Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: Cancer Res. 2018 Sep 17;79(1):159–170. doi: 10.1158/0008-5472.CAN-18-0981

Activation of B-1 cells promotes tumor cell killing in the peritoneal cavity

Marcela A Haro 1, Allison M Dyevoich 1, James P Phipps 1, Karen M Haas 1
PMCID: PMC6318009  NIHMSID: NIHMS1507354  PMID: 30224373

Abstract

Metastatic cancer involving spread to the peritoneal cavity is referred to as peritoneal carcinomatosis and has a very poor prognosis. Activating the anti-tumor immune response in the characteristically immune-suppressive peritoneal environment presents a potential strategy to treat this disease. In this study, we show that a toll-like receptor (TLR) and C-type lectin receptor (CLR) agonist pairing of monophosphoryl lipid A (MPL) and trehalose-6,6’-dicorynomycolate (TDCM) effectively inhibits tumor growth and ascites development in a mouse model of aggressive mammary cancer-induced peritoneal carcinomatosis. MPL/TDCM treatment similarly inhibited peritoneal EL4 tumor growth and ascites development. These effects were not observed in mice lacking B cells or mice lacking CD19, which are deficient in B-1a cells, an innate-like B cell population enriched in the peritoneal cavity. Remarkably, adoptive transfer of B-1a cells, but not splenic B cells from WT mice restored MPL/TDCM-induced protection in mice with B cell defects. Treatment induced B-1 cells to rapidly produce high levels of natural IgM reactive against tumor-associated carbohydrate antigens. Consistent with this, we found significant deposition of IgM and C3 on peritoneal tumor cells as early as 5 days post-treatment. Mice unable to secrete IgM or complement component C4 were not protected by MPL/TDCM treatment, indicating tumor killing was mediated by activation of the classical complement pathway. Collectively, our findings reveal an unsuspected role for B-1 cell-produced natural IgM in providing protection against tumor growth in the peritoneal cavity, thereby highlighting potential opportunities to develop novel therapeutic strategies for the prevention and treatment of peritoneal metastases.

Introduction

The majority of patients who succumb to cancer do so not from primary tumors, but from metastatic disease (1). In particular, the spread of malignant cells to the peritoneal cavity carries a grave prognosis, especially when associated with ascites development (2). The peritoneal surface and cavity may be affected by malignant epithelial (carcinomatosis), mesenchymal (sarcomatosis) and more rarely, lymphoid (lymphomatosis) cells (3). Peritoneal carcinomatosis due to cancers derived from malignant ovarian, colon, appendiceal, as well as breast (infiltrating ductal carcinoma) tissue (2,4), involves extensive spread and implantation of tumors and eventually, ascites development. The therapeutic options are limited and treatment plans are often palliative rather than curative, with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy currently representing the most common treatments (5). Immunotherapeutic approaches to treat peritoneal malignancies have been limited, although results obtained from some mouse models offer hope for future treatments (5).

Understanding the distinctive environment of the peritoneal cavity is key to devising optimal immunotherapies for peritoneal metastasis. The peritoneal space represents a unique immune environment (6). Monocytes and macrophages comprise the majority of leukocytes in the cavity under normal conditions. Innate-like B-1 cells, composed of CD5+ B-1a cells and CD5- B-1b cell populations, are the second most numerous (6,7). These B cells have been studied most in mice, but have been identified in non-human primate peritoneal and omental tissue (7,8) as well as in human blood (9). B-1 cells produce natural IgM and IgA as well as pathogen-specific antibodies, which are critical for host defense and clearance of apoptotic debris (10). Although they are known to have a critical role in protection against infectious diseases, their role in cancer is not well understood. Exchange of plasma components supplies the peritoneal fluid with many of the proteins found in the circulation, including B-1 cell-derived antibodies (6). However, additional soluble factors present in the peritoneal cavity, including IL-10 produced by B-1 cells and macrophage-produced prostaglandins, indoleamine 2,3-dioxygenase, and nitric oxide drive immunosuppression(11–15). Under normal conditions, both peritoneal B-1 cells and macrophages inhibit T cell activation and peritoneal macrophages additionally inhibit B cell proliferation and antibody production (11–13,15,16). Ascites from carcinomatosis patients contains high levels of IL-10, TGF-β, regulatory T cells, and immunosuppressive macrophages (17), suggesting suppression within the peritoneal cavity is maintained, if not augmented, in peritoneal metastases.

The use of pathogen-associated molecular pattern molecules (PAMPs) represents one strategy that is being investigated to overcome immune suppression and bolster anti-tumor responses (18). This is founded on the evidence of several successful therapeutic approaches in both pre-clinical models and patients employing bacterial-derived products. Indeed, over a century ago, studies documented that injecting ‘Coley’s toxin’ (consisting of heat-killed Streptococcus pyogenes and Serratia marcescens) into tumors lead to tumor regression and sometimes even cure. Killed Bacille Calmette-Guérin mycobacterium, one of the most widely used anti-tumor adjuvant therapies in humans, induces regression of transitional bladder cell carcinomas by stimulating a robust local immune response (18). Toll-like receptor (TLR) agonists, such as Imiquimod, have also been developed to treat several different forms of cancer (18). C-type lectin receptor (CLR) ligands such as fungal-derived beta-glucans (19) and cord factor (trehalose-6’,6-dimycolate) derived from mycobacteria (20) also have anti-tumor effects. Although both TLR and CLR agonists show promise as therapies for promoting anti-tumor responses, their mechanisms of action are not completely understood.

In the current study, we tested the effectiveness of monophosphoryl lipid A (MPL) and a synthetic cord factor analog, trehalose-6,6’-dicorynomycolate (TDCM) with 0.4% squalene in mouse models of peritoneal carcinomatosis and lymphomatosis. This treatment had potent and rapid effects in suppressing tumor growth and ascites development. Unexpectedly, the anti-tumor effects required B-1 cells, which harbor natural specificities for tumor associated carbohydrate antigens. Treatment elicited B-1 cell production of tumor-reactive IgM, which when combined with complement, elicited potent tumor cell killing. Collectively, our results highlight an unexpected role for the elicitation of tumor reactive-IgM through PAMP stimulation of innate B-1 cells. Our findings have implications for the development of future treatment strategies for peritoneal metastases in human patients.

Materials and Methods

Mice

Wild-type (WT) C57BL/6, CAF1, A/J, C3−/−, C4−/−, μMT (Ighmtm1Cgn), MHC class II−/−, IL-10−/−, and μS−/− mice were from Jackson Laboratories and Charles River. CD19−/− mice were as described (21). Studies were approved by Wake Forest Animal Care and Use Committee.

