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Supplementary Table 2 from NOTCH Is Part of the Transcriptional Network Regulating Cell Growth and Survival in Mouse Plasmacytomas

Supplementary Data 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells

Supplementary Table 1 from NOTCH Is Part of the Transcriptional Network Regulating Cell Growth and Survival in Mouse Plasmacytomas

We have compared histologic features and gene expression profiles of newly identified plasmacytomas from NFS.V+ congenic mice with plasmacytomas of IL6 transgenic, Fasl mutant, and SJL-β2M−/− mice. NFS.V+ tumors comprised an overlapping morphologic spectrum of high-grade/anaplastic, intermediate-grade/plasmablastic, and low-grade/plasmacytic cases with similarities to subsets of human multiple myeloma and plasmacytoma. Microarray and immunohistochemical analyses of genes expressed by the most prevalent tumors, plasmablastic plasmacytomas, showed them to be most closely related to immunoblastic lymphomas, less so to plasmacytomas of Fasl mutant and SJL mice, and least to plasmacytic plasmacytomas of IL6 transgenic mice. Plasmablastic tumors seemed to develop in an inflammatory environment associated with gene signatures of T cells, natural killer cells, and macrophages not seen with plasmacytic plasmacytomas. Plasmablastic plasmacytomas from NFS.V+ and SJL-β2M−/− mice did not have structural alterations in Myc or T(12;15) translocations and did not express Myc at high levels, regular features of transgenic and pristane-induced plasmacytomas. These findings imply that, as for human multiple myeloma, Myc-independent routes of transformation contribute to the pathogenesis of these tumors. These findings suggest that plasma cell neoplasms of mice and humans exhibit similar degrees of complexity. Mouse plasmacytomas, previously considered to be homogeneous, may thus be as diverse as their human counterparts with respect to oncogenic mechanisms of plasma cell transformation. Selecting specific types of mouse plasmacytomas that relate most closely to subtypes of human multiple myeloma may provide new opportunities for preclinical testing of drugs for treatment of the human disease. [Cancer Res 2007;67(6):2439–47]

Supplementary Figure 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells

Supplementary Figure Legends 1-2 from NOTCH Is Part of the Transcriptional Network Regulating Cell Growth and Survival in Mouse Plasmacytomas

Supplementary Figure 3 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells

Supplementary Figure 2 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells

Supplementary Figures 1-2 from NOTCH Is Part of the Transcriptional Network Regulating Cell Growth and Survival in Mouse Plasmacytomas

Aside from Myc-activating translocations characteristic of plasmacytomas (PCT), little is known about genetic factors and signaling pathways responsible for the development of spontaneous B-cell lineage lymphomas of mice. Here, we characterized the transcriptional profiles of PCT, centroblastic diffuse large B-cell lymphomas (CBL), and high-grade splenic marginal zone B-cell lymphoma (MZL++) using high-throughput quantitative reverse transcription-PCR. Expression profiles of CBL and MZL++ were strikingly similar and quite unlike that of PCT. Among the genes expressed at significantly higher levels by PCT were a number involved in NOTCH signaling, a finding supported by gene set enrichment analyses of microarray data. To investigate the importance of this pathway, NOTCH signaling was blocked in PCT cell lines by treatment with a γ-secretase inhibitor (GSI) or transduction of a dominant-negative mutant of MAML1. These treatments resulted in reduced expression of NOTCH transcriptional targets in association with impaired proliferation and increased apoptosis. GSI treatment of transformed plasma cells in a primary PCT also induced apoptosis. These results integrate NOTCH activation with oncogenic signaling pathways downstream of translocated Myc in the pathogenesis of mouse PCT, two signaling pathways also implicated in development of human multiple myeloma and T-cell lymphoblastic lymphoma. [Cancer Res 2008;68(22):9202–11]

