Vincent Hurez

Specific Btk inhibition suppresses B cell– and myeloid cell–mediated arthritis

Brutons tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

V Region-Mediated Selection of Autoreactive Repertoires by Intravenous Immunoglobulin (i.v.Ig)

In addition to its use as substitute therapy for primary and secondary immunodeficiencies, intravenous immunoglobulin (i.v.Ig) is increasingly being used as an immunomodulating therapy in the treatment of patients with a variety of autoimmune and systemic inflammatory disorders (Schwartz 1990, Dietrich et al. 1992b, Dwyer 1992, Ronda et al. 1993). The use of i.v.Ig in these situations is supported by a few randomized clinical trials and a large number of uncontrolled and smaller studies. Of relevance to this chapter are that the reported beneficial effects of i.v.Ig include those in autoantibody-mediated autoimmune diseases (e.g., autoimmune cytopenias, anti-factor VIII autoimmune disease) as well as diseases in which autoaggressive T cells are primarily involved in the pathogenesis (e.g., autoimmune uveitis) (Imbach et al. 1981, Bussel et al. 1983, Sultan et al. 1984, McGuire et al. 1987, LeHoang et al. 1994). Where it is a feature of the disease, successful outcome of i.v.Ig therapy is associated with an improvement in the patients systemic inflammatory condition. Modulation of B-cell and Tcell functions and of cytokine production has further been observed in animal models of autoimmune diseases following administration of human i.v.Ig or of normal homologous IgG (Forsgren et al. 1991, Rossi et al. 1991a, Saoudi et al. 1993, Hentati et al. 1994). The design of trials to establish the efficacy and appropriate therapeutic sched-

Intravenous immunoglobulins (IVIg) in the treatment of autoimmune diseases.

Intravenous immunoglobulin (IVIg) therapy is increasingly used in autoimmune diseases. Although its efficacy has only been established in a few specific antibody‐mediated autoimmune conditions, accumulating evidence on the regulatory role of circulating immunoglobulins in the selection of peripheral B cell repertoires makes it an attractive potential therapeutic option to clinical immunologists. This overview briefiy discusses the current use of IVIg in human autoimmune diseases with a particular emphasis on the possible mechanisms by which IVIg could suppress pathological autoimmune responses.

Notch2 Haploinsufficiency Results in Diminished B1 B Cells and a Severe Reduction in Marginal Zone B Cells

Recent studies have implicated a role for Notch in the generation of marginal zone (MZ) B cells. To further investigate the role of Notch in the B cell lineage, we have analyzed the effects of reduced Notch2 signaling in mice expressing one functional allele of Notch2 (Notch2+/−). Notch2+/− mice have reduced B1 B cells of the peritoneal cavity and show a severe reduction in MZ B cells of the spleen. The reduction in MZ B cells was not due to the disruption of splenic architecture, disregulated terminal differentiation, nor to increased apoptosis within the MZ B cell compartment. Rather, our data suggest that Notch2 haploinsufficiency leads to impaired development of MZ B cells, possibly by impacting the formation of immediate MZ B precursors. These results provide evidence that Notch2 plays a determining role in the development and/or the maintenance of B1 B and MZ B cells.

Intravenous Immunoglobulin Therapy of Autoimmune and Systemic Inflammatory Diseases

The proven beneficial effect of intravenous immunoglobulins (IVIg) in certain autoimmune disorders has led to the development of clinical trials in other autoimmune and systemic inflammatory diseases. In parallel, experimental studies are being carried out to better understand the mechanisms of action of IVIg. In this review, we discuss the clinical use of IVIg in autoimmune disorders and the possible mechanisms by which IVIg may be effective in the various diseases. A better understanding of the mechanisms of action of IVIg in autoimmune disease will allow optimization of their use as a therapeutic alternative to conventional immunosuppression.