Tumor challenges

TA3-Ha cells were obtained from Dr. Richard Lo-Man (Pasteur Institute, Paris, France) in 2010. This stock was tested for rodent pathogens and mycoplasma (IMPACT IV testing, IDEXX-RADIL). E0771 and EL4 cells (Wake Forest Cell and Viral Vector Core Laboratory) were also negative for mouse pathogens and mycoplasma and were tested in 2016 (Impact III, IDEXX). For TA3-Ha challenges, aliquots of one ascites stock were used. TA3-Ha cells were expanded in culture in cRPMI+10% FCS for 3 days prior to injection. EL4 and E0771 were grown in the same medium and were similarly expanded in vitro from a frozen stock immediately prior to injection. Additional cell line authentication was not performed. TA3-Ha cells stably expressing GFP-luciferase were generated in house using a lentiviral vector expression system with blasticidin selection and flow sorting on GFP+ cells followed by single cell subcloning. Mice were given 1–2 × 104 TA3-Ha or 2 × 103-2 × 104 TA3-Ha-GFP-luciferase cells as indicated, in 200 μl PBS i.p. For IL-10 blocking, mice were given 100 μg of IL-10 neutralizing monoclonal antibody (mAb) (JES5–2A5) or control rat IgG mAb (BioXcell) i.p. on days 0, 2, 5, and 8. Mice developing ascites with signs of distress (lethargy, dehydration, reduced/impaired movement, reduced grooming, labored breathing, etc.) were humanely euthanized. Mice were injected intraperitoneally (i.p.) with 10 μg LPS (Escherichia coli O111:B4, Sigma) or Sigma adjuvant system (containing 10 μg monophosphoryl lipid A (MPL) and 10 μg synthetic trehalose dicorynomycolate (TDCM) (mixed in 0.4% squalene; Sigma) in a 200 μl volume.

IVIS imaging

Mice challenged with TA3-Ha-GFP-luciferase cells were injected i.p. with 3 mg D-Luciferin (Promega) 15 minutes prior to bioluminescent imaging using an IVIS Lumina Series III (Perkin Elmer). All images compared within an experiment were captured using the same exposure time (either 1 or 2 seconds).

Cell Transfers

Unless otherwise indicated, peritoneal B-1a cells were purified using Thy 1.2-Dynal beads, and biotinylated F4/80, GR1, DX5, CD11c mAbs(Biolegend) in conjunction with magnetic bead negative depletion (Biotin-binder beads, Dynal) according to manufacturer’s instructions, followed by positive anti-Ly5.1 (CD5) bead selection according to manufacturer’s instructions (Miltenyi Biotec). CD43- spleen B cells were purified using anti-CD43 magnetic beads (Dynal). B cell purity was assessed by flow cytometry.

Flow cytometry

Peritoneal cells were harvested using 10 ml of PBS to lavage the peritoneal cavity. Cells were resuspended in wash buffer (PBS containing 2% newborn calf serum; 2 ×106/ml). Peritoneal cells were pre-incubated with 0.5 μg/ml Fc Block and stained with 2.5 ug/ml biotin-conjugated Helix Pomatia agglutinin (HPA; Sigma) and mAbs conjugated to fluorochromes (from Biolegend, BD Biosciences, and eBioscience): CD4(RM4–5), CD8(Ly-2), NK1.1(PK136), CD49b(DX5), CD25(PC61.5), MHC class I(34–2–12), CD5(53–7.3), Ly6G(Gr-1), CD86(GL-1), CD138(281–2), CD19(1D3), CD11b(M1/70), F4/80(BM8), and CD11c(N418). Cells were washed and stained with streptavidin-conjugated fluorochrome for 20 minutes, washed and fixed with 1.5% buffered formaldehyde. TA3-Ha cells were identified as HPA+CD11bnegCD138+MHC-I+CD19- (22). To detect Ig and C3 bound to tumor cells harvested from peritoneal cavities, cells were incubated with Fc Block for 15 minutes prior to addition of biotinylated HPA (2.5 μg/ml) and mAbs against CD138, MHC class I, CD19, CD11b, and goat anti-mouse Ig (H+L) (Invitrogen) and anti-mouse C3 IgG–FITC (Cappel). Cells were washed in wash buffer and streptavidin-BV510 was added to detect HPA. Isotype controls were used to define positive and negative populations. Serum IgM and IgG binding (1:10 dilution) to tumor cells was assessed as previously described (22). Bovine submaxillary mucin (MP Biologicals) was included as a blocking agent (100 μg/ml) in some experiments. Cells were fixed in 1.5% buffered formaldehyde and analyzed using a FACSCanto II or Fortessa-X20 cytometer (Becton Dickinson).

Statistical analysis

Data are shown as means ± SEM with differences assessed using unpaired Student’s t test. Differences in Kaplan-Meier survival curves were assessed using the Log Rank test.

Results

MPL/TDCM suppresses tumor growth and ascites development in the peritoneal cavity

We tested the effectiveness of MPL and a synthetic cord factor analog, TDCM, in 0.4% squalene (MPL/TDCM) against the aggressive mouse mammary adenocarcinoma, TA3-Ha, which models rapid tumor growth and ascites development in the peritoneal cavity as observed in peritoneal carcinomatosis (23–26). CAF1 (BALB/c x A/J F1) mice are susceptible to syngeneic (A/J) TA3-Ha cells, with 100% of mice succumbing to the tumor within 4 weeks following i.p. challenge with 2×103 TA3-Ha-GFP-luciferase cells, and 100% of mice succumbing to challenge with 2×104 cells within 2 weeks (Fig. 1A-B). However, CAF1 mice treated with MPL/TDCM had significantly prolonged survival (Fig. 1A-B). IVIS imaging revealed a notable reduction in TA3-Ha-GFP-luciferase as early as 6 days post challenge, with minimal detection of tumor cells at 10 days post challenge in MPL/TDCM-treated mice, whereas untreated mice showed high levels of bioluminescence with extensive ascites development by day 10 (Fig. 1C; 2×104). Combined MPL/TDCM and cyclophosphamide treatment modestly improved survival (Supplementary Fig. S1), although remaining survivors did not survive a rechallenge on d40. Similar results were observed in A/J mice, which are highly susceptible to challenge, with MPL/TDCM-treatment limiting tumor growth at 10 days post treatment (Fig. 1D) and significantly prolonging survival (Fig. 1E). This result is consistent with previous work demonstrating protective effects of natural Mycobacterium tuberculosis cord factor and lipopolysaccharide in the TA3-Ha model (27).