Epidermolysis Bullosa (EB) is a group of rare genetic disorders that compromise the structural integrity of the skin such that blisters and subsequent erosions occur after minor trauma. While primary genetic risk of all subforms of EB adhere to Mendelian patterns of inheritance, their clinical presentations and severities can vary greatly, implying genetic modifiers. TheLamc2jebmouse model of non-Herlitz junctional EB (JEB-nH) demonstrated that genetic modifiers can contribute substantially to the phenotypic variability of JEB and likely other forms of EB. The innocuous changes in an ‘EB related gene’,Col17a1, have shown it to be a dominant modifier ofLamc2jeb. This work identifies six additional Quantitative Trait Loci (QTL) that modify disease inLamc2jeb/jebmice. Three QTL include other known ‘EB related genes’, with the strongest modifier effect mapping to a region including the epidermal hemi-desmosomal structural gene dystonin (Dst-e/Bpag1-e). Three other QTL map to intervals devoid of known EB-associated genes. Of these, one contains the nuclear receptor coactivatorPpargc1aas its primary candidate and the others contain related genesPpargandIgf1, suggesting modifier pathways. These results, demonstrating the potent disease modifying effects of normally innocuous genetic variants, greatly expand the landscape of genetic modifiers of EB and therapeutic approaches that may be applied.

Albumin has an average plasma half-life of three weeks and is thus an attractive carrier to improve the pharmacokinetics of fused therapeutics. The half-life is regulated by FcRn, a cellular receptor that protects against intracellular degradation. To tailor-design the therapeutic use of albumin, it is crucial to understand how structural alterations in albumin affect FcRn binding and transport properties. In the blood, the last C-terminal residue (L585) of albumin may be enzymatically cleaved. Here we demonstrate that removal of the L585 residue causes structural stabilization in regions of the principal FcRn binding domain and reduces receptor binding. In line with this, a short half-life of only 3.5 days was measured for cleaved albumin lacking L585 in a patient with acute pancreatitis. Thus, we reveal the structural requirement of an intact C-terminal end of albumin for a long plasma half-life, which has implications for design of albumin-based therapeutics., Communications Biology, 3 (1), ISSN:2399-3642

Almost a decade has passed since the approval of belimumab, an mAb directed against B lymphocyte stimulation and the first targeted therapy approved for systemic lupus erythematous (SLE) in over 50 y. Although well tolerated, the efficacy of belimumab remains limited and is not labeled for patients suffering from nephritis, the leading cause of patient mortality. We sought to explore alternative targets of autoreactive B lymphocytes through manipulation of affinity maturation. The BXSB/MpJ mouse, a well-established model of human SLE, develops elevated antinuclear Abs and immune complex–mediated nephritis along with other manifestations of SLE-like disease. To limit interfering with critical background genetics, we used CRISPR-Cas9 to disrupt activation-induced cytidine deaminase (AID; Aicda) directly in BXSB zygotes. Homozygous null mice demonstrated significantly prolonged survival compared with wild-type. Although mice continued to develop plasma cells, splenic follicular structure was restored, and renal pathology was reduced. Mice developed expanded germinal center B lymphocyte populations as in other models of AID deficiency as well as increased populations of CD73+ B lymphocytes. Treatment with the small molecule inhibitor of RAD51, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, resulted in minimal changes in disease markers in BXSB mice. The prolonged survival in AID-deficient BXSB mice appears attributed primarily to the reduced renal pathology, warranting further exploration, as current therapeutics targeting lupus nephritis are limited and, thus, in great demand.

Heightened glycolysis is inherent to immune/inflammatory disorders, but little is known of its role in the pathogenesis of systemic lupus erythematosus (lupus). Here, we profile key autoimmune populations in acute and chronic lupus-prone models and their response to glycolytic inhibition. We demonstrate that glycolysis is specifically required for autoreactive germinal center B cells (GCB), but not for T follicular helper cells (Tfh) to survive. This augmented reliance on glucose oxidation to maintain ATP production in pathogenic GCB renders them highly susceptible to oxidative stress-induced apoptosis triggered by glycolysis blockade via 2-deoxyglucose (2DG). We show that 2DG can preferentially reduce GCB in lupus-prone mice, while sparing other autoreactive populations, including Tfh, but still significantly improving lifespan and kidney function. Furthermore, the subset of GCB expressing B-cell maturation antigen (BCMA) exhibits an exaggerated dependence on glycolysis to sustain their growth. Depletion of these cells with a proliferation-inducing ligand-based CAR T-cells leads to greatly prolonged lifespan of mice with severe autoimmune activation. These results reveal that glycolysis dependent GCB, especially those expressing BCMA, are key lupus mediators and highlight that they can be selectively targeted to improve disease outcomes for lupus patients.

