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Advances in multi-agent chemotherapy and supportive care have dramatically improved survival of children with B-cell acute lymphoblastic leukemia (B-ALL); however, patients with relapsed and refractory disease continue to represent a therapeutic challenge. Hematopoietic stem cell transplant was the first immunotherapeutic approach to be used in the treatment of patients with relapsed or refractory disease. However, novel therapies such as bispecific antibodies that engage T-cells and chimeric antigen receptor T-cells (CAR-T) therapy have emerged as novel FDA-approved options that have the potential to become the new standard of care for these difficult-to-treat leukemias. With multiple immunotherapeutic agents in the drug development pipeline, it is important for cancer researchers and oncologists to be familiar with these agents, including their mechanism of action, side effects and efficacy. In this paper, we review the role of the human immune system in the development and treatment of childhood ALL and provide an overview of current and upcoming immunotherapeutic treatment approaches.

TNFRSF13B encodes the transmembrane activator and CAML interactor (TACI) receptor, which drives plasma cell differentiation. Although TNFRSF13B supports host defense, dominant-negative TNFRSF13B alleles are common in humans and other species and only rarely associate with disease. We reasoned that the high frequency of disruptive TNFRSF13B alleles reflects balancing selection, the loss of function conferring advantage in some settings. Testing that concept, we investigated how a common human dominant-negative variant, TNFRSF13B A181E, imparts resistance to enteric pathogens. Mice engineered to express mono- or biallelic A144E variants of tnrsf13B, corresponding to A181E, exhibited a striking resistance to pathogenicity and transmission of Citrobacter rodentium, a murine pathogen that models enterohemorrhagic Escherichia coli, and resistance was principally owed to natural IgA deficiency in the intestine. In WT mice with gut IgA and in mutant mice reconstituted with enteric IgA obtained from WT mice, IgA induces LEE expression of encoded virulence genes, which confer pathogenicity and transmission. Taken together, our results show that C. rodentium and most likely other enteric organisms appropriated binding of otherwise protective antibodies to signal induction of the virulence program. Additionally, the high prevalence of TNFRSF13B dominant-negative variants reflects balancing selection.

The gut mounts secretory immunoglobulin A (SIgA) responses to commensal bacteria through nonredundant T cell-dependent (TD) and T cell-independent (TI) pathways that promote the establishment of mutualistic host-microbiota interactions. SIgAs from the TD pathway target penetrant bacteria, and their induction requires engagement of CD40 on B cells by CD40 ligand on T follicular helper cells. In contrast, SIgAs from the TI pathway bind a larger spectrum of bacteria, but the mechanism underpinning their production remains elusive. Here, we show that the intestinal TI pathway required CD40-independent B cell-activating signals from TACI, a receptor for the innate CD40 ligand-like factors BAFF and APRIL. TACI-induced SIgA responses targeted a fraction of the gut microbiota without shaping its overall composition. Of note, TACI was dispensable for TD induction of IgA in gut-associated lymphoid organs. Thus, BAFF/APRIL signals acting on TACI orchestrate commensal bacteria-specific SIgA responses through an intestinal TI program.

T cell acute lymphoblastic leukemia (T-ALL) is a serious hematologic malignancy that occurs in children and young adults. Current therapies include intensive chemotherapy and ionizing radiation that preferentially kill malignant cells. Unfortunately, they are frequently accompanied by unintended negative impacts, including the induction of cellular senescence and long-term toxicities in normal host tissues. Whether these senescent cells resulting from therapy increase the susceptibility to relapse or secondary cancers is unknown. Using transgenic and pharmacological approaches to eliminate doxorubicin-induced senescent cells in a Notch-driven T-ALL relapse mouse model, we find that these cells inhibit tumor recurrence, suggesting that senescence in response to treatment suppresses tumorigenesis. This finding, together with extensive evidence from others demonstrating that age-associated health problems develop dramatically earlier among childhood cancer survivors compared to age-matched counterparts, suggests a relationship between therapy-induced senescence and tumorigenesis. Although cancer risk is increased through accelerated premature-aging in the long run, therapy-induced senescence appears to protect survivors from recurrence, at least in the short run.

Cyclophilins (Cyp) are peptidil-prolyl-isomerases and the intracellular receptors for the immunosuppressant Cyclosporine-A (CsA), which produces epithelial-mesenchymal-transition (EMT) and renal tubule-interstitial fibrosis. Since CsA inhibits Cyp enzymatic activity, we hypothesized that Cyp could be involved in EMT and fibrosis. Here, we demonstrate that CypB is a critical regulator of tubule epithelial cell plasticity on the basis that: i) CypB silencing caused epithelial differentiation in proximal tubule-derived HK-2 cells, ii) CypB silencing prevented TGFβ-induced EMT in HK-2, and iii) CypB knockdown mice exhibited reduced UUO-induced inflammation and kidney fibrosis. By contrast, silencing of CypA induces a more undifferentiated phenotype and favors TGFβ effects. EMT mediators Slug and Snail were up-regulated in CypA-silenced cells, while in CypB silencing, Slug, but not Snail, was down-regulated; thus, reinforcing the role of Slug in kidney fibrosis. CypA regulates Slug through its PPIase activity whereas CypB depends on its ER location, where interacts with calreticulin, a calcium modulator which is involved in TGFβ signaling. In conclusion, this work uncovers new roles for CypA and CypB in modulating proximal tubular cell plasticity.

