Invented by Thomas F. Tedder, Koichi Yanaba, Jean-David Bouaziz, Duke University
The Duke University invention works as followsThe present invention relates a phenotypically different CD1dhighCD5+ subset of B cells that regulates T-cell mediated inflammatory response through the secretion interleukin-10. The invention also relates the use of these IL-10-producing regulatory B cells to manipulate immune and inflammatory response, as well as in the treatment and prevention of disease. The invention describes therapeutic approaches that involve adoptive transfer of regulatory B cells or the expansion of endogenous levels of these cells to control autoimmune and inflammatory conditions. This subset of regulatory cells can be eliminated or their IL-10 produced inhibited to regulate immunodeficiency conditions and/or treat cancer. “Diagnostic applications are also included.
Background for The use of regulatory B cells
The immune response is divided into two parts: the humoral response, which includes antibody production, and cell-mediated immunity, which involves activation of macrophages and natural killer cells (NK), antigen-specific T-lymphocytes and release of cytokines. B lymphocytes are usually characterized by the role they play in antibody production, whereas T cells are characterized for their cell-mediated immune system. “B cells also have additional immune functions such as the production of cytokines and the ability for them to act as antigen-presenting cells.
Once immune responses are generated, they need to be regulated in order to prevent responding effectors from causing harm. Immunoregulation was traditionally thought to be a T cell function. Subsets of regulatory T cells that are functionally distinct have been clearly defined. Helper T cells can up-regulate immune responses in a variety of ways. For instance, T helper types 1 (Th1) regulates cell-mediated immunity, while T helper types 2 (Th2) regulates humoral immunity. T regulatory cells are suppressor T cells that are crucial to the maintenance of immune tolerance. They include T regulatory 1 cells (Tr1), which produce IL-10, and T helper 3 (Th3), which produces TGF-?1. Recent studies on autoimmune diseases led to the idea that B cells could also be involved in immunoregulation. But regulatory B cell subsets are not well defined.
Multiple roles for B cells have been implicated in autoimmune diseases. B cells, a major immune cell population, can play a pathogenic role in acquired immune responses by producing autoantibodies that drive the development of autoimmune diseases. Certain therapies developed for treating autoimmunity are aimed at depleting pathogenic B cells. However, the tools currently available are not specific for the pathogenic B cells and deplete most B cells. For example, B cell depletion in humans using CD20 monoclonal antibody (mAb) can be effective in treating patients with various autoimmune disorders, such as rheumatoid arthritis and lupus (Edwards et al., 2001, Rheumatol. 40:205-11; Edwards et al., 2005, Rheumatol. 44:151-56; El Tal et al., 2006, J. Am. Acad. Dermatol. 55:449-59; Anolik et al., 2004, Arth. Rheum. 50:3580-90; Stasi et al., 2007, Blood 110:2924-30). CD20 is a B cell-specific marker that is first expressed on the cell surface during the pre-B to immature B cell transition, but is lost upon plasma cell differentiation (Tedder & Engel, 1994, Immunol. Today 15:450-54; Uchida et al., 2004, Int. Immunol. 16:119-29). A recent phase II trial using anti-CD20 antibodies indicates clinical efficacy in multiple sclerosis (MS) patients (Hauser et al., 2008, N. Engl. J. Med. 358:676-88). However, the mechanisms underlying the effect of B cell depletion on disease activity remains unknown. On the flip side, B cell depletion may exacerbate disease. Indeed, B cell depletion was recently found to exacerbate ulcerative colitis in human clinical trials (Goetz et al., 2007, Inflamm Bowel Dis. 13:1365-8) and may contribute to the development of psoriasis (Dass et al., 2007, Arthritis Rheum. 56:2715-8).
More than a decade ago Janeway and her colleagues (Wolf et. al., 1996 J. Exp. Med. Med. EAE is a central nervous system (CNS), autoimmune disease that mimics human multiple sclerosis. The results showed that eliminating B cells didn’t prevent autoimmunity. The absence of B cells appeared to worsen the disease, as the mice with B cell deficiencies did not recover fully compared to mice with wild-type B cells. These investigators concluded that, although B cells produce autoantibodies that are thought to cause disease, their presence was required to improve recovery. Recently, it has been reported that the production of IL-10 by B cells correlates with recovery from EAE (a Th1-mediated autoimmune disorder) (Fillatreau et. al., Nature Immunol, 2002). 3: 944-950). IL-10 is a cytokine that regulates immune responses. IL-10 suppresses cell-mediated immune responses and inflammation.
Other recent studies in mice models indicate that B-cells and IL-10 have a protective effect in T-cell-mediated inflammation. For example, in Th2-mediated inflammatory bowel diseases (Mizoguchi et. al. 2002, Immunity 16, 216-219) and contact hypersensitivity (CHS), a cutaneous inflammatory immunological reaction that is mediated primarily by T cells after subsequent contact with sensitized antigens (Enk 1997, Mol. Med. Today 3:423-8). Specifically, mice lacking CD19 expression on B cells (CD19 ?/?) CHS responses are enhanced in mice with B cells deficient for CD19 expression (CD19?/?) J. Pathol. 171:560-70). IL-10 is involved in the protection, since neutralizing IL-10 by mAb treatment increases CHS responses while systemic IL-10 therapy reduces CHS reactions (Ferguson et. al., J. Exp. Med. 179:1597-1604; Schwarz et al., 1994, J. Invest. Dermatol. 103:211-16).