Figure 1. MPL/TCDM protects against TA3-Ha tumor growth.

Figure 1.

Open in a new tab

A-E) CAF1 and A/J female mice were challenged i.p. with TA3-Ha-GFP-luciferase cells on d0 and either untreated or treated with MPL/TCDM i.p. on d1. Mice were monitored for tumor growth using IVIS imaging and visual inspection. Morbidity due to tumor growth requiring euthanasia was considered a death time point. A-B) Survival following challenge with 2×103 TA3-Ha cells (A; n=3–4/group) or 2×104 TA3-Ha cells (B; n=15/group, pooled results from 3 independent experiments). C) IVIS imaging results for CAF1 mice on d6 and d10 post challenge with 2×104 TA3-Ha cells (representative of 2 independent experiments, n=7 mice/group). D-E) IVIS imaging (d10) (D; n=4/group) and survival (E; n=5 mice/group) for A/J mice post challenge with 2×104 TA3-Ha cells.

The early clearance of TA3-Ha cells in CAF1 and A/J mice following MPL/TDCM treatment suggested a rapid and yet effective early innate-like response was playing a role in protection. Of note, the TA3-Ha cell line is highly invasive and mucinous and is so effective at evading the immune system that it is lethal in even non-syngeneic strains(23). To dissect the role of immune cells in MPL/TDCM-induced protection, we took advantage of the amenability of the TA3-Ha peritoneal carcinomatosis tumor model to growth in mice on a C57BL/6 background. 104 TA3-Ha cells injected i.p. into naïve WT C57BL/6 mice induces visible ascites between days 8–10 with mice rapidly succumbing to tumor growth and ascites development (22), similar to CAF1 and A/J mice (Fig. 2A). As observed in CAF1 and A/J mice, C57BL/6 mice were protected by MPL/TDCM treatment (Fig. 2A). Notably, E. coli-derived LPS elicited partial, yet significant protection (~50% survival; Fig. 2B), whereas MPL or TDB with squalene had only a partial effect (Supplementary Figure S2).

Figure 2. MPL/TCDM protects against TA3-Ha tumor growth and increases B cells in the peritoneal cavity.

Figure 2.

Open in a new tab

A-B) WT C57BL/6 female mice were challenged i.p. with 2×104 TA3-Ha cells and either untreated (n=32) or treated with MPL/TCDM (n=27) or LPS (n=19) i.p. on d0 and monitored for survival. Pooled data are representative of results obtained with MPL/TDCM in (A) and LPS in 4 independent experiments, with similar results (B). C-E) Leukocyte subset frequencies (C), B cell subset numbers (D), and CD138+ B cell frequencies (E) in peritoneal cavities on d5 (n=3 mice/group). Similar results were obtained in 2 independent experiments. F) Peritoneal TA3-Ha cell frequencies and numbers on d5 (n=3 mice/group). Similar results obtained in 3 independent experiments. Asterisks (*) indicate significant differences (p<0.05).

Alterations in leukocyte populations following MPL/TDCM treatment

Five days post treatment, CD11b+GR1+ myeloid cells and CD4+ and CD4+CD25+ T cell frequencies were decreased in the peritoneal cavity, whereas B cell frequencies were increased (Fig. 2C). This corresponded with a significant increase in the overall number of B cells, including B-1a, B-1b, and B-2 cells (Fig. 2D). Moreover, the frequency of Ab-secreting plasmablasts (a rare population in the peritoneal cavity) was significantly increased (Fig. 2E). Finally, consistent with IVIS imaging results, the frequency and number of TA3-Ha cells recovered from peritoneal cavities on day 5 were significantly reduced (≥6-fold) in MPL/TDCM-treated versus untreated mice (Fig. 2F).

B-1a cells are required for the anti-tumor effects of MPL/TDCM treatment

We next assessed the importance of B cells in MPL/TDCM-induced anti-tumor protection. As shown in Fig. 3A, MPL/TDCM treatment did not protect B-cell deficient (μMT) mice against TA3-Ha challenge. In addition, mice lacking CD19, a B cell signaling molecule critical for supporting B cell function and development of B-1a and marginal zone B cells, were not protected (Fig. 3B). Since CD19−/− mice are severely deficient in B-1a cells(21), we assessed whether transfer of bead-selected WT B-1a cells into CD19−/− and B-cell deficient mice could rescue the protective effects of MPL/TDCM treatment (Supplementary Fig. S3). As shown in Fig. 3C, transfer of WT B-1a cells into CD19−/− mice restored the protective effects of MPL/TDCM treatment, with 80% of B-1a cell recipient CD19−/− mice surviving challenge relative to 20% of non-reconstituted CD19−/− mice treated with MPL/TDCM. Similar effects were observed in μMT mice, with 60% survival achieved with B-1a cell reconstitution (Fig. 3D). Importantly, transfer of CD43- spleen (largely B-2 cells) B cells did not restore MPL/TDCM-induced protection in μMT mice (Fig. 3D), even with transfers of up to 107 B cells. FACS-sorted peritoneal B-1a, but not B-1b or B-2 cells, similarly protected μMT mice against tumor challenge (Supplementary Fig. S4). Nonetheless, transfer of peritoneal B-1a cells from MPL/TDCM-treated WT mice that had survived TA3-Ha challenge into μMT mice did not provide protection (Supplementary Fig. S5), indicating residual peritoneal “memory” B-1a cells (perhaps low-Ab secreting) are not sufficient for protection in the absence of MPL/TDCM stimulation. Collectively, these results demonstrate that B-1a cells are critical for the protective anti-tumor response elicited by MPL/TDCM.

Figure 3. B-1a cells promote MPL/TCDM-elicited anti-tumor protection.

Figure 3.

Open in a new tab

A-B) WT (n=5/group), μMT (A; n=5 naïve, n=7 treated), or CD19−/− (B; n=8 naïve, n=4 treated) mice were challenged i.p. with 2×104 TA3-Ha cells ± MPL/TDCM treatment (d0). C) Survival in untreated CD19−/− mice (n=8), MPL/TDCM-treated CD19−/− mice (n=10), and CD19−/− mice that had received 5×105 WT B-1a cells i.p. 1 week prior to challenge (n=5). D) Survival in untreated μMT mice (n=12), MPL/TDCM-treated μMT mice that had received 5×105 WT B-1a cells i.p. (n=10), and MPL/TDCM-treated μMT mice that had received CD43- spleen B cells i.p. (n=8; 4 received 1×106 and 4 received 1×107). Similar survival results were obtained with B-1a reconstitution in both CD19−/− and μMT mice in 3 independent experiments.