© 2014, The American Society for Clinical Investigation, The transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) controls differentiation of long-lived plasma cells, and almost 10% of individuals with common variable immunodeficiency (CVID) express either the C104R or A181E variants of TACI. These variants impair TACI function, and TACI-deficient mice exhibit a CVID-like disease. However, 1%-2% of normal individuals harbor the C140R or A181E TACI variants and have no outward signs of CVID, and it is not clear why TACI deficiency in this group does not cause disease. Here, we determined that TACI-deficient mice have low baseline levels of Ig in the blood but retain the ability to mutate Ig-associated genes that encode antigen-specific antibodies. The antigen-specific antibodies in TACI-deficient mice were produced in bursts and had higher avidity than those of WT animals. Moreover, mice lacking TACI were able to clear Citrobacter rodentium, a model pathogen for severe human enteritis, more rapidly than did WT mice. These findings suggest that the high prevalence of TACI deficiency in humans might reflect enhanced host defense against enteritis, which is more severe in those with acquired or inherited immunodeficiencies., This work was funded by NIH grants P01 HL079067-01 and R37 HL52297

Objective.To investigate B cell survival cytokine messenger RNA (mRNA) levels as biomarkers of idiopathic inflammatory myopathies (IIM).Methods.We measured and compared mRNA levels of B cell survival cytokines by quantitative real-time polymerase chain reaction in 98 patients with IIM, 38 patients with systemic lupus erythematosus, and 21 healthy controls. The cytokines were B cell-activating factor belonging to the tumor necrosis factor family (BAFF); ΔBAFF; and a proliferation-inducing ligand (APRIL); and their receptors BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor, and B cell maturation antigen (BCMA). We also identified autoantibodies, including anti-Sm, anti-RNP, anti-SSA/Ro, anti-SSB/La, anti-topoisomerase 1, anti-hystidyl-tRNA synthetase, anti-ribosomal P, and anti-chromatin. Clinical disease activity was assessed by the International Myositis Assessment and Clinical Studies core set tool. We examined correlation of mRNA with disease activity, medication use, and autoantibodies.Results.We found a positive correlation of BAFF and ΔBAFF expression with 3 disease activity measures, with ΔBAFF having a stronger correlation. Similarly, anti-SSA/Ro-52 and/or anti-SSA/Ro-60 had a strong positive correlation with mRNA levels of BAFF and ΔBAFF, and with relative ratios of BAFF/APRIL and BCMA/BAFF-R.Conclusion.These findings highlight the potential importance of BAFF, ΔBAFF, and BAFF-R in the pathogenesis of IIM, and suggest an important role in the assessment of disease activity.

Medulloblastoma arising from the cerebellum is the most common pediatric brain malignancy, with leptomeningeal metastases often present at diagnosis and recurrence associated with poor clinical outcome. In this study, we used mouse medulloblastoma models to explore the relationship of tumor pathophysiology and dysregulated expression of the NOTCH pathway transcription factor ATOH1, which is present in aggressive medulloblastoma subtypes driven by aberrant Sonic Hedgehog/Patched (SHH/PTCH) signaling. In experiments with conditional ATOH1 mouse mutants crossed to Ptch1+/− mice, which develop SHH-driven medulloblastoma, animals with Atoh1 transgene expression developed highly penetrant medulloblastoma at a young age with extensive leptomeningeal disease and metastasis to the spinal cord and brain, resembling xenografts of human SHH medulloblastoma. Metastatic tumors retained abnormal SHH signaling like tumor xenografts. Conversely, ATOH1 expression was detected consistently in recurrent and metastatic SHH medulloblastoma. Chromatin immunoprecipitation sequencing and gene expression profiling identified candidate ATOH1 targets in tumor cells involved in development and tumorigenesis. Among these targets specific to metastatic tumors, there was an enrichment in those implicated in extracellular matrix remodeling activity, cytoskeletal network and interaction with microenvironment, indicating a shift in transcriptomic and epigenomic landscapes during metastasis. Treatment with bone morphogenetic protein or SHH pathway inhibitors decreased tumor cell proliferation and suppressed metastatic tumor growth, respectively. Our work reveals a dynamic ATOH1-driven molecular cascade underlying medulloblastoma metastasis that offers possible therapeutic opportunities. Cancer Res; 77(14); 3766–77. ©2017 AACR.

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 (IC50 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 up-regulate CD69 surface expression and secrete interleukin-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 ...

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.

Calcium-modulating cyclophilin ligand (Caml) is a ubiquitously expressed cytoplasmic protein that is involved in multiple signaling and developmental path- ways. An observation in our laboratory of a protein-protein interaction between Caml and the cytoplasmic region of Cadherin23 led us to speculate that Caml might be important in the inner ear and play a role in the develop- ment and/or function of hair cells. To address this question, we generated a mouse line in which Caml expression was eliminated in Atoh1-expressing cells of the inner ear upon administration of tamoxifen. Tamoxifen was administered immediately after birth to neonates to assess the effect of loss of Caml in the inner ear during postnatal development. Hearing in treated animals was tested by auditory brain stem response (ABR) analysis and cochlear pathology was evaluated by light microscopy. Lack of Caml expression in the inner ear leads to severe loss of cochlear hair cells and complete deafness. Elucidating the role of Caml in the inner ear will aid our understanding of the molecular pathways important for auditory development and function.

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.

Antiviral Abs, for example those produced in response to influenza virus infection, are critical for virus neutralization and defense against secondary infection. While the half-life of Abs is short, Ab titers can last a lifetime due to a subset of the Ab-secreting cells (ASCs) that is long lived. However, the mechanisms governing ASC longevity are poorly understood. Here, we have identified a critical role for extrinsic cytokine signals in the survival of respiratory tract ASCs in a mouse model of influenza infection. Irradiation of mice at various time points after influenza virus infection markedly diminished numbers of lung ASCs, suggesting that they are short-lived and require extrinsic factors in order to persist. Neutralization of the TNF superfamily cytokines B lymphocyte stimulator (BLyS; also known as BAFF) and a proliferation-inducing ligand (APRIL) reduced numbers of antiviral ASCs in the lungs and bone marrow, whereas ASCs in the spleen and lung-draining lymph node were surprisingly unaffected. Mice deficient in transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a receptor for BLyS and APRIL, mounted an initial antiviral B cell response similar to that generated in WT mice but failed to sustain protective Ab titers in the airways and serum, leading to increased susceptibility to secondary viral challenge. These studies highlight the importance of TACI signaling for the maintenance of ASCs and protection against influenza virus infection.

Appropriate control of apoptosis during T lymphocyte differentiation is critical for destruction of T cells bearing potentially autoreactive or useless immuno-receptors and for survival of those T cells bearing antigen receptors that may recognize foreign proteins. Despite the well-established importance of thymocyte survival, the exact signals regulating thymocyte apoptosis have not been fully elucidated. Here, we show that thymocytes lacking the endoplasmic reticulum protein calcium-modulating cyclophilin ligand (CAML) failed to undergo normal T-cell development and exhibited dramatically increased rates of apoptosis. In vitro, CAML-deficient thymocytes accumulated high levels of reactive oxygen species (ROS) and underwent abnormally accelerated death in response to several cytotoxic stimuli, including treatment with etoposide, cytokine deprivation, or Fas ligation. Although neither p53 deletion nor loss of Fas rescued the survival and continued development of CAML-deficient thymocytes, removal of the pro-apoptotic BH3-only Bcl-2 family member Bim significantly restored their survival. This work reveals CAML to be a critically important regulator of ROS- and Bim-dependent thymocyte death.

Contains fulltext : 88453.pdf (Publisher’s version ) (Open Access) Somatic activating mutations in Notch1 contribute to the pathogenesis of T cell acute lymphoblastic lymphoma (T-ALL), but how activated Notch1 signaling exerts this oncogenic effect is not completely understood. Here we identify HIV-1 Rev-binding protein (Hrb), a component of the clathrin-mediated endocytosis machinery, as a critical mediator of Notch-induced T-ALL development in mice. Hrb was found to be a direct transcriptional target of Notch1, and Hrb loss reduced the incidence or delayed the onset of T-ALL in mouse models in which activated Notch1 signaling either contributes to or drives leukemogenesis. Consistent with this observation, Hrb supported survival and proliferation of hematopoietic and T cell precursor cells in vitro. We demonstrated that Hrb accelerated the uptake of transferrin, which was required for upregulation of the T cell protooncogene p21. Indeed, iron-deficient mice developed Notch1-induced T-ALL substantially more slowly than control mice, further supporting a critical role for iron uptake during leukemogenesis. Taken together, these results reveal that Hrb is a critical Notch target gene that mediates lymphoblast transformation and disease progression via its ability to satisfy the enhanced demands of transformed lymphoblasts for iron. Further, our data suggest that Hrb may be targeted to improve current treatment or design novel therapies for human T-ALL patients.

Capsular polysaccharides of encapsulated bacteria do not induce immune response in newborns and the mechanism for this unresponsiveness is not clear. In adults, transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI) is a TNFR family member molecule with a pivotal role in Ab responses against polysaccharide vaccines. We investigated the expression and the functions of the TNF family cytokines, B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL), and their receptors in newborn mice and found that TACI expression on B lymphocytes was dramatically reduced (p < 0.0001) in newborns as compared with adults. More importantly, TACI ligands BAFF or APRIL were unable to induce IgA/IgG/IgM secretion from newborn B lymphocytes. Additionally, TACI expression seems to be important in plasma cell development. Indeed, in contrast to adults, stimulation of newborn B lymphocytes with BAFF or APRIL did not result in up-regulation of CD138 expression. In vitro or in vivo exposure of newborn B lymphocytes to oligodeoxynucleotides (CpG ODN) led to up-regulation of TACI expression on newly formed, follicular, and marginal zone as well as B1 B lymphocyte populations, and rendered them responsive to BAFF- or APRIL-mediated CD138 expression and IgA/IgG secretion. Finally, immunization of newborn BALB/c mice but not TACI knockout mice with CpG ODN containing (4-hydroxy-3-nitrophenyl)acetyl-Ficoll led to development of IgG Abs against (4-hydroxy-3-nitrophenyl)acetyl. These findings demonstrate that low TACI expression may be a critical factor that determines the susceptibility of newborns to infections with encapsulated bacteria and the impaired immunogenicity of polysaccharide vaccines. Finally, CpG ODNs may correct deficient newborn response to polysaccharide vaccines by up-regulating TACI.

The HIV-1 Vpu protein is required for efficient viral release from human cells. For HIV-2, the envelope (Env) protein replaces the role of Vpu. Both Vpu and HIV-2 Env enhance virus release by counteracting an innate host-cell block within human cells that is absent in African green monkey (AGM) cells. Here we identify calcium-modulating cyclophilin ligand (CAML) as a Vpu-interacting host factor that restricts HIV-1 release. Expression of human CAML (encoded by CAMLG) in AGM cells conferred a strong restriction of virus release that was reversed by Vpu and HIV-2 Env, suggesting that CAML is the mechanistic link between these two viral regulators. Depletion of CAML in human cells eliminated the need for Vpu in enhancing HIV-1 and murine leukemia virus release. These results point to CAML as a Vpu-sensitive host restriction factor that inhibits HIV release from human cells. The ability of CAML to inhibit virus release should illuminate new therapeutic strategies against HIV.