Activated Notch2 Potentiates CD8 Lineage Maturation and Promotes the Selective Development of B1 B Cells

ABSTRACT Although studies have shown that the Notch2 family member is critical for embryonic development, little is known concerning its role in hematopoiesis. In this study, we show that the effects of an activated form of Notch2 (N2IC) on the T-cell lineage are dosage related. High-level expression of N2IC results in the development of T-cell leukemias. In contrast, lower-level expression of N2IC does not lead to transformation but skews thymocyte development to the CD8 lineage. Underlying this skew is a dramatic enhancement in positive selection and CD8SP maturation. N2IC permits early B-cell development but blocks the maturation of conventional B2 cells at the pre-B stage, which is the limit of endogenous Notch2 protein expression in developing B cells. Most strikingly, while B2 B cell development is blocked at the pre-B-cell stage, N2IC promotes the selective development of LPS-responsive B1 B cells. This study implicates a role for Notch2 in the maturation of the CD8 lineage and suggests a novel function for Notch2 in the development of the B1 B-cell subset.

B7-H1-dependent sex-related differences in tumor immunity and immunotherapy responses

CD4+CD25+Foxp3+ regulatory T cells (Tregs) are immunopathogenic in cancers by impeding tumor-specific immunity. B7-homologue 1 (B7-H1) (CD274) is a cosignaling molecule with pleiotropic effects, including hindering antitumor immunity. In this study, we demonstrate sex-dependent, B7-H1–dependent differences in tumor immunity and response to immunotherapy in a hormone-independent cancer, murine B16 melanoma. Antitumor immunity was better in B7-H1−/− females versus males as a result of reduced regulatory T cell function in the B7-H1−/− females, and clinical response following B7-H1 blockade as tumor immunotherapy was significantly better in wild-type females than in males, owing to greater B7-H1 blockade-mediated reduction of Treg function in females. Wild-type female Tregs expressed significantly lower B7-H1 versus males but were insensitive to estrogen in vitro. Female B7-H1−/− Tregs were exquisitely sensitive to estrogen-mediated functional reduction in vitro, suggesting that B7-H1 effects occur before terminal Treg differentiation. Immune differences were independent of known B7-H1 ligands. Sex-dependent immune differences are seldom considered in designing immune therapy or interpreting immunotherapy treatment results. Our data demonstrate that sex is an important variable in tumor immunopathogenesis and immunotherapy responses through differential Treg function and B7-H1 signaling.

Restricted Clonal Expression of IL-2 By Naive T Cells Reflects Differential Dynamic Interactions with Dendritic Cells

Limited frequencies of T cells express IL-2 in primary antigenic responses, despite activation marker expression and proliferation by most clonal members. To define the basis for restricted IL-2 expression, a videomicroscopic system and IL-2 reporter transgenic model were used to characterize dendritic cell (DC)–T cell interactions. T cells destined to produce IL-2 required prolonged interactions with DCs, whereas most T cells established only transient interactions with DCs and were activated, but did not express IL-2. Extended conjugation of T cells with DCs was not always sufficient to initiate IL-2 expression. Thus, there is intrinsic variability in clonal T cell populations that restricts IL-2 commitment, and prolonged engagement with mature DCs is necessary, but not sufficient, for IL-2 gene transcription.

Polyreactivity is a Property of Natural and Disease‐Associated Human Autoantibodies

Polyreactivity was earlier recognized as a feature of naturally expressed autoantibodies in serum. In the present study, we have compared the reactivity on a panel of self antigens of affinity‐purified anti‐DNA and anti‐thyroglobulin (TG) IgG autoantibodies from the serum of patients with systemic lupus erythematosus (SLE) and autoimmune thyroiditis with their affinity‐purified counterparts isolated from the serum of healthy individuals. Anti‐DNA autoantibodies exhibited a similar degree of polyreactivity whether originating from patients or from healthy adults. Natural anti‐TG autoantibodies were also found to be polyreactive. Anti‐TG autoantibodies from patients with Hashimotos thyroiditis showed little or no polyreactivity. Natural anti‐TG autoantibodies were equally polyreactive whether or not they belonged to a fraction of normal IgG that is connected through V regions with other IgG molecules from the same source. These results indicate that polyreactivity of autoantibodies is a feature that does not allow one to distinguish between natural and disease‐associated autoantibodies as well as between V‐region‐connected and unconnected autoantibodies.

Mitigating Age-Related Immune Dysfunction Heightens the Efficacy of Tumor Immunotherapy in Aged Mice

Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4(+)CD25(hi) regulatory T cells (Treg) exhibited equivalent in vitro T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti-Gr-1 antibody was immunologically and clinically more efficacious than anti-Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti-Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved.