Bram, Richard J.

Abstract Immune response to T cell independent type 2 (TI-2) Ags, such as bacterial polysaccharides, is severely impaired in X-linked immunodeficient (XID) mice. In this study, we investigated the involvement of a proliferation-inducing ligand (APRIL) or BAFF and their receptors in the unresponsiveness of XID mouse to TI-2 Ags. We discovered that whereas serum BAFF levels were increased, the expression of the APRIL and BAFF receptor transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) was severely reduced in XID B cells. Moreover, B cells from XID mouse were unable to secrete Igs in response to APRIL or BAFF. In correlation with reduced TACI expression and impaired TACI function, APRIL or BAFF did not activate the classical NF-κB pathway in XID cells. Also correlating with the unaltered expression of BAFF receptor, BAFF stimulation induced the activation of the alternative NF-κB pathway in XID cells. Moreover, activation of MAPK pathway was ablated in APRIL-stimulated XID cells. Prestimulation of XID B cells with the TLR9 agonist, CpG led to a significant increase in TACI expression and restored TACI-mediated functions. CpG prestimulation also restored TACI-mediated signaling in APRIL- or BAFF-stimulated XID B cells. Finally, immunization of XID mouse with the prototype TI-2 Ag NP-Ficoll induced IgG and IgM Abs when CpG was given with NP-Ficoll. Collectively, these results suggest that reduced TACI expression is responsible for the unresponsiveness of XID mouse to TI-2 Ags and BCR activation controls TACI expression.

Abstract Glioblastoma multiforme is an aggressive, treatment-refractory type of brain tumor for which effective therapeutic targets remain important to identify. Here, we report that cyclophilin B (CypB), a prolyl isomerase residing in the endoplasmic reticulum (ER), provides an essential survival signal in glioblastoma multiforme cells. Analysis of gene expression databases revealed that CypB is upregulated in many cases of malignant glioma. We found that suppression of CypB reduced cell proliferation and survival in human glioblastoma multiforme cells in vitro and in vivo. We also found that treatment with small molecule inhibitors of cyclophilins, including the approved drug cyclosporine, greatly reduced the viability of glioblastoma multiforme cells. Mechanistically, depletion or pharmacologic inhibition of CypB caused hyperactivation of the oncogenic RAS–mitogen-activated protein kinase pathway, induction of cellular senescence signals, and death resulting from loss of MYC, mutant p53, Chk1, and Janus-activated kinase/STAT3 signaling. Elevated reactive oxygen species, ER expansion, and abnormal unfolded protein responses in CypB-depleted glioblastoma multiforme cells indicated that CypB alleviates oxidative and ER stresses and coordinates stress adaptation responses. Enhanced cell survival and sustained expression of multiple oncogenic proteins downstream of CypB may thus contribute to the poor outcome of glioblastoma multiforme tumors. Our findings link chaperone-mediated protein folding in the ER to mechanisms underlying oncogenic transformation, and they make CypB an attractive and immediately targetable molecule for glioblastoma multiforme therapy. Cancer Res; 74(2); 484–96. ©2013 AACR.

ABSTRACT T cell-independent antibody responses develop rapidly, within 3 to 4 days, and are critical for preventing blood-borne pathogens from evolving into life-threatening infections. The interaction of BAFF, also known as BLyS, with its receptors BAFFR and TACI on B cells is critical for B cell homeostasis and function. Using a synthetic polysaccharide antigen, it has previously been shown that TACI is critical for T cell-independent antibody responses. To examine the role of BAFFR and TACI in T cell-independent antibody responses to an active infection, we utilized the Borrelia hermsii infection system. In this infection system, T cell-independent responses mediated by the B1b cell subset are critical for controlling bacteremia. We found that B1b cells express BAFFR and TACI and that the surface expression of both receptors is upregulated on B1b cells following exposure to whole B. hermsii cells. Surprisingly, we found that TACI −/− mice are not impaired either in specific antibody responses to B. hermsii or in controlling B. hermsii bacteremia. In contrast, TACI-deficient mice immunized with heat-killed type 3 serotype pneumococcus cells are impaired in generating pneumococcal polysaccharide-specific responses and succumb to challenge with live type 3 serotype pneumococcus, indicating that TACI is required for T cell-independent antibody responses to bacterial-associated polysaccharides. Although we have found that TACI is dispensable for controlling B. hermsii infection, mice deficient in BAFFR or BAFF exhibit impairment in B. hermsii -specific IgM responses and clearance of bacteremia. Collectively, these data indicate a disparity in the roles for TACI and BAFFR in primary T cell-independent antibody responses to bacterial pathogens.