MPL/TDCM treatment induces protection independent of MHC class II antigen (Ag) presentation and IL-10 production

B-1a cells produce a large amount of IL-10 (14,15), which in some cases, actually support anti-tumor immune responses(28). To determine if IL-10 was playing a role in treatment effects, WT mice were given either anti-IL-10 (neutralizing) or control Ab during tumor challenge. No significant difference in survival was observed between mice that received control Ab versus mice that received IL-10 blocking Ab (75% vs 100%, Fig. 4A) or in IL-10−/− mice (Supplementary Fig. S6). Thus IL-10 is not required for MPL/TDCM-induced anti-tumor effects.

Figure 4. B cell-elicited anti-tumor protection is associated with increased production of tumor-reactive IgM.

Figure 4.

Open in a new tab

A) WT mice were treated with an IL-10 neutralizing or control mAb following challenge with 2×104 TA3-Ha cells and MPL/TDCM treatment (d0) and monitored for morbidity. B) Survival in untreated (n=9) and MPL/TDCM-treated MHC class II−/− mice (n=10). C) Survival in CD19−/− mice (n=6), or MPL/TDCM-treated CD19−/− mice that had received 5×105 WT B-1a cells (n=11) or 5×105 MHC class II−/− B-1a cells i.p. (n=11) 7d prior to challenge. MHC class II−/− B-1a cell reconstituted vs. non-reconstituted mice (p=0.04; Log rank analysis). Results are pooled from 2 independent experiments. D) Ig bound to TA3-Ha cells harvested from peritoneal cavities of WT and CD19−/− mice on d5 (n=3 mice/group). Similar results obtained in 3 independent experiments. E-F) C57BL/6 WT and CD19−/− serum IgM and IgG binding to cultured TA3-Ha cells (E) and E0771 cells (F) (n=7/group) on d0 and 5 post TA3-Ha challenge (± MPL/TDCM treatment on d0). G) CAF1 and A/J serum IgM binding to cultured TA3-Ha cells from d0 and 6 post TA3-Ha challenge sera (± MPL/TDCM treatment on d1; n=4/group). H-I) Mean changes in serum IgM binding (MFI) to TA3-Ha cells with BSM included during serum incubation (n=4/group). Asterisks (*) indicate significant differences between indicated groups (p<0.05).

B-1a cells have also been shown to present MHC class II-restricted Ag to CD4+ T cells, which could promote anti-tumor effects (29). However, MPL/TDCM treatment significantly increased overall survival (~40%) and prolonged time to death in MHC class II−/− mice during TA3-Ha challenge (Fig. 4B). Nonetheless, the effect of MPL/TDCM treatment in MHC class II−/− mice was not as striking as in WT mice. We therefore assessed whether B-1a cells lacking MHC class II could provide protection in the context of MPL/TDCM treatment. Reconstitution of CD19−/− mice with MHC class II−/− B-1a cells provided a similar level of protection to reconstitution with WT B-1a cells (Fig. 4C). Thus, B-1a cells elicit anti-tumor protection independently of MHC class II presentation.

TA3-Ha-reactive IgM antibodies are produced following MPL/TDCM treatment

B-1a cells play a central role in producing natural Abs (nAb) important for clearance of cellular debris and surveillance against pathogens and tumor cells(10). These Abs are spontaneously produced in the absence of exogenous Ag stimulation and are weakly reactive with self carbohydrates, lipids, and proteins. PAMPs such as LPS and MPL potently stimulate increases in nAb production (30). We therefore assessed Ab levels bound to tumor cells harvested from the peritoneal cavities of WT and CD19−/− mice 5 days post TA3-Ha cell challenge. WT mice that received MPL/TDCM treatment had significantly higher levels (~8-fold) of Ig bound to recovered TA3-Ha cells relative to untreated mice (Fig. 4D). CD19−/− mice had very little Ig deposited on recovered TA3-Ha cells, regardless of MPL/TDCM treatment. Follow-up experiments demonstrated that increased IgM was bound to tumor cells recovered from treated WT mice (Supplementary Fig. S7).

Serum harvested from WT C57BL/6 mice 5 days post treatment similarly showed significantly increased IgM reactivity with tumor cells relative to untreated (10-fold; Fig. 4E), whereas tumor-reactive IgM was not induced in CD19−/− mice (Fig. 4E). In contrast, serum IgM reactivity against another mouse mammary adenocarcinoma, E0771, was only modestly increased (~2-fold) in treated WT mice (Fig. 4F). Increases in TA3-Ha-reactive serum IgG were not observed in either WT or CD19−/− mice following treatment (Fig. 4E-F). A/J and CAF1 mice treated with MPL/TDCM also exhibited significant (>12-fold) increases in TA3-Ha-reactive IgM (Fig. 4G). Thus, MPL/TDCM treatment significantly increases IgM levels bound to TA3-Ha cells in vivo and serum IgM binding to TA3-Ha cells in vitro. Notably, TA3-Ha–reactive serum IgM levels were also significantly higher in mice treated with MPL/TDCM in the absence of tumor challenge (Supplementary Fig. S8), and cultured peritoneal B cells activated with either MPL/TDCM or LPS produced significantly increased levels of IgM, but not IgG, reactive with TA3-Ha cells (Supplementary Fig. S9), indicating that TA3-Ha-reactive IgM rapidly arose from non-specific activation and differentiation of B cells.

B-1 cells produce carbohydrate-reactive IgM, including IgM reactive against tumor associated carbohydrate antigens (TACAs)(22). We therefore assessed whether increased IgM reactivity included increased specificity for TACAs using bovine submaxillary mucin (BSM) which shares tumor-associated antigens, such as TF, Tn, and sialyl Tn, with the epiglycanin mucin expressed at high levels by TA3-Ha cells(31,32). As shown in Fig. 4H-I, inclusion of BSM during staining significantly reduced serum IgM binding in WT mice treated with MPL/TDCM, but had no effect on serum IgM binding from untreated WT mice or treated CD19−/− mice. Thus, increased IgM reactivity induced by MPL/TDCM is in part, due to mucin-associated antigen reactivity.

