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The primary function of tissue factor (TF) resides in the vasculature as a cofactor of blood clotting; however, multiple solid tumors aberrantly express this transmembrane receptor on the cell surface. Here, we developed anti-TF antibody–drug conjugates (ADC) that did not interfere with the coagulation cascade and benchmarked them against previously developed anti-TF ADCs. After screening an affinity-matured antibody panel of diverse paratopes and affinities, we identified one primary paratope family that did not inhibit conversion of Factor X (FX) to activated Factor X (FXa) and did not affect conversion of prothrombin to thrombin. The rest of the antibody panel and previously developed anti-TF antibodies were found to perturb coagulation to varying degrees. To compare the anticancer activity of coagulation-inert and -inhibitory antibodies as ADCs, a selection of antibodies was conjugated to the prototypic cytotoxic agent monomethyl auristatin E (MMAE) through a protease-cleavable linker. The coagulation-inert and -inhibitory anti-TF ADCs both killed cancer cells effectively. Importantly, the coagulation-inert ADCs were as efficacious as tisotumab vedotin, a clinical stage ADC that affected blood clotting, including in patient-derived xenografts from three solid tumor indications with a need for new therapeutic treatments—squamous cell carcinoma of the head and neck (SCCHN), ovarian, and gastric adenocarcinoma. Furthermore, a subset of the anti-TF antibodies could also be considered for the treatment of other diseases associated with upregulation of membranous TF expression, such as macular degeneration. Mol Cancer Ther; 17(11); 2412–26. ©2018 AACR.

Supplementary Table S1. Screening Data; Supplementary Table S2. Binding Data; Supplementary Table S3. TGA Data; Supplementary Table S4. ADC Data With Ranking

Supplementary Materials and Methods

Despite widespread utilization of pneumococcal conjugate vaccines (PCVs) and the resultant disease reduction, the development of PCVs containing additional serotypes remains a public health priority due to serotype replacement and the resultant shift to non-vaccine containing serotypes. However, incorporating additional serotypes to existing PCVs using conventional technologies has proven problematic. Immune responses to individual serotypes have consistently decreased as more polysaccharide-conjugates are added due to carrier suppression. Using our proprietary cell-free protein synthesis (CFPS) platform, we have successfully produced eCRM® based on the CRM197 sequence for use as an enhanced carrier protein to develop a 24-valent PCV. The eCRM carrier protein contains multiple non-native amino acids (nnAAs) located outside of the primary T-cell epitope regions, thereby enabling site-specific covalent conjugation of the pneumococcal polysaccharides to the nnAAs to consistently expose the critical T-cell epitopes. eCRM also serves to reduce structural heterogeneity associated with classic reductive-amination conjugation while promoting formation of the conjugate matrix structures, the hallmark of PCVs. This process serves to increase the overall polysaccharide:protein ratio, enabling the inclusion of more serotypes while minimizing carrier-mediated immunological interference. The aim of this non-clinical study was to construct a 24-valent PCV and evaluate its immunogenicity. Using the XPressCF® CFPS platform, the eCRM carrier protein was separately conjugated through nnAAs to each of the 24 pneumococcal polysaccharides through click chemistry and mixed with aluminum phosphate to produce VAX-24, Vaxcyte's proprietary PCV preclinical candidate. VAX-24, Prevnar13® and Pneumovax®23 were administered to New Zealand White rabbits to compare the resulting opsonophagocytic activity (OPA) and anti-capsular IgG antibodies. VAX-24 showed conjugate-like immune responses to all 24 serotypes based on comparable OPA and IgG responses to Prevnar13 and higher responses than Pneumovax 23. This study demonstrates the utility of site-specific conjugation technology in a preclinical setting and the potential for a PCV with improved serotype coverage.

