It is often assumed that host immunity assembles its components to curb disease; however, the functional relationship between cellular immune system and tumor cells is far from clear. In mice, B-1 lymphocytes arise very early during the ontogeny of the immune system compared to the more evolved layers. Such cell lineage share several features with primitive B lymphocytes such as neonatal development, limited antibody repertoire, self-renewal capacity and regulatory feedback in adults. B-1 lymphocytes can be readily identified by their cell surface phenotype (IgMhigh
) and can be further subdivided in B-1a (CD5+
) and B-1b (CD5-
) subsets (9
We evaluated whether B-1 lymphocytes influence tumor progression in the well-established B16 melanoma mouse model. We found that B-1 lymphocyte depletion markedly decreases lung metastasis from melanoma cells. B-1 lymphocyte reconstitution specifically inhibits this phenomenon and restores metastasis to baseline levels. These results show that B-1 lymphocytes have a key role in melanoma growth and metastasis.
Given that B-1 lymphocytes are known to produce and release high amounts of cytokines, we originally designed experiments to evaluate whether a soluble factor such as, for instance, interleukin-10 (36
) might account for the observed metastasis enhancement. Unexpectedly, we found instead that a direct physical interaction between B-1 lymphocytes and melanoma cells, is needed for metastasis. These findings led to the hypothesis that cell surface molecules are likely to mediate the phenomenon.
To gain insight into the molecular mechanism of this cell-cell interaction and the resulting biological effects, we attempted to identify the functionally active molecules expressed at the surface of each cell type. To that end, we selected a combinatorial peptide library on melanoma cells after co-culture with B-1 lymphocytes. By comparing sequences of selected peptides with those available in protein databases, we found that ligand motifs were similar to the cell surface glycoprotein MUC18. Because MUC18 is an adhesion molecule that correlates with tumor growth and metastasis (32
), we next evaluated whether a MUC18-dependent cell interaction would influence melanoma metastasis. First, we established that MUC18 serves as the partner on the surface of B16 melanoma cells (“receptor”). Because MUC18-like peptides also bind to MUC18 itself, we reasoned that MUC18 could also serve as the partner on the surface of B-1 lymphocytes (“ligand”). If so, one would predict that the ligand-receptor system affecting B16 melanoma cells through B-1 lymphocytes is actually a MUC18-MUC18 interaction. Our data show that this is indeed the case because (i) MUC18-like peptides enhance phage binding to melanoma cells post co-culture, (ii) antibodies against MUC18 specifically inhibit phage targeting and (iii) MUC18 is also abundant on the surface of B-1 lymphocytes. Together, our findings indicate that an unrecognized biochemical interaction between MUC18 expressed on the surface of B16 melanoma cells and B-1 lymphocytes regulates metastatic potential. While the identification of a MUC18-like motif “in reverse” may originally have suggested an anti-parallel MUC18-MUC18 interaction, in vitro and in vivo experiments performed with a phage clone designed to display the corresponding native MUC18 sequence indicates that this particular molecular mimicry is not affected by peptide orientation. However, the same may not necessarily be true for the native MUC18 protein itself (due to potential steric hindrance). Therefore, whether this protein-protein interaction is influenced by orientation of the native protein expressed on the surface of melanoma and B-1 cells remains uncertain; a full understanding of the structural requirements for protein-protein or protein-peptide will likely have to wait for the elucidation of the X-ray crystal structures of MUC18-MUC18 and of MUC18-CLFMRLAWC complexes
To evaluate whether these observations are relevant in human disease, we investigated the distribution of B-1 lymphocytes in patients with melanoma. While the origin and characteristics of the “human B-1 lymphocyte” counterpart are still poorly defined, a considerable proportion of IgM+
B cells in the human peritoneal cavity are CD5+
, a phenotypic hallmark of mouse B-1 lymphocytes (48). Mice and human B-1 lymphocytes are both largely responsible for the production of auto-reactive IgM antibodies in patients with certain autoimmune diseases (34
We detected IgM+
B cells, presumably human B-1 lymphocytes, in 100% of the analyzed samples from a cohort of patients with metastatic melanoma (n=16) suggesting that B-1 lymphocytes likely play a functional role in human cancer. Importantly, the presence of B-1 lymphocytes within tumors directly correlates with increased expression of MUC18 in melanoma cells (38
), again supporting the relevance of a functional and active MUC18-dependent cross-talk between human B-1 lymphocytes and melanoma.
Notably, disruption of MUC18-dependent cell interactions may be of therapeutic value. In fact, (i) anti-MUC18 antibodies have shown promise in pre-clinical models (37
) and (ii) overexpression of MUC18 occurs in human melanoma among other tumor types (40
). One might speculate that if a MUC18-dependent interaction accounts for malignant melanoma homing to lung vasculature for instance, then anti-MUC18 antibodies and/or MUC18-based peptidomimetics might simultaneously block the functional protein-protein interaction from both partners. Considering that re-attachment of circulating tumor cells is a rate-limiting step in metastasis, our observations might help explain the difference of magnitude of effect we observed between the (mild-to-moderate) B16 tumor growth suppression relative to lung (marked) metastasis inhibition in B-1 lymphocyte-deficient mice.
In summary, we show that human and mouse melanoma cells can subvert a putatively defensive function of the immune system through a heterotypic cell-cell interaction with primitive B cells, and that this molecular crosstalk can influence tumor progression with adverse net effects to the host. New mechanism-based strategies such as physical elimination or functional modulation of human B-1 lymphocytes (through radiation or specific antibodies) may be considered as an experimental therapy against human melanomas; also, blocking MUC18 with antibodies or targeting MUC18 for ligand-directed delivery of agents in patients with melanoma may have translational potential.