Systemic lupus erythematosus (SLE) is the most common form of lupus; however, only one new drug was approved for SLE in the last 60 years. Inhibition of glycolysis via 2-deoxyglucose (2DG), a competitive inhibitor of glucose-6-phosphate, has recently shown efficacy in attenuating tumor growth and reducing autoimmune pathology; however, the mechanism of this efficacy is somewhat unclear. To elucidate how 2DG longitudinally modulates pathways within the context of systemic lupus erythematosus (SLE), BXSB.Yaa IL15−/−,CD8−/− mice, an SLE model, were treated orally with 2DG for 96-hours or 4-weeks, after which the transcriptional profiles of 9 tissues (heart, hippocampus, hypothalamus, kidney, liver, prefrontal cortex, skeletal muscle, small intestine, and spleen) were analyzed using unsupervised clustering and weighted gene correlation network analysis. Principal component analysis showed mice clustered by tissue. Tissues correlated to B cells and T follicular helper cells grouped together according to their summary gene expression for modules treatment or treatment, in a time dependent manner, and three pathway groups, transcription, immune, and glucose, emerged. However, within each tissue cluster, pathways for each module were uniquely altered by treatment or treatment, in a time dependent manner, according to ANOVA. Furthermore, while most pathways identified were grouped into either immune or metabolic, there was little overlap of specific pathways identified between tissues. These results show that 2DG has a systemic impact altering immune and metabolic pathways, but in a tissue-specific manner. Ongoing work includes identifying genes that are potentially central to pathway alterations for follow-up analysis. Supported by the AAI Intersect Fellowship

FcRn and CD32a form a ternary complex under acidic conditions and act together in determining responses to IgG immune complexes. The human autoimmune disease–associated CD32a-H131 variant more avidly forms this ternary complex, leading to greater FcRn-dependent immune responses to IgG., IgG immune complexes (ICs) promote autoimmunity through binding fragment crystallizable (Fc) γ-receptors (FcγRs). Of these, the highly prevalent FcγRIIa (CD32a) histidine (H)-131 variant (CD32aH) is strongly linked to human autoimmune diseases through unclear mechanisms. We show that, relative to the CD32a arginine (R)-131 (CD32aR) variant, CD32aH more avidly bound human (h) IgG1 IC and formed a ternary complex with the neonatal Fc receptor (FcRn) under acidic conditions. In primary human and mouse cells, both CD32a variants required FcRn to induce innate and adaptive immune responses to hIgG1 ICs, which were augmented in the setting of CD32aH. Conversely, FcRn induced responses to IgG IC independently of classical FcγR, but optimal responses required FcRn and FcγR. Finally, FcRn blockade decreased inflammation in a rheumatoid arthritis model without reducing circulating autoantibody levels, providing support for FcRn’s direct role in IgG IC-associated inflammation. Thus, CD32a and FcRn coregulate IgG IC-mediated immunity in a manner favoring the CD32aH variant, providing a novel mechanism for its disease association.

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers.

Follicular helper T (TFH) cells are expanded in systemic lupus erythematosus, where they are required to produce high affinity autoantibodies. Eliminating TFH cells would, however compromise the production of protective antibodies against viral and bacterial pathogens. Here we show that inhibiting glucose metabolism results in a drastic reduction of the frequency and number of TFH cells in lupus-prone mice. However, this inhibition has little effect on the production of T-cell-dependent antibodies following immunization with an exogenous antigen or on the frequency of virus-specific TFH cells induced by infection with influenza. In contrast, glutaminolysis inhibition reduces both immunization-induced and autoimmune TFH cells and humoral responses. Solute transporter gene signature suggests different glucose and amino acid fluxes between autoimmune TFH cells and exogenous antigen-specific TFH cells. Thus, blocking glucose metabolism may provide an effective therapeutic approach to treat systemic autoimmunity by eliminating autoreactive TFH cells while preserving protective immunity against pathogens., T cell functions depend on distinct metabolic fluxes. Here the authors show different metabolic requirements of humoral responses to self versus microbial antigens: while glucose is dispensable for antiviral Tfh and antibody responses, it is essential to mount these responses against autoantigens.

Targeting the early steps of the glycolysis pathway in cancers is a well-established therapeutic strategy; however, the doses required to elicit a therapeutic effect on the cancer can be toxic to the patient. Consequently, numerous preclinical and clinical studies have combined glycolytic blockade with other therapies. However, most of these other therapies do not specifically target cancer cells, and thus adversely affect normal tissue. Here we first show that a diverse number of cancer models – spontaneous, patient-derived xenografted tumor samples, and xenografted human cancer cells – can be efficiently targeted by 2-deoxy-D-Glucose (2DG), a well-known glycolytic inhibitor. Next, we tested the cancer-cell specificity of a therapeutic compound using the MEC1 cell line, a chronic lymphocytic leukemia (CLL) cell line that expresses activation induced cytidine deaminase (AID). We show that MEC1 cells, are susceptible to 4,4ʹ-Diisothiocyano-2,2ʹ-stilbenedisulfonic acid (DIDS), a specific RAD51 inhibitor. We then combine 2DG and DIDS, each at a lower dose and demonstrate that this combination is more efficacious than fludarabine, the current standard- of- care treatment for CLL. This suggests that the therapeutic blockade of glycolysis together with the therapeutic inhibition of RAD51-dependent homologous recombination can be a potentially beneficial combination for targeting AID positive cancer cells with minimal adverse effects on normal tissue. Implications: Combination therapy targeting glycolysis and specific RAD51 function shows increased efficacy as compared to standard of care treatments in leukemias.

Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4+ T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4+ T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4+ T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4+ T cells were introduced into Rag1−/− mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4+ T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4+ T cells in a nonredundant manner with myeloid/stromal cells.

Albumin has a long serum half-life due to its unique ability to bind the cellular neonatal Fc receptor (FcRn), which provides protection from intracellular degradation. The interaction can be capitalized to improve the efficacy of drugs by extending their serum persistence. However, species-specific binding of albumin to FcRn challenges preclinical development. The goal of this brief review is to provide insights into how FcRn and cross-species binding differences affect the pharmacokinetics of human serum albumin (HSA) in different animal models, and gives an overview of genetically modified mice that may serve as improved models for testing of albumin-based drugs.

Improved mouse models for type 1 diabetes (T1D) therapy development are needed. T1D susceptibility is restored to normally resistant NOD.β2m(−/−) mice transgenically expressing human disease–associated HLA-A*02:01 or HLA-B*39:06 class I molecules in place of their murine counterparts. T1D is dependent on pathogenic CD8(+) T-cell responses mediated by these human class I variants. NOD.β2m(−/−)-A2.1 mice were previously used to identify β-cell autoantigens presented by this human class I variant to pathogenic CD8(+) T cells and for testing therapies to attenuate such effectors. However, NOD.β2m(−/−) mice also lack nonclassical MHC I family members, including FcRn, required for antigen presentation, and maintenance of serum IgG and albumin, precluding therapies dependent on these molecules. Hence, we used CRISPR/Cas9 to directly ablate the NOD H2-K(d) and H2-D(b) classical class I variants either individually or in tandem (cMHCI(−/−)). Ablation of the H2-A(g7) class II variant in the latter stock created NOD mice totally lacking in classical murine MHC expression (cMHCI/II(−/−)). NOD-cMHCI(−/−) mice retained nonclassical MHC I molecule expression and FcRn activity. Transgenic expression of HLA-A2 or -B39 restored pathogenic CD8(+) T-cell development and T1D susceptibility to NOD-cMHCI(−/−) mice. These next-generation HLA-humanized NOD models may provide improved platforms for T1D therapy development.