Objectives We aimed to identify transcriptional gene signatures predictive of clinical response, for pharmacodynamic evaluation, and to provide mechanistic insight into JNJ-55920839, a human IgG1κ neutralizing mAb targeting IFN-α/IFN-ω, in participants with systemic lupus erythematosus (SLE). Methods Blood samples were obtained from SLE participants at baseline and up to Day 130, who received six 10 mg/kg IV doses of JNJ-55920839/placebo every 2 weeks. Participants with mild-to-moderate SLE who achieved clinical responses using SLE Disease Activity Index 2000 Responder Index 4-point change were considered responders. Transcriptional signatures from longitudinally collected blood were generated by RNA-Seq; signatures were generated by microarray from baseline blood samples exposed in vitro to JNJ-55920839 versus untreated. Results Two gene signatures (IFN-I Signaling and Immunoglobulin Immune Response) exhibited pharmacodynamic changes among JNJ-55920839 responders. The Immunoglobulin signature, but not the IFN-I signature, was elevated at baseline in JNJ-55920839 responders. A gene cluster associated with neutrophil-mediated immunity was reduced at baseline in JNJ-55920839 responders, substantiated by lower neutrophil counts in responders. An IFN-I signature was suppressed by JNJ-55920839 in vitro treatment versus untreated blood to a greater extent in responders before in vivo dosing. Conclusions These signatures may enable enrichment for treatment responders when using IFN-I-suppressing treatments in SLE.

Supplemental material, sj-pdf-1-lup-10.1177_0961203321995576 for Novel signatures associated with systemic lupus erythematosus clinical response to IFN-α/-ω inhibition by Loqmane Seridi, Matteo Cesaroni, Ashley Orillion, Jessica Schreiter, Marc Chevrier, Stanley Marciniak, Thi-Sau Migone, William Stohl, Walter Winn Chatham, Richard Alan Furie, Jacqueline Benson and Jarrat Jordan in Lupus

Additional file 1: Figure S1. Analysis of effect of methotrexate on TL1A levels from a second independent patient cohort. TL1A was measured in serum from an independent cohort of patients with RA treated with MTX for 4 months (66 good/moderate EULAR responders, 18 non-responders).

Additional file 2: Figure S2. Micro-CT Scoring system for erosions and joint damage (MCTS). A scoring system was devised for micro-CT measurements of hind paw joint scores incorporating a score for the ankle/tarsus, MTP, and distal joints with a total score of 0–30 points.

Additional file 3: Table S1. Demographic characteristics of SNUH cohort (data shown in Fig. 2 D-F and Fig. 3 B-D). Sr, serum; SF, synovial fluid; ESR, erythrocyte sedimentation rate; hsCRP, high sensitive C-reactive protein; RF, rheumatoid factor. Table S2. Demographic characteristics of patient subgroups from the SERA cohort.

Pre-existing anti-HLA allo-antibodies (allo-Abs) are a major barrier to successful kidney transplantation, resulting in an elevated risk for antibody-mediated rejection (AMR) and eventual graft loss. The cytokine B lymphocyte stimulator (BLyS) promotes B cell maturation and plasma cell survival; consequently, anti-BLyS therapy represents a potential therapeutic opportunity in diminishing pre-existing allo-Abs. Here we report that in our 1-year pilot trial, BLyS neutralization failed to reduce total anti-HLA allo-Ab levels in highly sensitized candidates awaiting kidney transplant in a clinically meaningful way. Additionally, we performed a post hoc analysis using sera from trial candidates which revealed selective depletion of anti-HLA class I and class II Abs in response to belimumab treatment, restricted to certain allele specificities and IgG subclasses. Altogether, we observed that BLyS blockade only results in selective depletion of anti-HLA Abs recognizing a few discrete HLA allele specificities.