Proteinuria contributes to chronic kidney disease by stimulating renal tubular epithelial cells to produce cytokines such as monocyte chemoattractant protein-1 (MCP-1). The present study determined whether cellular overexpression of heme oxygenase-1 (HO-1) can influence albumin-stimulated MCP-1 production. In response to bovine serum albumin, NRK-52E cells constitutively overexpressing HO-1 (HO-1 OE cells) exhibit less induction of MCP-1 mRNA and less production of MCP-1 protein compared with similarly treated, control NRK-52E cells (CON cells). In wild-type NRK-52E cells, and under these conditions, we demonstrate that the induction of MCP-1 is critically dependent on intact NF-κB binding sites in the MCP-1 promoter. In response to albumin, CON cells exhibit activation of NF-κB, and this is reduced in HO-1 OE cells. Albumin also activates ERK1/2 and increases ERK activity, both of which are exaggerated in HO-1 OE cells. Studies with an inhibitor of MAPK/ERK kinase (U0126) demonstrate that the inhibitory effects of U0126 on MCP-1 production are attenuated in HO-1 OE cells. We conclude that HO-1 overexpression in the proximal tubule reduces MCP-1 production in response to albumin, and this occurs, at least in part, by inhibiting an ERK-dependent, NF-κB-dependent pathway at a site that is distal to the activation of ERK. These findings suggest that the induction of HO-1 in the proximal tubule, as occurs in chronic kidney disease, may be a countervailing response that reduces albumin-stimulated production of cytokines such as MCP-1.

Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum resident protein that is widely expressed. Although it has been demonstrated to participate in the tail-anchored protein insertion pathway, its physiological role in the mature immune system is unknown. In this work, we show that mature, peripheral T cells require CAML for survival specifically following TCR-induced activation. In this study, we examined mature T cells from spleen and lymph nodes of tamoxifen-inducible CAML knockout mice (tCAML−/−). Whereas CAML-deficient T cells were able to express the early activation markers CD25 and CD69, and produce IL-2 normally upon stimulation, deficient cells proliferated less and died. Cells did not require CAML for entry into the S phase of the cell cycle, thus implicating its survival function at a relatively late step in the T cell activation sequence. In addition, CAML was required for homeostatic proliferation and for Ag-dependent cell killing in vivo. These results demonstrate that CAML critically supports T cell survival and cell division downstream of T cell activation.

The predicted structure of the autosomal recessive polycystic kidney disease protein, fibrocystin, suggests that it may function as a receptor, but its function remains unknown. To understand its function, we searched for proteins that interact with the intracellular C-terminus of fibrocystin using the yeast two-hybrid system. From the screening, we found calcium modulating cyclophilin ligand (CAML), a protein involved in Ca2+ signaling. Immunofluorescent analysis showed that both proteins are co-localized in the apical membrane, primary cilia, and the basal body of cells derived from the distal nephron Epitope-tagged expression constructs of both proteins were co-immunoprecipitated from COS7 cells. The intracellular C-terminus of fibrocystin interacts with CAML, a protein with an intracellular distribution that is similar to that of PKD2. Fibrocystin may participate in regulation of intracellular Ca2+ in the distal nephron in a manner similar to PKD1 and PKD2 that are involved in autosomal dominant polycystic kidney disease.

The cytokines B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) enhance autoimmune disease by sustaining B cell activation. In RA, B cells contribute to the formation of 3 functionally distinct types of lymphoid microarchitectures in the inflamed synovium: ectopic GCs; T cell-B cell aggregates lacking GC reactions; and unorganized, diffuse infiltrates. We examined 72 tissues representing the 3 types of synovitis for BLyS and APRIL production and for expression of APRIL/BLyS receptors. Biologic effects of BLyS and APRIL were explored by treating human synovium-SCID mouse chimeras with the APRIL and BLyS decoy receptor transmembrane activator and CAML interactor:Fc (TACI:Fc). GC+ synovitis had the highest levels of APRIL, produced exclusively by CD83+ DCs. BLyS was present in similar levels in all tissue types and derived exclusively from CD68+ macrophages. In GC+ synovitis, treatment with TACI:Fc resulted in GC destruction and marked inhibition of IFN-gamma and Ig transcription. In contrast, inhibition of APRIL and BLyS in aggregate and diffuse synovitis left Ig levels unaffected and enhanced IFN-gamma production. These differential immunomodulatory effects correlated with the presence of TACI+ T cells in aggregate and diffuse synovitis and their absence in GC+ synovitis. We propose that BLyS and APRIL regulate B cell as well as T cell function and have pro- and antiinflammatory activities in RA.

SummaryCalcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein implicated in T cell signaling, although its mechanism and physiologic role in the immune system are unknown. We show here that CAML is essential for peripheral T cell development. Inactivation of CAML in mouse thymocytes lowered the numbers of double-positive and single-positive thymocytes, concomitant with reduced positive and enhanced negative selection. We found that CAML interacts with p56Lck and appears to regulate subcellular localization of the kinase in both resting and T cell receptor (TCR)-stimulated cells. CAML-deficient cells displayed enhanced p56lck and ZAP-70 phosphorylation and increased IL2 production and cell death after TCR stimulation, suggesting that CAML may act as a negative regulator of p56lck. Our data establish a novel role for CAML as an essential mediator of T cell survival during thymopoiesis and indicate that its loss deregulates p56Lck signaling.

Long-lived humoral immunity is manifested by the ability of bone marrow plasma cells (PCs) to survive for extended periods of time. Recent studies have underscored the importance of BLyS and APRIL as factors that can support the survival of B lineage lymphocytes. We show that BLyS can sustain PC survival in vitro, and this survival can be further enhanced by interleukin 6. Selective up-regulation of Mcl-1 in PCs by BLyS suggests that this α-apoptotic gene product may play an important role in PC survival. Blockade of BLyS, via transmembrane activator and cyclophilin ligand interactor–immunoglobulin treatment, inhibited PC survival in vitro and in vivo. Heightened expression of B cell maturation antigen (BCMA), and lowered expression of transmembrane activator and cyclophilin ligand interactor and BAFF receptor in PCs relative to resting B cells suggests a vital role of BCMA in PC survival. Affirmation of the importance of BCMA in PC survival was provided by studies in BCMA−/− mice in which the survival of long-lived bone marrow PCs was impaired compared with wild-type controls. These findings offer new insights into the molecular basis for the long-term survival of PCs.

The kinase TBK1 participates in signaling pathways that induce antimicrobial responses. TBK1 is also necessary for the suppression of excessive production of immunoglobulin A by accelerating destruction of the kinase NIK.

On viral infection, infected cells can become the target of host immune responses or can go through a programmed cell death process, called apoptosis, as a defense mechanism to limit the ability of the virus to replicate. To prevent this, viruses have evolved elaborate mechanisms to subvert the apoptotic process. Here, we report the identification of a novel antiapoptotic K7 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) which expresses during lytic replication. The KSHV K7 gene encodes a small mitochondrial membrane protein, and its expression efficiently inhibits apoptosis induced by a variety of apoptogenic agents. The yeast two-hybrid screen has demonstrated that K7 targets cellular calcium-modulating cyclophilin ligand (CAML), a protein that regulates the intracellular Ca 2+ concentration. Similar to CAML, K7 expression significantly enhances the kinetics and amplitudes of the increase in intracellular Ca 2+ concentration on apoptotic stimulus. Mutational analysis showed that K7 interaction with CAML is required for its function in the inhibition of apoptosis. This indicates that K7 targets cellular CAML to increase the cytosolic Ca 2+ response, which consequently protects cells from mitochondrial damage and apoptosis. This is a novel viral antiapoptosis strategy where the KSHV mitochondrial K7 protein targets a cellular Ca 2+ -modulating protein to confer resistance to apoptosis, which allows completion of the viral lytic replication and, eventually, maintenance of persistent infection in infected host.

In this study, we investigated the mechanisms underlying cell death induced by the small molecule cyclophilin inhibitor NIM811. NIM811 effectively killed GBM cells through a non-apoptotic pathway, as there was no nuclear shrinkage or caspase activation. Instead, death was preceded by dramatic cytosolic vacuolization, stemming from enlargement of the endoplasmic reticulum (ER). RNA-seq revealed upregulation of genes pertinent to the unfolded protein response (UPR) and autophagy. Western blotting for UPR and autophagy mediators also supported a transient UPR signaling and compromised autophagy during prolonged NIM811 treatment. By utilizing a bicistronic reporter plasmid, we independently quantitated Cap-dependent and Cap-independent translation. We further assessed the long-term effects mediated by different short-term paraptosis inhibitors via colony formation assay. We find that NIM811 initiates paraptosis in GBM cells, due to an abnormal upregulation of protein translation that caused accumulation of misfolded or unfolded proteins in ER. Transient activation of pro-survival autophagy and UPR signals were shutdown during prolonged treatment with NIM811, thus allowing cell death to occur. Cycloheximide was previously reported by others to suppress paraptosis. In our studies, we find instead that it temporarily delayed vacuole formation, but actually enhanced paraptotic cell death in the long term. The mTOR inhibitors rapamycin or torin-2 rescued cells from NIM811 induced paraptosis by sustaining autophagy and the UPR, and specifically restrained cap-dependent translation. We propose that ER vacuolization could be a protective response generated by cells to isolate defective proteins within the ER in order to prevent misfolded aggregates to interfere with cellular homeostasis. For that reason, simply inhibiting vacuole formation without clearing protein accumulation in the ER was insufficient to prevent cell death. These findings aid our understanding of cellular mechanisms that contribute to non-apopotic death. Moreover, this work underlines the importance of evaluating cellular responses in cell death experiments over multiple time frames to judge the true impact. Citation Format: Lin Wang, Justin Gundelach, Richard Bram. Protein synthesis modulates paraptotic death induced by inhibition of cyclophilins in glioblastoma (GBM) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4313. doi:10.1158/1538-7445.AM2017-4313

Background The B-cell receptor transmembrane activator and calcium modulator ligand interactor (TACI) is important for T-independent antibody responses. One in 200 blood donors are heterozygous for the TACI A181E mutation. Objective We sought to investigate the effect on B-cell function of TACI A181E heterozygosity in reportedly healthy subjects and of the corresponding TACI A144E mutation in mice. Methods Nuclear factor κB (NF-κB) activation was measured by using the luciferase assay in 293T cells cotransfected with wild-type and mutant TACI. TACI-driven proliferation, isotype switching, and antibody responses were measured in B cells from heterozygous TACI A144E knock-in mice. Mouse mortality was monitored after intranasal pneumococcal challenge. Results Levels of natural antibodies to the pneumococcal polysaccharide component phosphocholine were significantly lower in A181E-heterozygous than TACI-sufficient Swedish blood donors never immunized with pneumococcal antigens. Although overexpressed hTACI A181E and mTACI A144E acted as dominant-negative mutations in transfectants, homozygosity for A144E in mice resulted in absent TACI expression in B cells, indicating that the mutant protein is unstable when naturally expressed. A144E heterozygous mice, such as TACI +/− mice, expressed half the normal level of TACI on their B cells and exhibited similar defects in a proliferation-inducing ligand–driven B-cell activation, antibody responses to TNP-Ficoll, production of natural antibodies to phosphocholine, and survival after intranasal pneumococcal challenge. Conclusion These results suggest that TACI A181E heterozygosity results in TACI haploinsufficiency with increased susceptibility to pneumococcal infection. This has important implications for asymptomatic TACI A181E carriers.

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.