The di‐2‐pyridylketone thiosemicarbazone Dp44mT is a metal‐chelating compound that has been demonstrated to have potent activity as an anticancer agent. Here we report that it also has a dramatic inhibitory effect on T‐cell activation in vitro. We found that 10 nM Dp44mT (IC 50 3.2 nM) prevented the up‐regulation of surface CD25, and completely suppressed the activation and proliferation of splenic T cells isolated from Mus musculus that were stimulated with either T‐cell receptor (TCR) cross‐linking antibodies or phorbol ester plus ionomycin. In contrast, Dp44mT had no adverse effects on the survival of resting T cells. In addition, T cells stimulated in the presence of Dp44mT maintained the ability to upregulate CD69 surface expression and secrete interleu‐kin‐2. Consistent with these observations, Dp44mT did not inhibit multiple canonical signals downstream of the TCR, including the nuclear factor of activated T cells. The effects of Dp44mT were easily mitigated by addition of nontoxic copper chelators or N ‐acetylcysteine, indicating a role for copper and reactive oxygen species in its actions. Together, these findings suggest that Dp44mT may serve as a potent immunosuppressive agent that could complicate its use as a cancer therapeutic agent, but might have utility in the treatment of autoimmunity.—Gundelach, J. H., Madhavan, A. A., Wettstein, P. J., Bram, R. J. The anticancer drug Dp44mT inhibits T‐cell activation and CD25 through a copper‐dependent mechanism. FASEB J. 27, 782–792 (2013). www.fasebj.org

Abstract Notch is activated by somatic mutation in more than 50% of cases of T-cell acute lymphoblastic lymphoma/leukemia (T-ALL). Exuberant notch signaling drives several oncogenic phenotypes, including enhanced proliferation, improved survival, and impaired differentiation. Inhibition of Notch is likely to yield improved treatment strategies for this disease and certain other malignancies, as well. However, because global inhibition of Notch may have intolerable toxicities, efforts are underway to identify the key downstream mediators of its neoplastic effects. We find that the clathrin-mediated receptor uptake adapter Hrb (HIV-rev binding protein) is a downstream target of Notch1. Hrb protein was induced to high levels by retroviral-mediated expression of constitutively active Notch (ICN1) in bone marrow stem cells and T cell precursors in vitro. To explore a potential role for Hrb downstream of Notch, we have examined the effects of ICN1 on bone marrow progenitor cells (BMPCs) from Hrb-deficient mice. Wild type BMPCs transduced with ICN1 grew to higher density in culture than did Hrbdeficient cells. There was no difference in distribution in cell cycle phases, and proliferative rates appeared to be the same. However, we found a significant increase in apoptosis in Hrb-deficient BMPCs in comparison to cells from littermate control mice. This increase in apoptosis could be reversed by reexpression of Hrb in the mutant cells, thus verifying that Hrb provides an enhanced survival signal to ICN1-expressing bone marrow cells. Potential downstream mediators of this survival signaling cascade are currently being identified. Because resistance to apoptosis is a key feature of leukemic cells, we hypothesize that the pro-survival effect of Hrb provides a portion of the oncogenic properties of Notch. Thus, because Hrb-knockout mice have no evident phenotype, other than male sterility, Hrb may be a useful molecular target for therapy of Notch-dependent malignancies, such as T-ALL. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B23.