Serum IgM reactivity against EL4 cells, a mouse thymoma, was also significantly increased (~6-fold) in MPL/TDCM-treated WT mice, but not CD19−/− mice (Fig. 5A). In contrast to the reduced binding observed with TA3-Ha cells, BSM had no effect on serum IgM binding to EL4 cells (Fig. 5B). This is not unexpected as this tumor is not described to express TACAs found on mucins, but expresses other TACAs, including the glycosphingolipid, GD2 (33), for which increased IgM reactivity was found in WT, but not CD19−/−-treated mice (Supplementary Fig. S10). Thus, MPL/TDCM rapidly and significantly increases the level of IgM reactive against some tumor cells known to express TACAs, including TA3-Ha and EL4 cells.

Figure 5. MPL/TDCM treatment significantly increases EL4-reactive IgM and elicits protection against EL4 challenge in a B-cell dependent manner.

Figure 5.

Open in a new tab

A) Sera from WT and CD19−/− C57BL/6 mice (n=7/group) harvested on d0 and 5 post TA3-Ha challenge (± MPL/TDCM treatment on d0) was assessed for IgM binding to cultured EL4 cells. B) Mean changes in serum IgM binding (MFI) to TA3-Ha cells and EL4 cells with BSM included during serum incubation step. C-D) Survival in WT and CD19−/− male mice (C) and WT C57BL/6 and μMT male mice (D) following i.p. challenge with 1×106 EL4 cells ± MPL/TDCM treatment on d2 (n=4–5 CD19−/− and μMT mice per group and n=7–12 WT mice/group; p=0.0029 WT treated vs. untreated). Results in WT mice were repeated in an independent experiment. E) Survival of WT female mice following i.p. challenge with 1×105 E0771 cells ± MPL/TDCM treatment on d1 (n=3 mice/group).

MPL/TDCM protects against peritoneal lymphomatosis and is dependent on B cells

We next assessed the efficacy of MPL/TDCM treatment on other tumors growing in the peritoneal cavity. EL4 is a C57BL/6-derived mouse thymoma line that also forms ascites in the peritoneal cavity (referred to as peritoneal lymphomatosis). WT male mice challenged with 1×106 EL4 cells i.p. became moribund with ascites around 20 days post challenge. However, WT mice treated with MPL/TDCM were completely protected from EL4 tumor growth and ascites development (Fig. 5C-D). As observed with TA3-Ha cell challenge, MPL/TDCM-induced protection against EL4 growth was dependent on B cells and functional CD19 expression, as MPL/TDCM had no effect in CD19−/− or μMT mice (Fig. 5C and D). Moreover, surviving mice that were given MPL/TDCM treatment following primary EL4 challenge were protected against EL4 rechallenge (Supplementary Fig. S11). In contrast, MPL/TDCM treatment, which had had modest effects in eliciting E0771-reactive IgM, had no protective efficacy against intraperitoneal E0771 challenge (Fig. 5E). Thus, MPL/TDCM induces significant IgM reactivity against EL4 cells, and induces B cell-dependent protection against EL4 tumor challenge.

MPL/TDCM-induced protection is dependent on complement

The significantly increased binding of IgM to TA3-Ha cells following MPL/TDCM treatment led us to investigate the possibility that IgM was eliciting classical complement activation to aid in tumor cell killing. WT mice treated with MPL/TDCM had significantly increased levels of C3 bound to tumor cells harvested from the peritoneal cavity compared to mice that received TA3-Ha cells only (Fig. 6A). Moreover, MPL/TDCM treatment offered little protection to C3−/− mice against TA3-Ha challenge (Fig. 6B). Similarly, MPL/TDCM treatment did not provide protection in C4−/− mice, which are unable to active classical and lectin-initiated complement pathways (Fig. 6C). Thus, C3 and C4 are required for MPL/TDCM-induced anti-tumor protection.

Figure 6. MPL/TCDM induces C3 deposition on tumor cells and requires C4 and C3 for protection against TA3-Ha tumor challenge.

Figure 6.

Open in a new tab

A) C3 levels bound to TA3-Ha cells harvested from peritoneal cavities of WT mice on d5 (± MPL/TDCM treatment on d0; n=3 mice/group). Similar results were obtained in 3 independent experiments. B-C) Survival in C3−/− (n=8–10/group) and C4−/− mice (n=7–10/group) following TA3-Ha challenge (± MPL/TDCM-treatment on d0). Similar results were obtained in 3 independent experiments.

MPL/TDCM-induced protection against peritoneal carcinomatosis is dependent on secreted IgM

We hypothesized production of tumor-reactive IgM was the key mechanism by which B-1 cells elicited anti-tumor protection. Results of a passive serum transfer experiment using day 5 sera from treated WT and CD19−/− mice demonstrated a mild protective effect with WT sera (33% survival) in contrast to CD19−/− sera (0% survival), although this did not reach significance (Supplementary Fig. S12). This result may have been due to the relatively short half-life of IgM. We therefore obtained μS−/− mice that lack secreted IgM. As shown in Fig. 7A, WT littermates treated with MPL/TDCM had high levels of Ig deposited on TA3-Ha cells 5 days post challenge relative to non-treated mice. In contrast, MPL/TDCM treatment did not increase Ig deposition on tumor cells recovered from μS−/− mice. Along with this, the frequency of CD138+ (Ab-producing) B cells were significantly increased (4-fold) in WT(μS+/+) but not in μS−/− littermates, following MPL/TDCM treatment (Fig. 7B). Tumor cells recovered from WT littermates treated with MPL/TDCM had significantly increased C3 deposition relative to non-treated mice (Fig. 7C). However, MPL/TDCM treatment in μS−/− mice did not induce C3 deposition on TA3-Ha cells. Thus, secreted IgM is required for increased C3 deposition on tumor cells in response to MPL/TDCM treatment.

Figure 7. Tumor protection elicited by MPL/TCDM requires secretory IgM.

Figure 7.

Open in a new tab

A) Ig bound to TA3-Ha cells harvested from peritoneal cavities of μS+/+ and μS−/− littermates 5 days post TA3-Ha challenge (n=5 mice/group). B) CD138+ B cell frequencies in peritoneal cavities of μS+/+ and μS−/− littermates 5 days post TA3-Ha challenge (n=5 mice/group). C) C3 bound to TA3-Ha cells harvested from peritoneal cavities of μS+/+ and μS−/− littermates 5 days post TA3-Ha challenge (n=5 mice/group). D) TA3-Ha cell frequencies in peritoneal cavities of μS+/+ and μS−/− littermates 5 days post TA3-Ha challenge (n=5 mice/group). E) Survival in μS+/+ (n=17–18/group) and μS−/− littermates (n=9/group) following TA3-Ha challenge. Pooled survival results from 3 independent experiments. In A-E, MPL/TDCM treatment was given on d0.