Background Due to the diversity of serotypes, exacerbated by the phenomenon of serotype replacement, there remains an unmet medical need for a pneumococcal conjugate vaccine (PCV) containing additional serotypes. Using a cell-free protein synthesis (CFPS) platform to produce an enhanced carrier protein (eCRM®) based on the CRM197 sequence, Vaxcyte is developing a PCV encompassing over 30 serotypes. The eCRM carrier protein contains multiple insertions of the non-native amino acid para-azidomethyl-L-phenylalanine (pAMF) that facilitates site-specific conjugation of the pneumococcal polysaccharides (PS) to eCRM. Unlike conventional methodologies, site-selective conjugation enhances process consistency and increases capacity for inclusion of additional serotypes in a PCV without promoting carrier suppression. Using this platform, the aim of the current study was to employ CFPS technology to construct a 31-valent PCV and evaluate its immunogenicity in New Zealand White (NZW) rabbits. Methods The eCRM carrier protein was individually conjugated to each of 31 selected pneumococcal PSs using copper-free click chemistry to produce 31 Conjugate Drug Substances (DS), which were then mixed with aluminum phosphate to produce the VAX-XP Drug Product. 24 of the DS conjugates in VAX-XP were generated at manufacturing scale. Two doses of VAX-XP were administered to NZW rabbits at 0 and 21 days to assess its ability to elicit anti-capsular IgG antibodies. Additionally, rabbits were also administered either Prevnar13 or a mixture of Pneumovax 23 and 8 incremental PS in isotonic saline, as comparators. Results VAX-XP showed conjugate-like immune responses for all 31 serotypes, as demonstrated by superior responses to PS-based vaccines and comparable responses to Prevnar13. IgG responses for VAX-XP compared with Prevnar13 and Pneumovax 23 at 14 days post dose 2 Conclusion These results demonstrate that increasing the number of pneumococcal serotypes does not result in immunological attenuation in any of the serotypes contained in VAX-XP relative to the current standard of care. Furthermore, the data confirm the scalability and reproducibility of the CFPS platform in the production of VAX-XP conjugates, creating the foundation for a next generation broad-valency PCV. Disclosures Chris Behrens, PhD, Vaxcyte, Inc. (Employee) Jeff Fairman, PhD, Vaxcyte, Inc. (Employee) Paresh Agarwal, PhD, Vaxcyte, Inc. (Employee) Shylaja Arulkumar, MS, Vaxcyte, Inc. (Employee) Sandrine Barbanel, MS, Vaxcyte, Inc. (Employee) Leslie Bautista, n/a, Vaxcyte, Inc. (Employee) Aym Berges, PhD, Vaxcyte, Inc. (Employee) John Burky, BS, Vaxcyte, Inc. (Employee) Peter Davey, MS, Vaxcyte, Inc. (Employee) Chris Grainger, PhD, Vaxcyte, Inc. (Employee) Sherry Guo, PhD, Vaxcyte, Inc. (Employee) Sam Iki, MS, Vaxcyte, Inc. (Employee) Mark Iverson, BS, Vaxcyte, Inc. (Employee) Neeraj Kapoor, PhD, Vaxcyte, Inc. (Employee) Olivier Marcq, PhD, Vaxcyte, Inc. (Employee) Thi-Sau Migone, PhD, Vaxcyte, Inc. (Employee) Lucy Pill, MS, Vaxcyte, Inc. (Employee) Mohammed Sardar, n/a, Vaxcyte, Inc. (Employee) Paul Sauer, MBA, Vaxcyte, Inc. (Employee) James Wassil, MS MBA, Vaxcyte, Inc. (Employee)

Background The tumor necrosis factor (TNF) superfamily cytokine TNF-like protein 1A (TL1A) and its receptor DR3 are essential for diverse animal models of autoimmune disease and may be pathogenic in rheumatoid arthritis (RA). However, the relationship of TL1A to disease duration, activity, and response to anti-TNF and other therapies in RA is not clear. Methods We measured soluble TL1A in synovial fluid (SF), serum, or plasma from RA first-degree relatives (FDRs) and in early RA and established disease. We measured the effects of anti-TNF and methotrexate (MTX) therapy on circulating TL1A from multiple independent RA treatment trials. We also determined the ability of a blocking anti-TL1A antibody to inhibit clinical disease and articular bone destruction in the murine collagen-induced arthritis (CIA) model of human RA. Results Soluble TL1A was specifically elevated in the blood and SF of patients with RA compared to patients with other diseases and was elevated early in disease and in at-risk anti-cyclic citrullinated peptide (CCP) (+) first-degree relatives (FDRs). Therapeutic TNF inhibition reduced serum TL1A in both responders and non-responders, whereas TL1A declined following MTX treatment only in responders. In murine CIA, TL1A blockade was clinically efficacious and reduced bone erosions. Conclusions TL1A is specifically elevated in RA from early in the disease course and in at-risk FDRs. The decline in TL1A after TNF blockade suggests that TL1A levels may be a useful biomarker for TNF activity in RA. These results support the further investigation of the relationship between TL1A and TNF and TL1A blockade as a potential therapeutic strategy in RA.