2-deoxy D-glucose (2DG) was tested for efficacy in treating alopecia areata using the C3H/HeJ skin graft model. 2DG has proven to be efficacious in treatment of various mouse models of autoimmunity with minimal serious side effects noted. This agent has been shown to normalize abnormally activated T-cell populations while also preventing cell surface expression of NKG2D; key factors defining alopecia areata disease progression. Daily oral ingestion of 2DG via drinking water to mice with patchy or diffuse alopecia areata for 16 weeks failed to prevent expansion of alopecia or cause regrowth of hair in treated mice. Histologically, there were no differences between treated and control groups. These results indicate that, while 2DG is effective for some autoimmune diseases, it was not efficacious for the cell-mediated autoimmune mouse disease, alopecia areata.

Summary Interleukin 21 (IL-21) plays key roles in humoral immunity and autoimmune diseases. It is known to function in mature CD4 + T follicular B cell helper (T FH ) cells, but its potential involvement in early T cell ontogeny is unclear. Here, we find that a significant population of newly activated thymic and peripheral CD4 + T cells functionally expresses IL-21 soon after birth. This naturally occurring population, termed natural (n)T H 21 cells, exhibits considerable similarity to mature T FH cells. nT H 21 cells originating and activated in the thymus are strictly dependent on autoimmune regulator (AIRE) and express high levels of NUR77, consistent with a bias toward self-reactivity. Their activation/expansion in the periphery requires gut microbiota and is held in check by FoxP3 + T REG cells. nT H 21 cells are the major thymic and peripheral populations of IL-21 + cells to expand in an IL-21-dependent humoral autoimmune disease. These studies link IL-21 to T cell ontogeny, self-reactivity, and humoral autoimmunity.

Mus pahari is a wild-derived, inbred mouse strain. M. pahari colony managers observed fragility of this strain’s skin resulting in separation of tail skin from the mouse if handled incorrectly. Tail skin tension testing of M. pahari resulted in significantly lowered force threshold for caudal skin rupture and loss in comparison to closely related inbred mouse species and subspecies and even more than a model for junctional epidermolysis bullosa. Histologically, the tail skin separated at the subdermal level with the dermis firmly attached to the epidermis, excluding the epidermolysis bullosa complex of diseases. The dermal collagen bundles were abnormally thickened and branched. Elastin fiber deposition was focally altered in the dermis adjacent to the hair follicle. Collagens present in the skin could not be differentiated between the species in protein gels following digestion with pepsin. Together these data suggest that M. pahari have altered extracellular matrix development resulting in separation of the skin below the level of the dermis with moderate force similar to the African spiny mouse (Acomys spp.).

Therapeutic blockade of FcRn in humans decreases IgG and IgG immune complex levels with the attendant immunologic implications., The neonatal crystallizable fragment receptor (FcRn) functions as an intracellular protection receptor for immunoglobulin G (IgG). Recently, several clinical studies have reported the lowering of circulating monomeric IgG levels through FcRn blockade for the potential treatment of autoimmune diseases. Many autoimmune diseases, however, are derived from the effects of IgG immune complexes (ICs). We generated, characterized, and assessed the effects of SYNT001, a FcRn-blocking monoclonal antibody, in mice, nonhuman primates (NHPs), and humans. SYNT001 decreased all IgG subtypes and IgG ICs in the circulation of humans, as we show in a first-in-human phase 1, single ascending dose study. In addition, IgG IC induction of inflammatory pathways was dependent on FcRn and inhibited by SYNT001. These studies expand the role of FcRn in humans by showing that it controls not only IgG protection from catabolism but also inflammatory pathways associated with IgG ICs involved in a variety of autoimmune diseases.

Inhibition of glycolysis via 2-deoxyglucose (2DG), a competitive inhibitor of glucose-6-phosphate, has recently shown efficacy in attenuating tumor growth and reducing autoimmune pathology; however, the mechanism of this efficacy is somewhat unclear. To elucidate how 2DG longitudinally modulates pathways without the influence of immunity in diseased status, wild-type C57BL/6J mice were treated orally with 2DG for 96-hours or 4-weeks, after which the transcriptional profiles of 9 tissues (heart, hippocampus, hypothalamus, kidney, liver, prefrontal cortex, skeletal muscle, small intestine, and spleen) were analyzed using unsupervised clustering and weighted gene correlation network analysis. Principal component analysis showed mice clustered by tissue. Tissues grouped together according to their summary gene expression for modules correlated to time, treatment and/or tissue, and two pathway groups, immune and metabolic, emerged. However, within each tissue cluster, pathways for each module were uniquely altered by time, treatment and/or tissue, according to ANOVA. Furthermore, while most pathways identified were grouped into either immune or metabolic, there was little overlap of specific pathways identified between tissues. These results show that 2DG has a systemic impact altering immune and metabolic pathways, but in a tissue-specific manner. Ongoing work includes identifying genes that are potentially central to pathway alterations for follow-up analysis.

Background Glucose is a primary substrate for cellular respiration. Glucose utilization increases in highly metabolic cells including activated, proliferating T cells and B cells as well as cancers. Lupus is a disorder in which autoreactive CD4 +T cell and B cells deviate from normal homeostasis by their uncontrolled proliferation and differentiation. Therapeutic limitation of glycolysis is therefore an attractive approach for attenuating such highly energetic, pathogenic processes inherent to lupus. Here we investigate the potential of the classic glycolysis inhibitor, 2-deoxyglucose (2DG), to prevent and reverse severe forms of autoimmune lupus-like disease in mice. As untoward side effects of 2DG therapy have also been reported, we further evaluate potential complications and biomarkers of 2DG therapy. Methods For autoimmune disease studies, the following mouse models for spontaneous lupus-like autoimmune disease were utilized: NZB × NZW F1 (BWF1) females (adult onset) and BXSB.Yaa Cd8-/- Il15-/- males (acute adolescent onset). 2DG was provided ad libitum in acidified drinking water (2DG 2–6 g/L) for intervals of 8 wks. Mice were monitored longitudinally for weight loss, proteinurea, and survival. To assess potential effects of 2DG in non-diseased mice, C57BL6/J mice treated with 2DG and controls were analyzed for a series of metabolic, serum chemistry, cardiac, behavioral, immunological and histopathological phenotypes after short and long term exposure. Results An 8 wk 2DG treatment of young BXSB.Yaa Cd8-/- Il15-/- mice prevented their development of prototypic cellular abnormalities and greatly extended lifespans. Given the strong normalizing effects of 2DG in disease prevention, we performed therapeutic interventions in which 2DG was intermittently supplied to 37 wk old BWF1 mice (figure 1A). This treatment abrogated proteinurea even in heavily diseased mice and extended the lifespan of many to beyond 80 wks of age (figure 1B and 1C). Safety assessments in C57BL6/J mice after 8 wks of treatment did not show any significant cardiac, behavioral, immunological and histopathological abnormalities. Alterations in serum chemistry analytes, including calcium and magnesium, but not markers of ketosis, were evident shortly after and persisted after 2DG exposure. Conclusions The results highlight the potent and remarkable normalizing effect of intermittent 2DG therapy in the prevention and treatment lupus-like autoimmune disease with differing genetic and mechanistic etiologies and in its most precipitous forms. This mode of therapy did not incur untoward side effects. We thus propose that therapeutic inhibition of early steps in glycolysis by 2DG has broad potential for the treatment of lupus as well as related autoimmune disorders.

The K/BxN mouse is a spontaneous model of arthritis driven by T cell receptor transgenic CD4+ T cells from the KRN strain that are activated by glucose-6-phosphate isomerase (GPI) peptides presented by the H-2g7 allele from the NOD strain. It is a model of autoimmune seropositive arthritis because the production of anti-GPI IgG is necessary and sufficient for joint pathology. The production of high levels of anti-GPI IgG requires on the expansion of CD4+ follicular helper T (Tfh) cells. The metabolic requirements of this expansion have never been characterized. Based on the therapeutic effects of the combination of metformin and 2-deoxyglucose (2DG) in lupus models that normalized the expansion of effector CD4+ T cells. We showed that the CD4+ T cells and to a lesser extent, the B cells from K/BxN mice are more metabolically active than the KRN controls. Accordingly, preventive inhibition of glycolysis with 2DG significantly reduced joint inflammation and the activation of both adaptive and innate immune cells, as well as the production of pathogenic autoantibodies. However, contrary to the lupus-prone mice, the addition of metformin had little beneficial effect, suggesting that glycolysis is the major driver of immune activation in this model. We propose that K/BxN mice are another model in which autoreactive Tfh cells are highly glycolytic and that their function can be limited by inhibiting glucose metabolism.

The neonatal Fc receptor (FcRn) has been demonstrated to contribute to a high bioavailability of monoclonal antibodies (mAbs). In this study, we explored the cellular sites of FcRn-mediated protection after subcutaneous (SC) and intravenous (IV) administration. SC absorption and IV disposition kinetics of a mAb were studied in hFcRn transgenic (Tg) bone marrow chimeric mice in which hFcRn was restricted to radioresistant cells or hematopoietic cells. SC bioavailabilities close to 90% were observed in hFcRn Tg mice and chimeric mice with hFcRn expression in hematopoietic cells, whereas SC bioavailabilities were markedly lower when FcRn was missing in hematopoietic cells. Our study demonstrates: 1) FcRn in radiosensitive hematopoietic cells is required for high SC bioavailability, indicating first-pass catabolism after SC administration by hematopoietic cells; 2) FcRn-mediated transcytosis or recycling by radioresistent cells is not required for high SC bioavailability; and 3) after IV administration hematopoietic and radioresistent cells contribute about equally to clearance of the mAb. A pharmacokinetic model was devised to describe a mixed elimination via radioresistent and hematopoietic cells from vascular and extravascular compartments, respectively. Overall, the study indicates a relevant role of hematopoietic cells for first-pass clearance of mAbs after SC administration and confirms their role in the overall clearance of mAbs.

Infection with the helminth Schistosoma mansoni results in hepatointestinal granulomatous inflammation mediated by CD4 T cells directed against parasite eggs. The severity of disease varies greatly in humans and mice; however, the genetic basis of such a heterogenous immune response remains poorly understood. Here we show that, despite their close genetic relationship, C57BL/10SnJ (B10) mice developed significantly more pronounced immunopathology and higher T helper 17 cell responses than C57BL/6J (B6) mice. Similarly, live egg-stimulated B10-derived dendritic cells (DCs) produced significantly more IL-1β and IL-23, resulting in higher IL-17 production by CD4 T cells. Gene expression analysis disclosed a heightened proinflammatory cytokine profile together with a strikingly lower expression of Ym1 in B10 versus B6 mice, consistent with failure of B10 DCs to attain alternative activation. To genetically dissect the differential response, we developed and analyzed congenic mouse strains that capture major regions of allelic variation, and found that the level of inflammation was controlled by a relatively small number of genes in a locus mapping to chromosome 4 117-143 MB. Our study has thus identified novel genomic regions that regulate the severity of the schistosome infection by way of controlling the mode of DC activation and consequent CD4 T-cell subset development.