Finally, we assessed the extent to which secreted IgM was involved in MPL/TDCM-mediated protection against TA3-Ha tumor challenge. Five days post treatment, WT mice had a significant reduction (>20-fold) in the frequency of TA3-Ha cells present in the peritoneal cavity relative to untreated mice (Fig. 7D). Relative to these results, MPL/TDCM treatment had a modest effect in reducing the tumor burden in μS−/− mice. Consistent with these findings, MPL/TDCM treatment protected WT mice during TA3-Ha challenge, but did not induce significant protection in μS−/− mice (Fig. 7E). Thus, MPL/TDCM-mediated protection against TA3-Ha cell peritoneal carcinomatosis is mediated by secretion of tumor-reactive IgM by B-1 cells.

Discussion

The current study reveals an unexpected role for B-1 cells in the protective anti-tumor response stimulated by PAMPs. Intraperitoneal injection of the TLR4 agonist, MPL, combined with the Mincle/MCL agonist, TDCM, significantly reduced tumor growth, ascites development, and morbidity and mortality associated with peritoneal carcinomatosis and lymphomatosis. Treatment resulted in a significant increase in peritoneal B cells, including antibody-secreting cells, as well as a significant increase (10–20-fold) in tumor-reactive IgM. Importantly, B-1a cells, but not splenic or peritoneal B-2 cells, were able to reconstitute protection in B-cell deficient mice. B-1 cells rapidly secrete high levels of natural IgM in response to PAMPs(34) and it is known that some of these antibodies are reactive with carbohydrate antigens expressed by tumor cells (22). Consistent with this, MPL/TDCM treatment significantly increased IgM reactivity with tumor cells known to express TACAs. The elicited IgM was critical for C3 complement deposition on tumor cells, and both secreted IgM and the classical complement pathway were essential for MPL/TDCM-induced anti-tumor protection. Collectively, our results highlight a major role for B-1a cell-produced IgM in combating peritoneal metastases.

The role of B cells in cancer is only beginning to be understood (35,36). Early studies using mouse models initially suggested B cells inhibit T cell anti-tumor responses and thus, promote tumor growth(36). However, some studies using mouse models support a protective role for B cells in cancer (35,36). Along with these data are divergent reports regarding the pro-tumorigenic and anti-tumorigenic functions of B cell-produced antibodies in cancer (35). Nonetheless, in human patients, the presence of tumor infiltrating B cells often correlates with improved survival (35,36). B-1 cells also appear to play dual roles in cancer. Through the production of tumor-reactive natural IgM, B-1 cells likely play a significant role in immune surveillance. Increased production of these antibodies, through non-antigen specific activation signals, may promote direct tumor cell killing as well as promote the induction of adaptive antitumor responses and memory, as supported by our EL4 rechallenge results. Through the production of antigen-induced TACA-reactive antibodies, B-1b cells can also participate in the protective adaptive humoral response to tumors (22). Nonetheless, due to their production of IL-10 and other tumor-promoting soluble factors (37), B-1a (and potentially B-1b) cells may also exert a strong pro-tumorigenic role in cancer. Our current study illustrates that the role of B-1 cells in cancer may be in fact, dynamic and context dependent. Indeed, our findings raise the possibility that PAMPs may skew pro-tumorigenic B-1 cells towards potent drivers of anti-tumor immune responses.

Many human cancer patients generate tumor-reactive antibody responses. These antibodies are often germ-line encoded and largely belong to the IgM subclass (38). Included within these specificities are TACAs found on glycoproteins and glycolipids, such as Tn, sialyl Tn, TF, LeY, GM2, GD2, and GD3 (38–40). Natural antibodies reactive with TACA have demonstrated oncolytic effects(38,41). Additional studies indicate the production of TACA-specific antibodies are associated with increased survival rates in patients (42–45) and in mice (22). Although the breadth and fine specificity of PAMP-elicited IgM is not yet elucidated, our results indicate mucin-related TACAs expressed by BSM (sTn, Tn, TF, sTF; (46,47)), and shared by the epiglycanin mucin that contributes to the aggressive nature of TA3-Ha cells (31,32), represent at least a portion of the TA3-Ha-, but not EL4-, reactive IgM elicited by treatment. EL4 cells, in contrast, express high concentrations of the tumor-associated glycolipid, ganglioside D2 (GD2) (33). Similar to the results described herein, an IgM mAb specific for GD2 works in concert with complement to protect mice against EL4 tumor growth and metastases (33). More detailed analyses of the tumor-protective IgM reactivities elicited by PAMPs are certainly warranted. Research into the efficacy of tumor-reactive IgM and complement in penetrating and eliciting efficacy against solid tumor masses is also needed.

Although we have determined PAMP-elicited tumor protection requires rapidly secreted IgM and classical complement activation, it is not yet clear what precise mechanism(s) are involved in tumor cell killing. It seems plausible that complement mediated cytotoxicity (CDC) plays a role in tumor cell death given the evidence of C3 deposition as well as the finding that treated A/J mice (naturally impaired in CDC due to C5 deficiency) succumb to tumor challenge much more rapidly than CAF1 mice. Nonetheless, it has previously been suggested that mucin-bearing tumors are resistant to this mechanism of killing (48). Importantly, expression of cell surface complement regulators may further limit CDC, yet still allow complement mediated killing by IgM (33). Under these circumstances, complement dependent cellular cytotoxicity (CDCC) may play a role in eliciting killing via recognition of complement fragments by effector cells, such as NK cells and macrophages (49). Although complement has been shown to have tumor-promoting potential (49), its role in PAMP-elicited protection against peritoneal carcinomatosis supports that, similar to the divergent roles described for B cells in cancer, complement can be tumor-promoting or –protective. Further studies are required to determine the mechanisms by which complement and IgM eradicate tumor cells in the unique environment of the peritoneal cavity.