The primary function of tissue factor (TF) resides in the vasculature as a cofactor of blood clotting; however, multiple solid tumors aberrantly express this transmembrane receptor on the cell surface. Here, we developed anti-TF antibody–drug conjugates (ADC) that did not interfere with the coagulation cascade and benchmarked them against previously developed anti-TF ADCs. After screening an affinity-matured antibody panel of diverse paratopes and affinities, we identified one primary paratope family that did not inhibit conversion of Factor X (FX) to activated Factor X (FXa) and did not affect conversion of prothrombin to thrombin. The rest of the antibody panel and previously developed anti-TF antibodies were found to perturb coagulation to varying degrees. To compare the anticancer activity of coagulation-inert and -inhibitory antibodies as ADCs, a selection of antibodies was conjugated to the prototypic cytotoxic agent monomethyl auristatin E (MMAE) through a protease-cleavable linker. The coagulation-inert and -inhibitory anti-TF ADCs both killed cancer cells effectively. Importantly, the coagulation-inert ADCs were as efficacious as tisotumab vedotin, a clinical stage ADC that affected blood clotting, including in patient-derived xenografts from three solid tumor indications with a need for new therapeutic treatments—squamous cell carcinoma of the head and neck (SCCHN), ovarian, and gastric adenocarcinoma. Furthermore, a subset of the anti-TF antibodies could also be considered for the treatment of other diseases associated with upregulation of membranous TF expression, such as macular degeneration. Mol Cancer Ther; 17(11); 2412–26. ©2018 AACR.

Although antibiotics treat bacteremia in inhalational anthrax, pathogenesis is mainly driven by bacterial exotoxins. Raxibacumab, an IgG1 monoclonal antibody, binds the protective antigen (PA) of Bacillus anthracis , thus blocking toxin effects and leading to improved survival in the rabbit and monkey models of inhalational anthrax. To assess raxibacumab's added benefit over levofloxacin (LVX) alone, rabbits surviving to 84 h after a challenge with 200 times the median (50%) lethal dose of B. anthracis spores were randomized to receive 3 daily intragastric LVX doses of 50 mg/kg of body weight, with the first LVX dose administered just prior to administration of a single intravenous dose of placebo or 40 mg/kg raxibacumab. The percentages of animals alive at 28 days following the last LVX dose were compared between the 2 treatment groups using a two-sided likelihood-ratio chi-square test. The 82% survival rate for the LVX-raxibacumab combination was higher than the 65% survival rate for LVX alone ( P = 0.0874). There were nearly 2-fold fewer deaths for the combination (7 deaths; n = 39) than for LVX alone (13 deaths; n = 37), and the survival time was prolonged for the combination ( P = 0.1016). Toxin-neutralizing-activity titers were similar for both treatment groups, suggesting that survivors in both groups were able to mount a toxin-neutralizing immune response. Microscopic findings considered consistent with anthrax were present in animals that died or became moribund on study in both treatment groups, and there were no anthrax-related findings in animals that survived. Overall, raxibacumab provided a meaningful benefit over antibiotic alone when administered late in the disease course.