Abstract Calcium-modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein that is important during thymopoiesis. However, whether it serves a function in mature lymphocytes is unknown. In this article, we show that CAML is essential for survival of peripheral follicular (Fo) B cells. Conditional deletion of CAML in CD19-Cre transgenic mice caused a significant reduction in Fo cell numbers and increased rates of homeostatic proliferation. CAML-deficient Fo cells showed increased cellular turnover and normal proliferative ability. Although CAML-deficient Fo cells responded to AgR stimulation and to B cell activating factor, they displayed decreased survival and increased apoptosis following stimulation with LPS and IL-4 in vitro. Failure to survive was not due to aberrant B cell development in the absence of CAML, because induced deletion of the gene in mature cells resulted in a similar phenotype. These data establish an essential and ongoing role for CAML in the long-term survival of mature B cells.

Abstract Mechanistic understanding of RP105 has been confounded by the fact that this TLR homolog has appeared to have opposing, cell type-specific effects on TLR4 signaling. Although RP105 inhibits TLR4-driven signaling in cell lines and myeloid cells, impaired LPS-driven proliferation by B cells from RP105−/− mice has suggested that RP105 facilitates TLR4 signaling in B cells. In this article, we show that modulation of B cell proliferation by RP105 is not a function of B cell-intrinsic expression of RP105, and identify a mechanistic role for dysregulated BAFF expression in the proliferative abnormalities of B cells from RP105−/− mice: serum BAFF levels are elevated in RP105−/− mice, and partial BAFF neutralization rescues aberrant B cell proliferative responses in such mice. These data indicate that RP105 does not have dichotomous effects on TLR4 signaling and emphasize the need for caution in interpreting the results of global genetic deletion.

Abstract Deficiencies in transmembrane activator and CAML interactor (TACI) result in common variable immune deficiency, a syndrome marked by recurrent infections with encapsulated microorganisms, impaired production of antibodies, and lymphoproliferation. How TACI promotes antibody production and inhibits lymphoproliferation is not understood. To answer this question, we studied the generation of immunity to protein antigens in both TACI-deficient and TACI-proficient mice. We show that TACI promotes sustained Blimp-1 expression by B cells responding to antigen, which in turn limits B-cell clonal expansion and facilitates differentiation of long-lived antibody-secreting cells. Short-term IgG secretion occurs independently of TACI as DNA double-strand breaks associated with isotype class switching induce Blimp-1 transiently, independently of TACI. Our results showing that TACI induces and maintains Blimp-1 provide, for the first time, a unified molecular and cellular mechanism explaining the primary features of common variable immune deficiency, exquisite vulnerability to infection with encapsulated organisms, lymphoproliferation, and hypogammaglobulinemia.

AbstractTNFRSF13B, which encodes TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), is mutated in 10% of patients with common variable immune deficiency (CVID). One of the 2 most common TACI mutations in CVID, A181E, introduces a negative charge into the transmembrane domain. To define the consequence of the A181E mutation on TACI function, we studied the effect of its murine equivalent, mTACI A144E, on TACI signaling in transfected cells and on TACI function in transgenic mice. The mTACI A144E mutant, like its human TACI A181E counterpart, was expressed on the surface of 293T transfectants and was able to bind ligand, but exhibited impaired constitutive and ligand-induced NFκB signaling. In addition, constitutive and ligand-induced clustering of the intracellular domain was deficient for A144E as measured by fluorescence resonance energy transfer. Transgenic mice expressing the A144E mutant on TACI−/− background had low serum IgA levels and significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll. B cells from A144E transgenic mice were impaired in their capacity to proliferate and secrete IgG1 and IgA in response to TACI ligation. These results suggest that mTACI A144E mutation and its human counterpart, A181E, disrupt TACI signaling and impair TACI-dependent B-cell functions.