammary carcinoma models demonstrate reduced or absent growth in B-cell deficient mice (BCDM) compared to WT mice [4, 6, 63]. The anti-tumor effect conferred by B cell deficiency in all three models was associated with increased T cell and NK cell infiltration and more vigorous Th1 cytokine and cytolytic T cell responses, and in EMT-6 was also associated with decreased proliferation of CD4⁺FoxP3⁺ Tregs. Adoptive transfer of B cells from WT mice into tumor-bearing BCDM restored tumor growth and Treg proliferation and diminished CD8⁺IFN-?⁺ and NK cell infiltration into the tumor bed. Interestingly, in contrast to several autoimmune models the adoptive transfer of either WT or IL-10^?/? B cells was capable of rescuing tumor growth in the EMT-6 model [63]. In the MC38 model, adoptive transfer of OX40L^?/? B cells was less efficient in rescuing tumor growth than WT B cell transfer, demonstrating a role for cognate interactions between OX40L and OX40 on B cells and T cells respectively in modulating the anti-tumor response [64].

Pulmonary metastasis of murine 4 T1 breast cancer requires inactivation of antitumor NK cells and expansion of Treg cells [5, 65]. Olkhanud et al. [5] identified a CD25⁺CD19⁺B220⁺ B cell subset designated as tumor-evoked Bregs (tBregs), that constitutively expressed Stat3 and was expanded in 4 T1 tumor-bearing mice. In vitro, mouse B cells treated with cancer cell-derived cultured media (CM) but not control CM was able to suppress T cell proliferation. Furthermore, T cell inhibitory activity was restricted to CD25⁺, but not CD25^? cancer-CM treated B cells. Interestingly, peripheral blood CD19⁺ B cells from healthy human donors treated with ovarian- and colon-cancer cell-derived CM upregulated CD25 and suppressed T cell proliferation in vitro as well, suggesting that human tumors may also induce a suppressive CD25⁺ Breg population.

EMT-6 mammary tumors are rejected or show markedly diminished growth in BCDM but growth is restored in BCDM reconstituted with B cells [63]. Using the EMT-6 model Zhang et al. [69] demonstrated that tumor-infiltrating B cells (TIL-B) develop increased expression of LAP/TGF-?, CD80, CD86, and PD-L1 in vivo compared to splenic B cells. Development of a similar B cell immunosuppressive phenotype also occurred when B cells were co-cultured in vitro with EMT-6 cells, and was dependent on direct physical B cell: tumor cell contact. Functionally, these TIL-B cells demonstrated greater ability to suppress CD4⁺ and CD8⁺ T cell proliferation in response to anti-CD3/anti-CD28 co-stimulation, and also markedly suppressed NK cell proliferation in response to IL-15 compared to splenic B cells. Monoclonal antibodies directed against TGF-? or PD-L1 dramatically suppressed EMT-6 tumor growth in WT mice, suggesting a potential therapeutic strategy for targeting this specific B cell subpopulation. In addition, TIL-B but not splenic B cells were capable of secreting IL-10 following stimulation, suggesting that an IL-10 secreting subpopulation was predominantly contained within the TIL-B population. These results point to the local acquisition of immunosuppressive properties by B cells migrating into the tumor bed through intimate contact with the tumor cells. Mechanism(s) underlying this functional transition and migration, as well as evidence for similar activity in human tumors are being actively investigated.

1. IV)

   T2-MZP B cells accumulate in tumor-draining lymph nodes and promote metastasis independently of IL-10

MDSCs are key regulators of tumor growth and metastasis in the tumor microenvironment [71]. Using the B16 melanoma model in which tumor growth is inhibited in BCDM but restored with adoptive B cell transfer, Bodogai et al. [72] showed that adoptive transfer of MDSCs from B16-implanted WT mice into tumor-bearing BCDM restored tumor growth to the same degree as did adoptive transfer of B cells from B16-implanted WT mice. Conversely, adoptive transfer of MDSCs from B16-implanted BCDM into tumor-bearing BCDM did not restore metastasis.

In human cell lines, myeloid cells sort-purified from healthy donor PBMCs depleted of T and NK cells and treated with CM from MDA-MB-231 breast cancer cells were able to suppress T-cell proliferation in vitro. However, myeloid cells from donor PBMCs depleted of T, NK, and B cells and treated with cancer-derived CM failed to suppress T cell proliferation.