Using PAMPs to activate B-1 cells to secrete tumor-reactive natural IgM may offer yet another therapeutic strategy that when combined with other immune activating agents, improve patient outcomes. Bacterial products as well as other PAMPs have previously been demonstrated to elicit anti-tumor effects in mouse tumor models as well as in cancer patients (18). However, in most cases, the mechanisms involved have not been clearly elucidated. Thus, our novel findings specifically demonstrating that the TLR and CLR agonist combination of MPL and a cord factor analog elicit innate-like B cell-dependent anti-tumor protection in the unique environment of the peritoneal cavity may stimulate additional research aimed at developing new therapeutic strategies for prevention and treatment of peritoneal metastases, and perhaps other TACA-expressing cancers in distinct anatomical locations. In order for such opportunities to be realized, it is critical that the mechanisms by which PAMPs elicit anti-tumor killing are elucidated. Thus, it remains to be established whether MPL and/or TDCM directly stimulate B-1 cells to produce tumor reactive IgM, and whether these agonists yield synergistic signals through TLR4 and Mincle/MCL, as has been described for other TLR/CLR agonist pairings (50). Moreover, whether CDC or CDCC is a primary mechanism of tumor cell killing, and whether other effector or accessory cells are involved remains to be determined. It is our hope that our study on mouse B-1a cells will offer insights into elicitation of protective TACA-reactive IgM in humans. Nonetheless, as there is much to learn about the newly characterized human B-1 population, additional work on human TACA-reactive B cells is necessary before therapeutic approaches specifically aimed at activating natural IgM production and potentially other tumor-promoting effects can be designed for human patients.

Supplementary Material

1

Acknowledgements

This work was supported by DOD grant W81XWH-15–1-0585 and an American Cancer Society research scholar grant (RSG-12–170-01-LIB) awarded to K. M. Haas. Shared resources support was provided by NCI-CCSG grant P30CA012197 awarded to B. C. Pasche. M. A. Haro was supported by NIH/NCI F31CA183567 and A. M. Dyevoich was supported in part by National Institutes of Health Training Grant AI007401 awarded to M. A. Alexander-Miller.

Footnotes

Conflict of Interest Statement: The authors have no conflicts to disclose.

