Catalina Lee-Chang

Inhibition of Breast Cancer Metastasis by Resveratrol-Mediated Inactivation of Tumor-Evoked Regulatory B Cells

We reported previously that tumor-evoked regulatory B cells (tBregs) play an essential role in breast cancer lung metastasis by inducing TGF-β–dependent conversion of metastasis-promoting Foxp3+ regulatory T cells (Tregs). In this article, we show that resveratrol (RSV), a plant-derived polyphenol, at low and noncytotoxic doses for immune cells, can efficiently inhibit lung metastasis in mice. The mechanism of this process is that RSV inactivates Stat3, preventing the generation and function of tBregs, including expression of TGF-β. As a result, it frees antitumor effector immune responses by disabling tBreg-induced conversion of Foxp3+ Tregs. We propose that low doses of RSV may also benefit humans by controlling cancer escape–promoting tBregs/Tregs without nonspecific inactivation of effector immune cells.

Cancer-Produced Metabolites of 5-Lipoxygenase Induce Tumor-Evoked Regulatory B Cells via Peroxisome Proliferator–Activated Receptor α

Breast cancer cells facilitate distant metastasis through the induction of immunosuppressive regulatory B cells, designated tBregs. We report in this study that, to do this, breast cancer cells produce metabolites of the 5-lipoxygenase pathway such as leukotriene B4 to activate the peroxisome proliferator–activated receptor α (PPARα) in B cells. Inactivation of leukotriene B4 signaling or genetic deficiency of PPARα in B cells blocks the generation of tBregs and thereby abrogates lung metastasis in mice with established breast cancer. Thus, in addition to eliciting fatty acid oxidation and metabolic signals, PPARα initiates programs required for differentiation of tBregs. We propose that PPARα in B cells and/or tumor 5-lipoxygenase pathways represents new targets for pharmacological control of tBreg-mediated cancer escape.

Immunosuppressive and Prometastatic Functions of Myeloid-Derived Suppressive Cells Rely upon Education from Tumor-Associated B Cells

Myeloid-derived suppressive cells (MDSC) have been reported to promote metastasis, but the loss of cancer-induced B cells/B regulatory cells (tBreg) can block metastasis despite MDSC expansion in cancer. Here, using multiple murine tumor models and human MDSC, we show that MDSC populations that expand in cancer have only partially primed regulatory function and limited prometastatic activity unless they are fully educated by tBregs. Cancer-induced tBregs directly activate the regulatory function of both the monocyte and granulocyte subpopulations of MDSC, relying, in part, on TgfβR1/TgfβR2 signaling. MDSC fully educated in this manner exhibit an increased production of reactive oxygen species and NO and more efficiently suppress CD4(+) and CD8(+) T cells, thereby promoting tumor growth and metastasis. Thus, loss of tBregs or TgfβR deficiency in MDSC is sufficient to disable their suppressive function and to block metastasis. Overall, our data indicate that cancer-induced B cells/B regulatory cells are important regulators of the immunosuppressive and prometastatic functions of MDSC.

Inhibition of lung metastasis by chemokine CCL17-mediated in vivo silencing of genes in CCR4+ Tregs.

Despite significant attractiveness of antisense oligonucleotide/RNAi technology, its clinical application has been precluded by a lack of methods for targeted delivery and transduction of primary immune cells in vivo. Here, we devised a chemokine CCL17-based strategy (TARC-arp) that transiently silences expression of genes in immune cells by delivering inhibitory oligonucleotides through their chemokine receptors. In modeling studies using mice with established 4T1.2 breast cancer, we show that IL10 produced by CCR4+ cells, in particular FoxP3+ regulatory T cells (Tregs), plays an important role in lung metastasis. As such, TARC-arp-mediated silencing of IL10 or FoxP3 in CCR4+ Tregs is sufficient to block lung metastasis. Thus, we provide a simple solution that circumvents the problems of RNAi use in vivo, indicating that a disease outcome can be successfully controlled by delivering inhibitory oligonucleotides with chemokines to inactivate a selective subset of immune cells.

Accumulation of 4-1BBL+ B cells in the elderly induces the generation of granzyme-B+ CD8+ T cells with potential antitumor activity

Although the accumulation of highly-differentiated and granzyme B (GrB)-expressing CD8(+)CD28(-) T cells has been associated with aging, the mechanism for their enrichment and contribution to immune function remains poorly understood. Here we report a novel B-cell subset expressing 4-1BBL, which increases with age in humans, rhesus macaques, and mice, and with immune reconstitution after chemotherapy and autologous progenitor cell transplantation. These cells (termed 4BL cells) induce GrB(+)CD8(+) T cells by presenting endogenous antigens and using the 4-1BBL/4-1BB axis. We found that the 4BL cells increase antitumor responses in old mice, which may explain in part the paradox of retarded tumor growth in the elderly. 4BL cell accumulation and its capacity to evoke the generation of GrB(+)CD8(+) T cells can be eliminated by inducing reconstitution of B cells in old mice, suggesting that the age-associated skewed cellular immune responses are reversible. We propose that 4BL cells and the 4-1BBL signaling pathway are useful targets for improved effectiveness of natural antitumor defenses and therapeutic immune manipulations in the elderly.