Activation of the nuclear factor of activated T cells transcription factor (NF-AT) is a key event underlying lymphocyte action. The CAML (calcium-modulator and cyclophilin ligand) protein is a coinducer of NF-AT activation when overexpressed in Jurkat T cells. A member of the tumor necrosis factor receptor superfamily was isolated by virtue of its affinity for CAML. Cross-linking of this lymphocyte-specific protein, designated TACI (transmembrane activator and CAML-interactor), on the surface of transfected Jurkat cells with TACI-specific antibodies led to activation of the transcription factors NF-AT, AP-1, and NFκB. TACI-induced activation of NF-AT was specifically blocked by a dominant-negative CAML mutant, thus implicating CAML as a signaling intermediate.

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.

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 CAMLG gene encodes a novel cyclophilin B-binding protein called calcium-modulating cyclophilin ligand, which appears to be involved in the regulation of calcium signaling in T lymphocytes and other cells. The murine homolog, Caml, was localized by interspecific backcross analysis to the middle of chromosome 13. By fluorescence in situ hybridization, this gene was localized to human chromosome 5 in a region (q23) known to be syntenic to mouse chromosome 13. These results provide further evidence supporting the extensive homology between human chromosome 5q and mouse chromosome 13. In addition, the results will provide a basis for further evaluation of cytogenetic anomalies that may contribute to inherited defects in calcium signaling or immune system function.

Calcium and calcium-binding proteins play important roles in the signaling cascade leading from the initial engagement of TCRs on T cells to the fully activated state. To undertake a molecular dissection of this cascade, we first isolated a Jurkat T cell line derivative containing the NF-AT promoter element driving transcription of the diphtheria toxin A chain gene ( dipA ), resulting in rapid cell death. Selecting viable cells that fail to activate NF-AT-dependent transcription, we isolated two independent cell lines possessing defects in capacitative Ca 2+ entry. NF-AT-dependent transcription can be restored in these cells by expression of a constitutively active calcineurin, but not by overexpression of the Ca 2+ regulatory protein CAML, which can normally replace the Ca 2+ signal. The defect in these cell lines probably lies between CAML and calcineurin in the T cell activation cascade.

We have shown previously that multiple rounds of a systemic treatment of GM-CSF, followed by intravenous reovirus, leads to effective treatment of subcutaneous melanomas (Ilett et al., Mol. Ther. 2015). We show here that a similar regimen is also effective at treating both melanoma (B16) and glioma (GL261) tumors growing intra-cranially. As a result of these pre-clinical studies, we initiated a Phase I clinical trial in paediatric patients with gliomas of GM-CSF and reovirus therapy. To date, three patients have been treated. In two of three of these patients there were possible indications of pseudoprogression and intra-cranial inflammation, both of which were clinically resolved upon treatment with dexamethasone. Whilst this trial continues to recruit, we have investigated additional treatments for paediatric brain tumors, especially those in which direct virus injection may allow greater local access to tumor. In this respect, there is a constant tension between the pro-inflammatory, anti-tumor nature of oncolytic viroimmunotherapy and the need to reduce potentially toxic local inflammatory reactions within the brain. Our overall hypothesis is that it will be possible to balance the anti tumor effects of viral oncolysis, in part caused by inflammatory reactions to the virus, with the associated toxicity. Nowhere is this more relevant than for the treatment of Diffuse Pontine Gliomas (DIPG), which grow in the brain stem. Our experiments show that it is indeed possible to treat tumors growing in the pons/and or medulla, with inflammatory, immune based therapies (both T cell mediated and locally cytotoxic) without increasing toxicity over that caused by tumor growth alone. Therefore, we have screened multiple oncolytic virus types for their ability to kill DIPG cell lines both in vitro and in vivo. In particular, we have compared viruses with different speeds of oncolysis and different inflammatory properties to investigate how the efficacy can be balanced with local toxicity, using standard of care anti inflammatory treatments (with dexamethasone) as well as with novel agents and viruses designed to suppress local inflammatory reactions.

Background TNFRSF13B , which encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), is mutated in 10% of patients with common variable immunodeficiency. One of the 2 most common TACI mutations in common variable immunodeficiency, C104R, abolishes ligand binding and is found predominantly in the heterozygous state. The murine TACI mutant C76R is the equivalent of the human TACI mutant C104R. Objective We sought to define the consequence of the C76R mutation on TACI function in mice that express both wild-type TACI and the murine C76R mutant. Methods Transgenic mice that express murine TACI C76R, the counterpart of human TACI C104R, on the TACI +/− B6/129 background (C76R/TACI +/− mice) were constructed. Serum immunoglobulins and antibody responses to the type II T-independent antigen trinitrophenylated (TNP)-Ficoll were determined by means of ELISA. B-cell proliferation in response to a proliferation-inducing ligand was determined based on tritiated thymidine incorporation into DNA. IgG1 secretion by B cells in response to a proliferation-inducing ligand plus IL-4 was determined by means of ELISA. Results C76R/TACI +/− mice had significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll compared with TACI +/+ B6/129 control animals, and their B cells were impaired in their capacity to proliferate and secrete IgG1 in response to TACI ligation. Unexpectedly, TACI +/− mice had similarly impaired B-cell function as C76R/TACI +/− littermates. Impaired TACI function caused by haploinsufficiency was confirmed in TACI +/− mice on the C57BL/6 background. Conclusion These results suggest that the human TACI mutant C104R might impair TACI function in heterozygotes through haploinsufficiency.

TNFRSF13B, 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.