Objective To determine the necessity for any individual BAFF receptor in the development of systemic lupus erythematosus (SLE). Methods Bcma-, Taci-, and Br3-null mutations were introgressed into NZM 2328 mice. NZM.Bcma−/−, NZM.Taci−/−, and NZM.Br3−/− mice were evaluated for lymphocyte phenotype and BAFF receptor expression by flow cytometry; for B cell responsiveness to BAFF by in vitro culture; for serum levels of BAFF and total IgG and IgG anti–double-stranded DNA (anti-dsDNA) by enzyme-linked immunosorbent assay; for renal immunopathology by immunofluorescence and histopathology; and for clinical disease. Results BCMA, TACI, and B lymphocyte stimulator receptor 3 (BR3) were not surface-expressed in NZM.Bcma−/−, NZM.Taci−/−, and NZM.Br3−/− mice, respectively. Transitional and follicular B cells from NZM.Br3−/− mice were much less responsive to BAFF than were the corresponding cells from wild-type, NZM.Bcma−/−, or NZM.Taci−/− mice. In comparison with wild-type mice, NZM.Bcma−/− and NZM.Taci−/− mice harbored an increased number of spleen B cells, T cells, and plasma cells, whereas serum levels of total IgG and IgG anti-dsDNA were similar to those in wild-type mice. Despite their paucity of B cells, NZM.Br3−/− mice had an increased number of T cells, and the numbers of plasma cells and levels of IgG anti-dsDNA were similar to those in wild-type mice. Serum levels of BAFF were increased in NZM.Taci−/− and NZM.Br3−/− mice but were decreased in NZM.Bcma−/− mice. Despite their phenotypic differences, NZM.Bcma−/−, NZM.Taci−/−, and NZM.Br3−/− mice had renal immunopathology and clinical disease that were at least as severe as that in wild-type mice. Conclusion Any single BAFF receptor, including BR3, is dispensable for the development of SLE in NZM mice. Development of disease in NZM.Br3−/− mice demonstrates that BAFF–BCMA and/or BAFF–TACI interactions contribute to SLE, and that a profound, life-long reduction in the numbers of B cells does not guarantee protection against SLE.

Objective To determine the role of APRIL in the development of systemic lupus erythematosus (SLE) in mice. Methods Wild-type (WT) NZM 2328, NZM. April–/–, NZM.Baff–/–, and NZM.Baff–/–.April–/– mice were evaluated for lymphocyte phenotype by flow cytometry, for serum total IgG and IgG autoantibody levels by enzyme-linked immunosorbent assay, for glomerular deposition of IgG and C3 by immunofluorescence, for renal changes by histopathology, and for clinical disease by laboratory assessment (severe proteinuria). Results In comparison to WT mice, NZM.April–/– mice harbored increased spleen B cells, T cells, and plasma cells (PCs), increased serum levels of IgG antichromatin antibodies, and decreased numbers of bone marrow (BM) PCs. Glomerular deposition of IgG and C3 was similar in NZM.April–/– mice and WT mice, renal changes on histopathology tended to be more severe in NZM.April–/– mice than in WT mice, and development of clinical disease was identical in NZM.April–/– mice and WT mice. BM (but not spleen) PCs and serum IgG antichromatin and anti–double-stranded DNA antibody levels were lower in NZM.Baff–/–.April–/– mice than in NZM.Baff–/– mice, whereas renal immunopathology in each cohort was equally mild. Conclusion APRIL is dispensable for the development of full-blown SLE in NZM mice. Moreover, the elimination of both APRIL and BAFF had no discernible effect on the development of renal immunopathology or clinical disease beyond that of elimination of BAFF alone. The reduction in BM PCs in hosts doubly deficient in APRIL and BAFF beyond that in hosts deficient only in BAFF raises concern that combined antagonism of APRIL and BAFF may lead to greater immunosuppression without a concomitant increase in therapeutic efficacy.

Background CCR5 is a main co-receptor for HIV, but also homes lymphocytes to sites of inflammation. We hypothesized that inhibition of CCR5 signaling would reduce HIV-associated chronic immune activation. Methods To test this hypothesis, we administered an antagonistic anti-CCR5 monoclonal antibody (HGS101) to five uninfected rhesus macaques (RMs) and monitored lymphocyte dynamics in blood and tissue. Results CCR5 blockade resulted in decreased levels of CCR5+ T cells in blood and, at later timepoints, in lymph nodes. Additionally, the levels of CD25+ T cells increased in lymph nodes, but decreased in blood, bone marrow, and rectal mucosa. Finally, a profile of gene expression from HGS101-treated RMs revealed a subtle, but consistent, in vivo signature of CCR5 blockade that suggests a mild immune-modulatory effect. Conclusions Treatment with anti-CCR5 antibody induces changes in the tissue distribution of CCR5+ and CD25+ T cells that may impact on the overall levels of immune activation during HIV and SIV infection.