These results indicate that the immune-suppressive and tumor-promoting functions of MDSCs may be acquired through “education” by Bregs. The nature of B cell mediated education of MDSC is unclear, but clearly indicates a role for Breg in modulating myeloid suppressor cell activity, in addition to aforementioned effects on T and NK cells, thereby shaping the overall immune microenvironment.

1. VI)

   The role of Stat3 activated Bregs in promoting tumor growth and metastasis via generation of Treg cells and promotion of angiogenesis

Consistent with the finding that CD19⁺CD25⁺ tumor-evoked Bregs (tBregs) constitutively express Stat3 and promote tumor growth and metastasis by TGF?-dependent conversion of non-Tregs to Tregs [5], Lee-Chang et al. [73] demonstrated that treatment with non-cytotoxic doses of resveratrol (RSV), a potent inhibitor of Stat3 phosphorylation, suppressed proliferation of tBregs and FoxP3⁺ Tregs in vitro, and inhibited growth of B16 and 4 T1 murine tumors in vivo. In in vitro experiments using naïve B cells treated with 4 T1 cell-derived CM, low-dose RSV blocked tBreg generation, Treg generation, and reversed tBreg/Treg-mediated suppression of T cell proliferation. tBregs treated with RSV also had decreased expression of phosphorylated Stat3 (pStat3) and TGF? compared to mock-treated tBregs. Furthermore, RSV-treated tBregs adoptively transferred into 4 T1 tumor-bearing mice lacked the ability to expand FoxP3⁺ Tregs in vivo and promote lung metastasis as compared to mock-treated tBregs. These findings suggest that inhibition of Stat3 phosphorylation in Bregs, by RSV or other methods, may inhibit tumor growth by preventing the downstream local elaboration of TGF-? and subsequent promotion of FoxP3⁺ Tregs.

JSI-124 (cucurbitacin I), a potent Stat3 inhibitor, also inhibited growth of 4 T1 murine tumors. 4 T1-implanted mice treated with JSI-124 suppressed tumor growth compared to non-treated mice, and B cells isolated from treated mice had decreased expression of Stat3. Consistent with the hypothesis that inhibition of B cell-expressed Stat3 was responsible for the anti-tumor effect of JSI-124, Stat3^low B cells from treated mice exhibited a tumor-suppressive effect when injected into 4 T1-bearing mice, while injection of Stat3^high B cells from non-treated mice promoted tumor growth [74].

Yang et al. [75] showed that increased tumor growth mediated by Stat3 expression in B cells was associated with increased tumor angiogenesis. In T- and B-cell deficient Rag1^?/? mice, growth of B16 and Lewis Lung cancer (LLC) tumors was augmented following the adoptive transfer of Stat3^+/+ B cells, while growth was diminished with adoptive transfer of Stat3^?/? B cells. Augmented tumor growth with Stat3^+/+ B cell adoptive transfer was associated with increased tumor angiogenesis in vivo by Matrigel assay and in vitro in B cell: endothelial cell co-culture assays, and increased expression of pro-angiogenic genes by RNA in vivo. TIL-B cells with persistently-activated Stat3 has also been identified in several human tumors including melanoma, gastric, lung, liver, and prostate cancers [75]. Furthermore, Stat3 activity in human tumor tissues was associated with increased density of TIL-B cells and significantly increased intratumoral angiogenesis.

Zhang et al. [57] recently showed that CD5 positivity among CD19 cells strongly correlated with levels of Stat3 expression in human lung and prostate tumor tissues and in corresponding TDLN, indicating that Stat3-expressing Bregs may be contained within a CD5⁺ B cell population in human tumors.

Collectively these studies indicate an important role for Stat3 in conferring an immunosuppressive phenotype to Breg cells, mediated in part through local elaboration of TGF-? and through the induction of pro-angiogenic gene expression. Inhibition of Stat3 signaling may therefore be a useful means of reducing or reversing Breg promotion of tumor growth.