Accumulation of GABA(A) receptors (GABA(A)Rs) at GABAergic synapses requires the cytoplasmic loop region and C-terminal transmembrane domain of the receptor gamma2 subunit. We here report a novel interaction of gamma2 with Calcium-Modulating cyclophilin Ligand (CAML), an integral membrane protein that regulates this mechanism. Interaction of GABA(A)Rs with CAML depends on both the cytoplasmic region and fourth transmembrane domain of the gamma2 subunit, CAML immunoprecipitates with GABA(A)Rs from transfected cells and brain lysates and colocalizes with gamma2 in ER vesicles in soma and dendrites of neurons. CAML shRNA treatment results in reduced expression of postsynaptic GABA(A)Rs, along with significant reductions in GABA-evoked whole cell currents and GABAergic synaptic function, while glutamatergic transmission is unaffected. Reduced surface expression of GABA(A)Rs in CAML mutant neurons is associated with selective deficits in recycling of endocytosed GABA(A)Rs to the cell surface. Our results indicate a specific role of CAML in functional expression and endocytic recycling of postsynaptic GABA(A)Rs.

Through their differential interactions with B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), the three BLyS family receptors play central roles in B cell survival and differentiation. Recent evidence indicates BLyS receptor levels shift following BCR ligation, suggesting that activation cues can alter overall BLyS receptor profiles and thus ligand sensitivity. In this study, we show that TLR stimuli also alter BLyS receptor expression, but in contrast to BCR ligation, TLR9 and TLR4 signals, preferentially increase transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) expression. Although both of these TLRs act through MyD88-dependent mechanisms to increase TACI expression, they differ in terms of their downstream mediators and the B cell subset affected. Surprisingly, only TLR4 relies on c-Rel and p50 to augment TACI expression, whereas TLR9 does not. Furthermore, although all follicular and marginal zone B cells up-regulate TACI in response to TLR9 stimulation, only marginal zone B cells and a subset of follicular B cells respond to TLR4. Finally, we find that both BLyS and APRIL enhance viability among quiescent and BCR-stimulated B cells. However, although BLyS enhances viability among TLR stimulated B cells, APRIL does not, suggesting that TACI but not BLyS receptor 3 may share survival promoting pathways with TLRs.

BLyS, recently shown to be critical for survival of normal B cells, has been found to be elevated in a number of immune disease models. A role for BLyS in the survival of malignant B cells has also been revealed and we therefore sought to identify a role for BLyS and its receptors in non-Hodgkin lymphoma (NHL). We found that tumor cells from all NHL histologic subtypes expressed one or more of 3 known receptors (BCMA, TACI, and BAFF-R) for BLyS; however, the pattern of expression was variable. We provide evidence that BLyS is expressed in tumors from patients with NHL and that BLyS levels increase as tumors transform to a more aggressive phenotype. Additionally, we provide evidence that serum BLyS levels are elevated in a subgroup of patients with NHL. In patients with de novo large B-cell lymphoma, a high BLyS level correlated with a poorer median overall survival, the presence of constitutional symptoms, and elevated values of lactic dehydrogenase. When BLyS levels were correlated with response to therapy in all patients, responding patients had a significantly lower BLyS level than those with progressive disease. In summary, we found that BLyS and its receptors represent a potentially important therapeutic target in B-cell lymphoma.

B-cell chronic lymphocytic leukemia (B-CLL) is defined by the accumulation of CD5+ B cells in the periphery and bone marrow. This disease is not characterized by highly proliferative cells but rather by the presence of leukemic cells with significant resistance to apoptosis and, therefore, prolonged survival. B-lymphocyte stimulator (BLyS) is a newly identified tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis and it shares significant homology with another TNF superfamily member, APRIL. The striking effects of BLyS on normal B-cell maintenance and survival raises the possibility that it may be involved in pathogenesis and maintenance of hematologic malignancies, including B-CLL. In this study, we investigated the status of APRIL and BLyS expression, as well as their receptors, in this disease. All B-CLL patient cells studied expressed one or more of 3 known receptors for BLyS; however, the pattern of expression was variable. In addition, we demonstrate for the first time that B-CLL cells from a subset of patients aberrantly express BLyS and APRIL mRNA, whereas these molecules were not detectable in normal B cells. Furthermore, we provide in vitro evidence that BLyS protects B-CLL cells from apoptosis and enhances cell survival. Because these molecules are key regulators of B-cell homeostasis and tumor progression, leukemic cell autocrine expression of BLyS and APRIL may be playing an important role in the pathogenesis of this disease.

The Ca2+-modulating cyclophilin ligand (CAML) protein causes stimulation of transcription factors via activation of a store-operated Ca2+ entry pathway. Since CAML is widely expressed in mammalian tissues, it may be an important regulator of Ca2+ store function. In the present study, we investigated the consequence of CAML overexpression on Ca2+ signaling using rapid confocal imaging of Fluo3-loaded NIH3T3 fibroblasts. Control and CAML-expressing cells gave concentration-dependent responses to the Ca2+ mobilizing agonist ATP. CAML expression reduced the sensitivity of the cells so that higher concentrations of ATP were needed to achieve global Ca2+ waves. The amplitudes of Ca2+ waves were significantly reduced in CAML expressing cells, consistent with earlier suggestions that CAML causes depletion of internal Ca2+ stores. With low ATP concentrations, only local Ca2+ release events were observed. CAML did not affect the characteristics of these local Ca2+ signals, suggesting that it does not directly affect Ca2+ release channels.

