Microenvironment and Immunology Anti-CD20 Antibody Promotes Cancer Escape via Enrichment of Tumor-Evoked Regulatory B Cells Expressing Low Levels of CD20 and CD137L

The possible therapeutic benefits of B-cell depletion in combating tumoral immune escape have been debated. In support of this concept, metastasis of highly aggressive 4T1 breast cancer cells in mice can be abrogated by inactivationof tumor-evoked regulatoryB cells (tBreg). Here,we report the unexpectedfinding thatB-cell depletion by CD20 antibodywill greatly enhance cancer progression andmetastasis. Bothmurine and human tBregs express low levels of CD20 and, as such, anti-CD20 mostly enriches for these cells. In the 4T1 model of murine breast cancer, this effect of enriching for tBregs suggests that B-cell depletion by anti-CD20 may not be beneficial at all in some cancers. In contrast, we show that in vivo–targeted stimulation of B cells with CXCL13-coupled CpG oligonucleotides (CpG-ODN) canblock cancermetastasis by inhibitingCD20 tBregs.Mechanistic investigations suggested that CpG-ODN upregulates low surface levels of 4-1BBL on tBregs to elicit granzyme B–expressing cytolytic CD8þ T cells, offering some explanative power for the effect. These findings underscore the immunotherapeutic importance of tBreg inactivation as a strategy to enhance cancer therapy by targeting both the regulatory and activating arms of the immune system in vivo. Cancer Res; 73(7); 2127–38. 2013 AACR. Introduction Cancer metastasis involves an active hijacking of regulatory immune cells to suppress antitumor effector immune responses. As a result, the increase in myeloid and myeloid-derived suppressor cells (MSC and MDSC) and regulatory T cells (Treg) is often a signof poordisease outcome inbothmiceandhumans with cancer (1–3). However, the role of B cells, in particular regulatory B cells (Breg), in this process remains poorly understood and is still debatable, although the existence of suppressive B cells has been known for more than 30 years. Protection from autoimmune diseases in mice and humans is shown to be mediated by unique subsets of Bregs [the definition first used by Mizoguchi and colleagues (4)], such as murine interleukin (IL)-10–producing B10 and B1b Bregs (4, 5); human IL-10– producing memory CD24HighCD27þ B cells (6), CD25 CD27CD86CD1d B cells (7), and CD19þCD24High CD38 B cells (8). As such, the inactivation of B cells or Bregs exacerbates ulcerative colitis in patients with non-Hodgkin's lymphoma, colitis, and Graves disease and increases the incidence of psoriasis in patients with psoriatic arthropathy (9–11). B cells also facilitate carcinogenesis of methylcholanthreneinduced (12, 13) and transplanted tumors (14). In humans with metastatic ovarian carcinoma, infiltration of CD19þ B cells was associated withworse disease outcome (15). Unless replenished with B220þ B cells, orthotopic tumors progress poorly in syngeneic mice deficient in B cells (16, 17). B cells promote a tumorsuppressive milieu through the production of immunoglobulin or/and immunomodulatory factors and cytokines or inducing the generation of Tregs (18). For example, B-cell–expressed lymphotoxin-a/b was linked with the androgen-independent growth of prostate cancer cells (19); and B-cell–expressed immunoglobulinwas linkedwith inflammation inpremalignant tissues and growth of HPV16-induced tumors (20). B cells can alsomediate T helper 2 cell (TH2) polarization and inhibition of antitumor cytotoxic activity of CD8þ T and natural killer (NK) cells by producing IL-10 (21) and TNF-a (22). To the best of our knowledge, only 2 clearly defined examples of cancer escape-promoting Bregs are reported. First, B10 cells reduce the therapeutic efficacy of anti-CD20 antibody against lymphoma by inhibiting monocyte activity and surface expression of FcgR in an IL-10–dependent fashion (23). Second, we recently found that 4T1 carcinoma cells actively convert normal B cells into TGF-b–producing Bregs, designated tumor-evoked Bregs (tBregs) to successfully metastasize (17). tBregs differ from the immune tolerance-inducing IL10–producing Bregs and B cells (24–26). Phenotypically, they are poorly proliferative B2-like cells (IgD) that express constitutively active Stat3 and surface markers CD25B7Authors' Affiliations: Immunotherapeutic Section; Clinical Research Branch; and Flow Cytometry Unit, National Institute on Aging, Baltimore; Translational Surgical Pathology; Experimental Transplantation and Immunology Branch, National Cancer Institute, Bethesda, Maryland; and Department of Immunology, Genentech, Inc., South San Francisco, California Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Arya Biragyn, National Institute on Aging, 251 BayviewBlvd, Suite 100, Baltimore,MD21224. Phone: 410-558-8680; Fax: 410-558-8284; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-4184 2013 American Association for Cancer Research. Cancer Research www.aacrjournals.org 2127 on April 12, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 30, 2013; DOI: 10.1158/0008-5472.CAN-12-4184