1. VII)

   CD19^±CD1d^highCD5^± Bregs infiltrate pancreatic neoplasms and mediate tumor growth via IL-35 signaling

Pylayeva-Gupta et al. [76] recently showed that prominent B cell infiltrates are frequently present in human pancreatic intraepithelial neoplasia (PanIN) and in pancreata of mice harboring Kras-driven pancreatic neoplasms. In addition, implantation of pancreatic ductal epithelial cells expressing oncogenic Kras (Kras^G12D) into WT mice pancreata leads to infiltration of B cells adjacent to the newly developing tumor. Kras^G12D pancreatic tumor growth is diminished in BCDM but tumor growth is restored following B cell adoptive transfer, which is accompanied by de novo tumor infiltration with the adoptively transferred B cells.

Since the CD1d^hiCD5⁺ B cell population has been shown to mediate immune-suppression via IL-10 secretion [37, 38] and IL-35 [77] in mouse autoimmune and tumor models, Pylayeva-Gupta et al. investigators hypothesized that CD1d^hiCD5⁺ B cells would also regulate immune-suppression and promote tumor growth in the mouse pancreatic ductal adenocarcinoma (PDAC) model. Adoptive transfer of CD19⁺CD1d^hiCD5⁺ B cells but not CD19⁺CD1d^loCD5^? B cells rescued tumor growth in BCDM inoculated with Kras^G12D. Adoptive transfer of IL-10^?/? B cells was capable of rescuing tumor growth in BCDM inoculated with Kras^G12D to the same extent as WT B cells, however tumor growth remained suppressed with adoptive transfer of IL-12?^?/? B cells, indicating that promotion of tumor growth by infiltrating CD1d^hiCD5⁺ B cells in this PDAC model is mediated by IL-35, a heterodimer comprised of p35 and EBI3 subunits encoded by the genes IL12? and EBI3, respectively.

Previous findings by Affara et al. [80] and Andreu et al. [81] suggested that TIL-B cells promote tumor growth in squamous cell carcinoma (SCC) models through interactions with Ig receptor FcR??+?myeloid cells and subsequent repolarization of TAM towards an immune-suppressive type (“M2”-type). Gunderson et al. [82] showed that mouse PDAC tumors were heavily infiltrated with B cells and FcR??+?myeloid cells, and furthermore that tumor growth was diminished in BCDM and Ig-receptor null FcR?^?/? mice. Based on these observations, investigators hypothesized that signaling pathways common to both B cells and macrophages, such as BTK signaling, may be involved in the suppression of antitumor immunity by infiltrating lymphocytes in PDAC tumors. In accordance with this hypothesis, activated BTK (pBTK) was identified in murine PDAC tumors in single-cell suspensions and was most prominent in CD19⁺ B cells and CD11b⁺ myeloid cells.

In in vitro assays using PDAC-derived B cells co-cultured with macrophages, PDAC-derived B cells enhanced macrophage expression of Th2 cytokines. In contrast, the pre-treatment of PDAC-derived B cells with the BTK inhibitor ibrutinib instead enhanced macrophage expression of Th1 cytokines. These results indicate that inhibition of BTK in tumor-infiltrating B cells may promote macrophage repolarization from an immune-suppressive, tumorigenic M2-type toward a pro-inflammatory, anti-tumor M1-type.

In vivo, early stage PDAC tumor-bearing mice treated with ibrutinib exhibited significantly diminished tumor growth. Treatment of late-stage tumor-bearing mice treated with ibrutinib plus gemcitabine resulted in significantly diminished tumor growth compared to gemcitabine alone. BTK may therefore represent a novel potential target for reduction of Breg cell activity in cancer, and furthermore BTK inhibition may augment the efficacy of chemotherapy.

Interestingly, depletion of B cells with an anti-CD20 antibody (?-CD20 mAb) in WT mice prior to inoculation with 4 T1 cells blocked metastasis of 4 T1 tumor, consistent with findings observed in genetically B-cell deficient mice [85]. However, administration of ?-CD20 mAb to WT mice with established 4 T1 tumors unexpectedly resulted in significantly enhanced tumor growth and metastasis.

Bodogai et al. [85] reconciled this paradoxical result by demonstrating that ?-CD20 mAb treatment of 4 T1 tumor-bearing mice enriches for B cells that are phenotypically CD20^low and have enhanced T cell-suppressive effects ex vivo compared to B cells from control IgG2a-treated tumor-bearing mice. Consistent with these findings in mice, co-culture of human peripheral blood B cells with human breast cancer or colon cancer cells resulted in emergence of a CD20^low Breg population that suppressed T-cell activity and was sustained after treatment with ?-CD20 mAb in vitro.