Serum albumin is the major determinant of blood colloidal osmotic pressure acting as a depot and distributor of compounds including drugs. In humans, serum albumin exhibits an unusually long half-life mainly due to protection from catabolism by neonatal Fc receptor (FcRn)-mediated recycling. These properties make albumin an attractive courier of therapeutically-active compounds. However, pharmaceutical research and development of albumin-based therapeutics has been hampered by the lack of appropriate preclinical animal models. To overcome this, we developed and describe the first mouse with a genetic deficiency in albumin and its incorporation into an existing humanized FcRn mouse model, B6.Cg-Fcgrt(tm1Dcr) Tg(FCGRT)32Dcr/DcrJ (Tg32). Albumin-deficient strains (Alb(-/-)) were created by TALEN-mediated disruption of the albumin (Alb) gene directly in fertilized oocytes derived from Tg32 mice and its non-transgenic background control, C57BL/6J (B6). The resulting Alb(-/-) strains are analbuminemic but healthy. Intravenous administration of human albumin to Tg32-Alb(-/-) mFcRn(-/-) hFcRn(Tg/Tg)) mice results in a remarkably extended human albumin serum half-life of ∼24 days, comparable to that found in humans, and in contrast to half-lives of 2.6-5.8 d observed in B6, B6-Alb(-/-) and Tg32 strains. This striking increase can be explained by the absence of competing endogenous mouse albumin and the presence of an active human FcRn. These novel albumin-deficient models provide unique tools for investigating the biology and pathobiology of serum albumin and are a more appropriate rodent surrogates for evaluating human serum albumin pharmacokinetics and albumin-based compounds.

Select humans and animals control persistent viral infections via adaptive immune responses that include production of neutralizing antibodies. The precise genetic basis for the control remains enigmatic. Here, we report positional cloning of the gene responsible for production of retrovirus-neutralizing antibodies in mice of the I/LnJ strain. It encodes the beta subunit of the non-classical Major Histocompatibility Complex class II (MHC-II)-like molecule H2-O, a negative regulator of antigen presentation. The recessive and functionally null I/LnJ H2-Ob allele supported the production of virus-neutralizing antibodies independently of the classical MHC haplotype. Subsequent bioinformatics and functional analyses of the human H2-Ob homologue, HLA-DOB revealed both loss- and gain-of-function alleles, which could affect the ability of their carriers to control infections with Human Hepatitis B (HBV) and C (HCV) viruses. Thus, understanding of the previously unappreciated role of H2-O (HLA-DO) in immunity to infections may suggest new approaches in achieving neutralizing immunity to viruses.

Albumin and IgG have remarkably long serum half-lives due to pH-dependent FcRn-mediated cellular recycling that rescues both ligands from intracellular degradation. Furthermore, increase in half-lives of IgG and albumin-based therapeutics has the potential to improve their efficacies, but there is a great need for robust methods for screening of relative FcRn-dependent recycling ability. Here, we report on a novel human endothelial cell-based recycling assay (HERA) that can be used for such pre-clinical screening. In HERA, rescue from degradation depends on FcRn, and engineered ligands are recycled in a manner that correlates with their half-lives in human FcRn transgenic mice. Thus, HERA is a novel cellular assay that can be used to predict how FcRn-binding proteins are rescued from intracellular degradation., The development of IgG and albumin-based therapeutics with increased half-lives needs more efficient screening procedures. Here the authors report a human endothelial cell-based recycling assay enabling screening of IgG and albumin variants without chemical labelling and prior to animal testing.

The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood. Here we demonstrate that mice with global or liver-specific FcRn deletion exhibit hypoalbuminemia, albumin loss into the bile, and increased albumin levels in the hepatocyte. In vitro models with polarized cells illustrate that FcRn mediates basal recycling and bidirectional transcytosis of albumin and uniquely determines the physiologic release of newly synthesized albumin into the basal milieu. These properties allow hepatic FcRn to mediate albumin delivery and maintenance in the circulation, but they also enhance sensitivity to the albumin-bound hepatotoxin, acetaminophen (APAP). As such, global or liver-specific deletion of FcRn results in resistance to APAP-induced liver injury through increased albumin loss into the bile and increased intracellular albumin scavenging of reactive oxygen species. Further, protection from injury is achieved by pharmacologic blockade of FcRn–albumin interactions with monoclonal antibodies or peptide mimetics, which cause hypoalbuminemia, biliary loss of albumin, and increased intracellular accumulation of albumin in the hepatocyte. Together, these studies demonstrate that the main function of hepatic FcRn is to direct albumin into the circulation, thereby also increasing hepatocyte sensitivity to toxicity.