A New Paradigm for an Old Story: The Role of Regulatory B Cells in Cancer

A common feature between cancer escape and autoimmune diseases is an inappropriate involvement of the regulatory immune system, albeit for opposing purposes. While autoimmune disease is a reflection of the failure to control responses to self, cancer is a result of an exaggerated use of these controls to abrogate antitumor effector responses. Although the importance of regulatory B cells [Bregs, the definition first used by Mizoguchi to describe B cells exerting protection from colitis in mice (Mizoguchi et al., 1997)] in protection from autoimmunity is now accepted, their involvement in cancer escape remains poorly understood. The conundrum of Bregs is that, if their numbers are low (in analogy with Tregs), their existence and importance may be concealed by the overwhelming response of effector B cells. For example, aberrant activation of B cells promotes autoimmune diseases, such as rheumatoid arthritis (RA), type 1 diabetes mellitus (T1D), multiple sclerosis (MS), and systemic lupus erythematosus (SLE). As such, the depletion of B cells with anti-CD20 antibody rituximab impairs antigen-specific CD4+ T cell activation (Bouaziz et al., 2007) and ameliorates RA, MS, and T1D (Townsend et al., 2010). Yet, treatment with rituximab can also exacerbate the disease in some patients with ulcerative colitis, or even induce other diseases, such as psoriasis with psoriatic arthropathy and colitis in patients with Graves disease and non-Hodgkin lymphoma, respectively (Dass et al., 2007; Goetz et al., 2007; Mielke et al., 2008). The increased numbers of B cells in peripheral blood of transplant patients is positively associated with a rare but long-term drug-free clinical tolerance (Newell et al., 2010; Pallier et al., 2010; Sagoo et al., 2010). Although these clinical examples clearly indicate the importance of B cells, a current issue is how to segregate the role of Bregs from suppressive activity of B cells that has been known for more than 30 years. As first proposed by Morris and Moller in late 1960s (Morris and Moller, 1968), B cell-produced immunoglobulin can elicit immune suppression by directly triggering ITIM-mediated suppressive signaling in target cells upon binding with inhibitory FcγRIIB (Ravetch and Bolland, 2001) or by indirectly modulating dendritic cells (DCs) via activating FcγR (Morris and Moller, 1968).

Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers

B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL+ MHC class-IHi CD86Hi B cells of unknown origin. In this article, we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result, activated B1a/4BL cells induce expression of granzyme B in CD8+ T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus, for the first time, to our knowledge, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8+ T cells.

Generation and identification of tumor-evoked regulatory B cells.

The involvement of Bregs in cancer remains poorly understood despite their well-documented regulation of responses to the self and protection from harmful autoimmunity. We recently discovered a unique regulatory B cell subset evoked by breast cancer to mediate protection of metastasizing cancer cells. These results together with the wealth of findings of the last 40 years on B cells in tumorigenesis suggest the existence of additional cancer Bregs modulating anticancer responses. To facilitate the search for them, here we provide our detailed protocol for the characterization and generation of tumor-evoked regulatory B cells. Wherever applicable, we also discuss nuances and uniqueness of a Breg study in cancer to warn potential pitfalls.

Tumor-Evoked Regulatory B Cells as Important Mediators of Cancer Escape

Our understanding of immune regulation was drastically changed by Sakaguchi’s seminal discovery of regulatory T cells (Tregs). To date, the cells that exert regulatory activity are found among almost every type of immune cells. Their primary function is to control immune homeostasis and prevent aberrant autoimmune responses, making them an attractive target of cancer. In fact, cancer actively hijacks Tregs and myeloid-derived suppressive cells (MDSCs) by corrupting them in order to abrogate antitumor effector responses and to promote a cancer-benefiting milieu. However, the role of regulatory B cells (Bregs) in this process is poorly understood, despite the existence of protective IL-10-producing B cells and Bregs in autoimmune diseases. Here in this review, we discuss the pros and cons and the latest evidence of the importance of Bregs in mediation of immunosuppression required in cancer escape and metastasis.

B Cells and Regulatory B Cells in Cancer Escape

In order to evade antitumor effector immune cells and metastasize, cancer often employs a diverse group of immune cells with suppressive and immunomodulatory functions. In this process, the role of regulatory B cells (Bregs) remains poorly understood despite their ability to modulate immune responses. The search for Bregs is complicated by the complexity of cancer, lack of unique markers, and functional overlap with B cells expressing suppressive factors. To date, cancer is shown to generate at least two types of Bregs, including tumor-evoked (t)Bregs and granzyme (GrB) + Bregs, although it may also use other Bregs that protect from autoimmunity.