References

  • 1.Fidler IJ, Kripke ML. The challenge of targeting metastasis. Cancer Metastasis Rev 2015;34:635–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Lambert LA, Wiseman J. Palliative Management of Peritoneal Metastases. Ann Surg Oncol 2018. [DOI] [PubMed] [Google Scholar]
  • 3.Cabral FC, Krajewski KM, Kim KW, Ramaiya NH, Jagannathan JP. Peritoneal lymphomatosis: CT and PET/CT findings and how to differentiate between carcinomatosis and sarcomatosis. Cancer Imaging 2013;13:162–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Carcoforo P, Raiji MT, Langan RC, Lanzara S, Portinari M, Maestroni U, et al. Infiltrating lobular carcinoma of the breast presenting as gastrointestinal obstruction: a mini review. J Cancer 2012;3:328–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Morano WF, Aggarwal A, Love P, Richard SD, Esquivel J, Bowne WB. Intraperitoneal immunotherapy: historical perspectives and modern therapy. Cancer Gene Ther 2016;23:373–81. [DOI] [PubMed] [Google Scholar]
  • 6.Capobianco A, Cottone L, Monno A, Manfredi AA, Rovere-Querini P. The peritoneum: healing, immunity, and diseases. J Pathol 2017;243:137–47. [DOI] [PubMed] [Google Scholar]
  • 7.Haas KM. B-1 lymphocytes in mice and nonhuman primates. Ann N Y Acad Sci 2015;1362:98–109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Yammani RD, Haas KM. Primate B-1 cells generate antigen-specific B cell responses to T cell-independent type 2 antigens. J Immunol 2013;190:3100–08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Griffin DO, Holodick NE, Rothstein TL. Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20+ CD27+ CD43+ CD70. J Exp Med 2011;208:67–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Baumgarth N The double life of a B-1 cell: self-reactivity selects for protective effector functions. Nat Rev Immunol 2011;11:34–46. [DOI] [PubMed] [Google Scholar]
  • 11.Matlack R, Yeh K, Rosini L, Gonzalez D, Taylor J, Silberman D, et al. Peritoneal macrophages suppress T-cell activation by amino acid catabolism. Immunology 2006;117:386–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Stein SH, Phipps RP. Macrophage-secreted prostaglandin E2 potentiates immune complex-induced B cell unresponsiveness. Eur J Immunol 1990;20:403–7. [DOI] [PubMed] [Google Scholar]
  • 13.Hamilton MJ, Antignano F, von Rossum A, Boucher JL, Bennewith KL, Krystal G. TLR agonists that induce IFN-beta abrogate resident macrophage suppression of T cells. J Immunol 2010;185:4545–53. [DOI] [PubMed] [Google Scholar]
  • 14.O’Garra A, Chang R, Go N, Hastings R, Haughton G, Howard M. Ly-1 B (B-1) cells are the main source of B cell-derived interleukin 10. Eur J Immunol 1992;22:711–17. [DOI] [PubMed] [Google Scholar]
  • 15.Maseda D, Candando KM, Smith SH, Kalampokis I, Weaver CT, Plevy SE, et al. Peritoneal cavity regulatory B cells (B10 cells) modulate IFN-gamma+CD4+ T cell numbers during colitis development in mice. J Immunol 2013;191:2780–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Goldman N, Valiuskyte K, Londregan J, Swider A, Somerville J, Riggs JE. Macrophage regulation of B cell proliferation. Cell Immunol 2017;314:54–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kipps E, Tan DS, Kaye SB. Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research. Nat Rev Cancer 2013;13:273–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Pradere JP, Dapito DH, Schwabe RF. The Yin and Yang of Toll-like receptors in cancer. Oncogene 2014;33:3485–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Yoon TJ, Koppula S, Lee KH. The effects of beta-glucans on cancer metastasis. Anticancer Agents Med Chem 2013;13:699–708. [DOI] [PubMed] [Google Scholar]
  • 20.Bekierkunst A, Wang L, Toubiana R, Lederer E. Immunotherapy of Cancer with Nonliving BCG and Fractions Derived from Mycobacteria: Role of Cord Factor (Trehalose-6, 6’-Dimycolate) in Tumor Regression. Infect Immun 1974;10:1044–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Haas KM, Poe JC, Steeber DA, Tedder TF. B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae. Immunity 2005;23:7–18. [DOI] [PubMed] [Google Scholar]
  • 22.Haro MA, Littrell CA, Yin Z, Huang X, Haas KM. PD-1 Suppresses Development of Humoral Responses That Protect against Tn-Bearing Tumors. Cancer Immunol Res 2016;4:1027–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Hagmar B, Ryd W. Site dependency of TA3 Ha allotransplantability. Transplantation 1977;23:93–7. [DOI] [PubMed] [Google Scholar]
  • 24.Nagy JA, Herzberg KT, Dvorak JM, Dvorak HF. Pathogenesis of malignant ascites formation: initiating events that lead to fluid accumulation. Cancer Res 1993;53:2631–43. [PubMed] [Google Scholar]
  • 25.Singhal A, Fohn M, Hakomori S. Induction of alpha-N-acetylgalactosamine-O-serine/threonine (Tn) antigen-mediated cellular immune response for active immunotherapy in mice. Cancer Res 1991;51:1406–11. [PubMed] [Google Scholar]
  • 26.Nagy JA, Masse EM, Herzberg KT, Meyers MS, Yeo KT, Yeo TK, et al. Pathogenesis of ascites tumor growth: vascular permeability factor, vascular hyperpermeability, and ascites fluid accumulation. Cancer Res 1995;55:360–8. [PubMed] [Google Scholar]
  • 27.Butler JC, Nowotny A. Combined immunostimulation in the prevention of tumor take in mice using endotoxins, their derivatives, and other immune adjuvants. Cancer Immunol Immunother 1979;6:55–262. [Google Scholar]
  • 28.Oft M IL-10: master switch from tumor-promoting inflammation to antitumor immunity. Cancer Immunol Res 2014;2:194–9. [DOI] [PubMed] [Google Scholar]
  • 29.Popi AF, Longo-Maugeri IM, Mariano M. An Overview of B-1 Cells as Antigen-Presenting Cells. Front Immunol 2016;7:138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Gunti S, Messer RJ, Xu C, Yan M, Coleman WG, Jr., Peterson KE, et al. Stimulation of Toll-Like Receptors profoundly influences the titer of polyreactive antibodies in the circulation. Sci Rep 2015;5:15066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Fung PY, Longenecker BM. Specific immunosuppressive activity of epiglycanin, a mucin-like glycoprotein secreted by a murine mammary adenocarcinoma (TA3-HA). Cancer Res 1991;51:1170–6. [PubMed] [Google Scholar]
  • 32.Van den Eijnden DH, Evans NA, Codington JF, Reinhold V, Silber C, Jeanloz RW. Chemical structure of epiglycanin, the major glycoprotein of the TA3-Ha ascites cell. The carbohydrate chains. J Biol Chem 1979;254:12153–9. [PubMed] [Google Scholar]
  • 33.Imai M, Landen C, Ohta R, Cheung NK, Tomlinson S. Complement-mediated mechanisms in anti-GD2 monoclonal antibody therapy of murine metastatic cancer. Cancer Res 2005;65:10562–8. [DOI] [PubMed] [Google Scholar]
  • 34.Genestier L, Taillardet M, Mondiere P, Gheit H, Bella C, Defrance T. TLR agonists selectively promote terminal plasma cell differentiation of B cell subsets specialized in thymus-independent responses. J Immunol 2007;178:7779–86. [DOI] [PubMed] [Google Scholar]
  • 35.Tsou P, Katayama H, Ostrin EJ, Hanash SM. The Emerging Role of B Cells in Tumor Immunity. Cancer Res 2016;76:5597–601. [DOI] [PubMed] [Google Scholar]
  • 36.Nelson BH. CD20+ B cells: the other tumor-infiltrating lymphocytes. J Immunol 2010;185:4977–82. [DOI] [PubMed] [Google Scholar]
  • 37.Zhang C, Xin H, Zhang W, Yazaki PJ, Zhang Z, Le K, et al. CD5 Binds to Interleukin-6 and Induces a Feed-Forward Loop with the Transcription Factor STAT3 in B Cells to Promote Cancer. Immunity 2016;44:913–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Vollmers HP, Brandlein S. Natural antibodies and cancer. N Biotechnol 2009;25:294–98. [DOI] [PubMed] [Google Scholar]
  • 39.Kieber-Emmons T, Monzavi-Karbassi B, Hutchins LF, Pennisi A, Makhoul I. Harnessing benefit from targeting tumor associated carbohydrate antigens. Hum Vaccin Immunother 2017;13:323–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Diaz-Zaragoza M, Hernandez-Avila R, Viedma-Rodriguez R, Arenas-Aranda D, Ostoa-Saloma P. Natural and adaptive IgM antibodies in the recognition of tumor-associated antigens of breast cancer (Review). Oncol Rep 2015;34:1106–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Varambally S, Bar-Dayan Y, Bayry J, Lacroix-Desmazes S, Horn M, Sorel M, et al. Natural human polyreactive IgM induce apoptosis of lymphoid cell lines and human peripheral blood mononuclear cells. Int Immunol 2004;16:517–24. [DOI] [PubMed] [Google Scholar]
  • 42.Takahashi T, Johnson TD, Nishinaka Y, Morton DL, Irie RF. IgM anti-ganglioside antibodies induced by melanoma cell vaccine correlate with survival of melanoma patients. J Invest Dermatol 1999;112:205–09. [DOI] [PubMed] [Google Scholar]
  • 43.Hsueh EC, Gupta RK, Qi K, Morton DL. Correlation of specific immune responses with survival in melanoma patients with distant metastases receiving polyvalent melanoma cell vaccine. J Clin Oncol 1998;16:2913–20. [DOI] [PubMed] [Google Scholar]
  • 44.Livingston PO, Wong GY, Adluri S, Tao Y, Padavan M, Parente R, et al. Improved survival in stage III melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with GM2 ganglioside. J Clin Oncol 1994;12:1036–44. [DOI] [PubMed] [Google Scholar]
  • 45.Fremd C, Stefanovic S, Beckhove P, Pritsch M, Lim H, Wallwiener M, et al. Mucin 1-specific B cell immune responses and their impact on overall survival in breast cancer patients. Oncoimmunology 2016;5:e1057387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Tsuji T, Osawa T. Carbohydrate structures of bovine submaxillary mucin. Carbohydr Res 1986;151:391–402. [DOI] [PubMed] [Google Scholar]
  • 47.Manimala JC, Roach TA, Li Z, Gildersleeve JC. High-throughput carbohydrate microarray profiling of 27 antibodies demonstrates widespread specificity problems. Glycobiology 2007;17:17C–23C. [DOI] [PubMed] [Google Scholar]
  • 48.Ragupathi G, Liu NX, Musselli C, Powell S, Lloyd K, Livingston PO. Antibodies against tumor cell glycolipids and proteins, but not mucins, mediate complement-dependent cytotoxicity. J Immunol 2005;174:5706–12. [DOI] [PubMed] [Google Scholar]
  • 49.Reis ES, Mastellos DC, Ricklin D, Mantovani A, Lambris JD. Complement in cancer: untangling an intricate relationship. Nat Rev Immunol 2018;18:5–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Geijtenbeek TB, Gringhuis SI. Signalling through C-type lectin receptors: shaping immune responses. Nat Rev Immunol 2009;9:465–79. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1507354-supplement-1.pdf (887.6KB, pdf)

RESOURCES