Abstract 3349 Introduction: The protease ADAMTS13 (A Disintegrin and Metalloprotease with Thrombo Spondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder Thrombotic Thrombocytopenic Purpura (TTP). TTP has been in particular observed in transplant patients following treatment with Cyclosporin A (CsA), a drug which is frequently administered in patients to suppress allogeneic immune response. However, a mechanistic explanation for such a link has remained unclear. Here, we report that cyclophilin-B mediated peptidyl-prolyl cis–trans isomerase (PPIase) activity, which is also targeted by CsA, plays an important role in the production of active ADAMTS13. Methods and Results: An in vitro transient expression system was used to study the effects of CsA on the synthesis and activity of ADAMTS13. We found that exposing HEK293 cells to CsA results in a dose-dependent reduction in the level of secreted ADAMTS13, but not the ubiquitously-expressed housekeeping protein Hsp70. Moreover, treatment with other immunosuppressive drugs, such as rapamycin or tacrolimus/FK506 did not impact the secretion of ADAMTS13. CsA is a known inhibitor of several cellular substrates, including cyclophilin B (CypB), which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities. We show a direct, functional interaction between CypB and ADAMTS13 using co-immunoprecipitation analysis. Specifically, ADAMTS13 and CypB could be detected in association together in untreated cells, but not in those treated with CsA, where only ADAMTS13 could be detected. ADAMTS13 levels are also reduced in the setting of siRNA-mediated CypB knockdown, suggesting a direct link between CypB expression/activity and ADAMTS13 maturation. We have found that CsA leads to intracellular conformational changes in the ADAMTS13 protein, evidenced by a unique trypsin digestion pattern of ADAMTS13 from CsA-treated cells relative to untreated cells. Additionally we found that treatment of cells with N-acetyl-Leu-Leu-norleucinal (ALLN), a proteasome inhibitor, resulted in about a 2.5 fold rescue in ADAMTS13 expression in the setting of CsA treatment. This observation suggests that CsA treatment leads to an enhanced proteasomal degradation of ADAMTS13, possibly due altered conformation. Although CsA did not influence the overall level of intracellular ADAMTS13, it resulted in diminished specific proteolytic activity, further suggesting improper ADAMTS13 folding. Finally, we show that plasma samples from CypB knockout mice have reduced levels of circulating ADAMTS13. Conclusions: Our findings indicate that cyclophilin-mediated activity is an important factor in ADAMTS13 secretion and activity. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization process mediated by CypB in its proper folding and maturation. We suggest that CsA treatment disrupts the interaction between ADAMTS13 and CypB. Although this may not be the sole mechanism by which CsA ultimately lowers levels of secreted ADAMTS13, this work altogether provides a novel mechanistic explanation for CsA-induced TTP in transplant patients. Disclosures: No relevant conflicts of interest to declare.

Osteogenesis Imperfecta (OI) is a human syndrome characterized by exquisitely fragile bones due to osteoporosis. The majority of autosomal dominant OI cases result from point or splice site mutations in the type I collagen genes, which are thought to lead to aberrant osteoid within developing bones. OI also occurs in humans with homozygous mutations in Prolyl-3-Hydroxylase-1 (LEPRE1). Although P3H1 is known to hydroxylate a single residue (pro-986) in type I collagen chains, it is unclear how this modification acts to facilitate collagen fibril formation. P3H1 exists in a complex with CRTAP and the peptidyl-prolyl isomerase cyclophilin B (CypB), encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been a complete mystery. To study the role of mammalian CypB, we generated mice lacking this protein. Early in life, Ppib-/- mice developed kyphosis and severe osteoporosis. Collagen fibrils in Ppib-/- mice had abnormal morphology, further consistent with an OI phenotype. In vitro studies revealed that in CypB–deficient fibroblasts, procollagen did not localize properly to the golgi. We found that levels of P3H1 were substantially reduced in Ppib-/- cells, while CRTAP was unaffected by loss of CypB. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1. Consistent with these changes, post translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB–deficient cells and tissues from CypB–knockout mice. These data provide significant new mechanistic insight into the pathophysiology of OI and reveal how the members of the P3H1/CRTAP/CypB complex interact to direct proper formation of collagen and bone., Author Summary Osteogenesis Imperfecta (OI), also known as “brittle bone disease,” is an inherited condition with multiple defects in collagen-containing structures, including the bones, skin, and other connective tissues. Patients with OI suffer from short stature, scoliosis, thin skin, hearing loss, and, most notably, fragile bones that break with little or no trauma. Although many cases are due to dominantly inherited point mutations in the collagen genes, autosomal recessive forms have been described due to defects in the genes for Prolyl-3-Hydroxylase-1 (LEPRE1) and Cartilage-Associated Protein (CRTAP), proteins that modify newly synthesized procollagen. Some patients with OI do not have mutations in any of the known disease-related genes. Here, through the use of newly generated knockout mice, we identify the endoplasmic-reticulum resident prolyl-isomerase cyclophilin B (CypB) as a new autosomal recessive OI gene in mice. CypB, P3H1, and CRTAP were shown to have interrelated effects in maintaining their respective protein levels and ability to bind to collagen. These studies enhance our understanding about how collagen, the most abundant protein in the body, becomes properly assembled to form bones with adequate strength.

CYCLOSPORINA and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response1–3. In T lymphocytes these drugs disrupt an unknown step in the trans-mission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response4–6. The putative intracellular receptors for FK506 and cyclosporin arecis-trans prolyl isomerases7–11. Binding of the drug inhibits isomerase activity8,10,11, but studies with other prolyl isomerase inhibitors12 and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase13,14, and not from inhibi-tion of isomerase activity. A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-κB (refs 15–17). Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT. FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit.

TACI is a TNFR homolog expressed by mature B lymphocytes that has been implicated in the positive regulation of B cell growth and antibody production, as well as in the development of autoimmune disease. Its biology is complex due to the existence of two ligands, BLyS and APRIL, and a homologous receptor, BCMA, that similarly binds both ligands. To determine its critical biological role, we generated TACI knockout mice. Surprisingly, these mice demonstrated a 2-fold increase in numbers of circulating and splenic B cells, apparently due to increased proliferation rate. Maturation of B cells and T-dependent antibody production was normal, but responses to T-independent type II antigens were almost completely abolished. It appears that TACI provides an essential costimulatory signal for the T-independent humoral response.