Screening for TLR ligands capable of inactivating Bregs revealed that the TLR9 ligand CpG-ODN was able to upregulate CD20 and block the T cell-suppressive effects of human and murine tBregs in vitro. Treatment of 4 T1 tumor-bearing mice with CpG-ODN abrogated lung metastasis, and ex vivo B cells derived from CpG-ODN treated tumor-bearing mice induced T cell proliferation and expansion of GrB⁺CD8⁺ cytolytic T cells. Enhancement of metastasis by ?-CD20 mAb treatment was completely reversed in vivo when ?-CD20 mAb-treated tumor-bearing mice were adoptively transferred with CpG-ODN-pretreated B cells from syngeneic naïve mice. In contrast, adoptive transfer of mock-treated B cells minimally reduced the enhanced lung metastasis following ?-CD20 mAb treatment.

4-1BBL (CD137L) is an immune co-stimulatory receptor and a member of the TNF receptor family, which activates CD8⁺ T cells and NK cells via cross-linking of 4-1BB (CD137) expressed primarily on T cells [86]. Bodogai et al. [85] investigators observed that human and murine CD20^low Bregs also had reduced 4-1BBL expression compared to normal B cells. Treatment with CpG-ODN reversed the reduced expression of 4-1BBL on murine and human Bregs in vitro as well as in vivo in tumor-bearing mice, resulting in emergence of a CD20^high4-1BBL^high B cell subset.

Consistent with the results seen in the 4 T1 murine model, the depletion of B cells using ?-CD20 mAb in mice inoculated with EMT-6 did not result in tumor rejection as expected, and was associated with a paradoxical increase of CD4⁺FoxP3⁺ Tregs in the spleens of treated mice, although a role for 4-1BBL was not investigated in the EMT-6 model [63].

In both the TRAMP and Myc-Cap (MC) mouse models of metastatic prostate carcinoma (PC), large tumors (0.7 g, 350–400 mm³ respectively) were resistant to treatment with low-dose oxaliplatin in WT mice but were sensitive to treatment in BCDM [88]. The anti-tumor effect conferred by B cell deficiency was associated with enhanced CD8+ cell infiltration in both tumor types. Treatment of tumor-bearing WT mice with oxaliplatin was associated with emergence of a tumor-infiltrating CD19⁺CD138⁺IgA⁺ plasma cell population that expressed IL-10, PD-L1, phosphorylated Stat3, and Fas-L. Reconstitution of tumor-bearing BCDM with WT B cells, but not PD-L1^?/? or IL-10^?/? B cells, could restore tumor growth in oxaliplatin-treated mice, implicating both PD-L1 and IL-10 in mediating the Breg suppressive effects. In addition, ablation of B cell TGF?R2 inhibited oxaliplatin-induced IgA⁺ plasma cell generation, blocked induction of tumoral PD-L1⁺ by oxaliplatin and was associated with increased tumoral CTL density and IFN-? production, thus implicating TGF-? signaling in the generation of this Breg subpopulation. Large MC tumors resistant to treatment with oxaliplatin monotherapy showed diminished tumor growth when treated with oxaliplatin plus anti-PD-L1, but not with anti-PD-L1 alone.

In human prostatectomy samples, CD8⁺ and CD20⁺ cell density was higher in patients with PC compared to healthy controls, and correlated significantly with advanced stage and treatment failure [88]. IL-10-producing IgA⁺CD138⁺ plasma cells were also present in human PC samples but were more abundant in metastatic PC and therapy-resistant PC compared to early stage disease.

These results indicate that treatment with an immunogenic chemotherapeutic agent, oxaliplatin, may induce a CD138⁺IgA⁺PD-L1⁺IL-10⁺ Breg population generated via a TGF?-dependent pathway that limits the effectiveness of oxaliplatin by inhibiting intratumoral CTL infiltration. This further suggests that the successful treatment of large prostate tumors with oxaliplatin may require the elimination of these immunosuppressive IgA⁺ plasma cells that are present in both mouse and human PC, and suggests a rational approach to augmenting beneficial chemotherapy effects on the anti-tumor immune response. Targeting of the PD-L1/PD-1 axis may be an especially useful strategy in this regard.

A summary of reported B reg phenotypic markers and functional attributes is presented in Table 1, and several of the key reported interactions reviewed above are schematically represented in Fig. 1.

Breg markers of immune suppression and tumorigenesis