The results of these studies and others have led to the hypothesis that B cells, like T-cells, can be divided into subsets that are functionally distinct and capable of inhibiting inflammatory reactions and inducing immunity tolerance through mechanisms such as IL-10 and TGF? Production, secondary antigen presentations, and interactions with immune cells directly or via secreted antibody. For reviews of the topic, see Mauri & Ehrenstein 2007, TRENDS In Immunol. (For reviews on the subject, see Mauri & Ehrenstein, 2007, TRENDS in Immunol. 176:705-710).
However it is unclear whether regulatory B-cells represent a unique lineage of regulatory cells that are tasked with maintaining tolerance in the same way as naturally occurring regulatory T-cells.” In multiple mouse models of chronic inflammatory disease, the generation of regulatory B-cells has been observed, but their presence in normal mice is unknown (Mizoguchi & Bhan 2006, J. Immunol. 176:705-10). Although a murine regulatory B cell subset was identified in these mouse models (Mauri and Ehrenstein, 2007, The Trends in Immunity 29:34-40), no definitive murine B cell phenotype could be established. In fact, the only list of possible cell-surface markers that are associated with regulatory B cells is a general one. The existence of regulatory cells in humans is still a subject of speculation. Potential phenotypic indicators for human regulatory cells are also unknown (Mauri and Ehrenstein, 2007 Trends Immun 29.34-40). Inoue et. al. (2006) Cancer Res. 66:7741-7747). Nevertheless, it is not yet known whether CD40 can directly be targeted with anti-CD40 antibody as a way to generate regulatory B cell in vivo. (Mauri & Ehrenstein 2007, Trends Immun, 29:34-40).
Asadullah et. al. 2003, Pharmacol. Rev. 55:241-69). It is not known whether the regulatory B cell population in humans exists, whether it can be expanded or activated, or whether it has potent activity in vivo. Subsets of immune-regulatory B cell subsets need to better defined, and their phenotype needs to be closely correlated with the function in vivo.
The present invention relates a phenotypically different CD1dhighCD5+ subset of B cells that regulates immune and inflammatory responses mediated by T cells through the secretion IL-10. The invention also involves harnessing this regulatory subset of B cells for manipulating immune and inflammatory reactions in humans and other mammal. “Treatments for diseases with decreased IL-10, such autoimmune and inflammatory diseases, are described. As well as treatments for disease with elevated IL-10, such immunosuppression or cancer.
The invention describes “Cellular compositions that are enriched with the CD1dhighCD5+ subset of B cells, as well as methods for their preparation.” The invention is based on the discovery that a cellular population that has been enriched with both CD1dhigh (as a cellular marker) and CD5 (as a cellular marker) will have a higher percentage IL-10-producing B cells than a cellular population enriched only using one of these markers. By adoptive transfer, these cellular compositions may be used to treat autoimmune and/or inflammatory conditions. As an alternative, therapeutic regimens that are designed to increase the endogenous CD1dhighCD5+ subset of B cells in patients who need such treatment can also be used to treat autoimmune and/or inflammatory conditions or diseases. This approach can use antibodies that stimulate or activate the expansion of regulatory B cells, or increase IL-10 production.
In an alternative embodiment, methods for treating conditions or diseases involving immunosuppression and/or cancer are described by removing or ablating CD1dhighCD5+ subsets of regulatory B cells in the subjects who need them. This approach can use antibodies that either kill or inhibit the regulatory B-cell subset or their proliferation or production of IL-10.
In yet another embodiment, methods are described to identify the regulatory B-cell subset within patients or patient samples for the purpose of diagnosing the immunity status of the affected individual.
The invention is based in part on the identification of an uncommon regulatory B cell subset which controls T cell-mediated immunity and inflammation in vivo. In the examples below, the principles of the invention were demonstrated in animal models. They resolve contradictions previously reported in literature regarding the role of B-cells in diseases such as EAE and arthritis. “The examples infra show:
The invention encompasses a human regulatory B-cell subset that is phenotypically distinct from other B cell populations. This regulatory B-cell subset is phenotypically different from other B-cell populations. It can be identified using transcription factors that display the same cell surface marker, i.e. CD1dhighCD5+.
FIG. “FIG. mice. To identify B cells, Splenocytes are cultured for five hours with LPS and PMA. Ionomycin and monensin are added. After permeabilization the cells were stained by anti-IL10 mAb, and flow cytometry was used to assess them. The histograms represent results from single mice, while the bar graph represents results for all samples. The values represent the percentages of IL-10 producing cells in total non-B-cells. “All data represent 3 independent experiments, with 3 mice per group.Click here to view the patent on Google Patents.