The peripheral B cell prosurvival cytokine BAFF/B lymphocyte stimulator (BLyS) has been proposed to participate in the regulation of immunological tolerance. Selective elimination or reconstitution of B cells expressing transgene-encoded, autoreactive BCRs upon systemic BLyS depletion or supplementation, respectively, was observed in two separate studies. Such findings led to a model positing a higher dependency of autoreactive B cells on BLyS. We tested this model by exploiting two targeted IgH transgenic mice (H chain knock-in [HKI]) that produce large numbers of follicular (FO) B cells that are either weakly or strongly autoreactive with nuclear autoantigens. Even though HKI B cells do not exhibit classical features of anergy, we found that mature, naive, autoreactive HKI B cells are outcompeted for representation in the periphery by a polyclonal B cell population. However, this is not due to a higher dependency of HKI B cells on BLyS for survival. Additionally, excess BLyS does not rescue HKI B cells from selective elimination. These findings suggest that some autoreactive FO B cells can fully develop while in competition with non-autoreactive cells for BLyS, but remain at a competitive disadvantage for other trophic factors that regulate peripheral stability. As such, our data indicate the existence of peripheral tolerance mechanisms that regulate the frequency of autoreactive FO B cells independent of the BLyS pathway.

The tumor necrosis factor (TNF)-family cytokine TL1A (TNFSF15) costimulates T cells through its receptor DR3 (TNFRSF25) and is required for autoimmune pathology driven by diverse T-cell subsets. TL1A has been linked to human inflammatory bowel disease (IBD), but its pathogenic role is not known. We generated transgenic mice that constitutively express TL1A in T cells or dendritic cells. These mice spontaneously develop IL-13-dependent inflammatory small bowel pathology that strikingly resembles the intestinal response to nematode infections. These changes were dependent on the presence of a polyclonal T-cell receptor (TCR) repertoire, suggesting that they are driven by components in the intestinal flora. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) were present in increased numbers despite the fact that TL1A suppresses the generation of inducible Tregs. Finally, blocking TL1A-DR3 interactions abrogates 2,4,6 trinitrobenzenesulfonic acid (TNBS) colitis, indicating that these interactions influence other causes of intestinal inflammation as well. These results establish a novel link between TL1A and interleukin 13 (IL-13) responses that results in small intestinal inflammation, and also establish that TL1A-DR3 interactions are necessary and sufficient for T cell-dependent IBD.

Current strategies for immunotherapy after transplantation are primarily T-lymphocyte directed and effectively abrogate acute rejection. However, the reality of chronic allograft rejection attests to the fact that transplantation tolerance remains an elusive goal. Donor-specific antibodies are considered the primary cause of chronic rejection. When naive, alloreactive B-cells encounter alloantigen and are activated, a resilient "sensitized" state, characterized by the presence of high-affinity antibody, is established. Here, we will delineate findings that support transient B-lymphocyte depletion therapy at the time of transplantation to preempt sensitization by eliminating alloreactive specificities from the recipient B-cell pool (ie, "repertoire remodeling"). Recent advances in our understanding of B-lymphocyte homeostasis provide novel targets for immunomodulation in transplantation. Specifically, the tumor necrosis factor-related cytokine BLyS is the dominant survival factor for "tolerance-susceptible" transitional and "preimmune" mature follicular B-cells. The transitional phenotype is the intermediate through which all newly formed B-cells pass before maturing into the follicular subset, which is responsible for mounting an alloantigen-specific antibody response. Systemic BLyS levels dictate the stringency of negative selection during peripheral B-cell repertoire development. Thus, targeting BLyS will likely provide an opportunity for repertoire-directed therapy to eliminate alloreactive B-cell specificities in transplant recipients, a requirement for the achievement of humoral tolerance and prevention of chronic rejection. In this review, the fundamentals of preimmune B-cell selection, homeostasis, and activation will be described. Furthermore, new and current B-lymphocyte-directed therapy for antibody-mediated rejection and the highly sensitized state will be discussed. Overall, our objective is to propose a rational approach for induction of humoral transplantation tolerance by remodeling the primary B-cell repertoire of the allograft recipient.

Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the Gαq subunit of trimeric G proteins (Gnaq−/− mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq−/− B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive. Gnaq−/− chimeric mice rapidly develop arthritis as well as other manifestations of systemic autoimmune disease. Importantly, we demonstrate that the development of the autoreactive B cell compartment is the result of an intrinsic defect in Gnaq−/− B cells, resulting in the aberrant activation of the prosurvival factor Akt. Together, these data show for the first time that signaling through trimeric G proteins is critically important for maintaining control of peripheral B cell tolerance induction and repressing autoimmunity.

Peripheral B-cell numbers are tightly regulated by homeostatic mechanisms that influence the transitional and mature B-cell compartments and dictate the size and clonotypic diversity of the B-cell repertoire. B-lymphocyte stimulator (BLyS, a trademark of Human Genome Sciences, Inc.) plays a key role in regulating peripheral B-cell homeostasis. CD22 also promotes peripheral B-cell survival through ligand-dependent mechanisms. The B-cell subsets affected by the absence of BLyS and CD22 signals overlap, suggesting that BLyS- and CD22-mediated survival are intertwined. To examine this, the effects of BLyS insufficiency following neutralizing BLyS mAb treatment in mice also treated with CD22 ligand-blocking mAb were examined. Combined targeting of the BLyS and CD22 survival pathways led to significantly greater clearance of recirculating bone marrow, blood, marginal zone and follicular B cells than either treatment alone. Likewise, BLyS blockade further reduced bone marrow, blood and spleen B-cell numbers in CD22(-/-) mice. Notably, BLyS receptor expression and downstream signaling were normal in CD22(-/-) B cells, suggesting that CD22 does not directly alter BLyS responsiveness. CD22 survival signals were likewise intact in the absence of BLyS, as CD22 mAb treatment depleted blood B cells from mice with impaired BLyS receptor 3 (BR3) signaling. Finally, enforced BclxL expression, which rescues BR3 impairment, did not affect B-cell depletion following CD22 mAb treatment. Thus, the current studies support a model whereby CD22 and BLyS promote the survival of overlapping B-cell subsets but contribute to their maintenance through independent and complementary signaling pathways.

Inhalational anthrax caused by Bacillus anthracis is associated with high mortality primarily due to toxin-mediated injury. Raxibacumab is a human IgG1lambda monoclonal antibody directed against protective antigen, a component of the anthrax toxin.We evaluated the efficacy of raxibacumab as a prophylactic agent and after disease onset in a total of four randomized, placebo-controlled studies conducted in rabbits and monkeys. Animals were exposed to an aerosolized target exposure of B. anthracis spores that was approximately 100 times (in the prophylactic studies) and 200 times (in the therapeutic-intervention studies) the median lethal dose. In the therapeutic-intervention studies, animals were monitored for the onset of symptoms. Animals with detectable protective antigen in serum, a significant increase in temperature, or both received a single intravenous bolus of placebo or raxibacumab at a dose of either 20 mg per kilogram of body weight or 40 mg per kilogram. The primary end point was survival at day 14 (in rabbits) or at day 28 (in monkeys). Safety studies were conducted with intravenous raxibacumab (40 mg per kilogram) in 333 healthy human volunteers.In both rabbits and monkeys, the time to detection of protective antigen correlated with the time to bacteremia (r=0.9, P0.001). In the therapeutic-intervention studies, the survival rate was significantly higher among rabbits that received raxibacumab at a dose of 40 mg per kilogram (44% [8 of 18]) than among rabbits that received placebo (0% [0 of 18]; P=0.003). Raxibacumab treatment also significantly increased survival in monkeys (64% [9 of 14], vs. 0% [0 of 12] with placebo; P0.001). In human subjects, intravenous raxibacumab at a dose of 40 mg per kilogram had a half-life of 20 to 22 days and provided a maximum concentration of the drug in excess of levels that are protective in animals. Concentrations of raxibacumab provide a surrogate end point that should be predictive of clinical benefit.A single dose of raxibacumab improved survival in rabbits and monkeys with symptomatic inhalational anthrax. (ClinicalTrials.gov number, NCT00639678.)

The pharmacodynamics of albinterferon alfa-2b (alb-IFN), a novel recombinant protein consisting of interferon-α-2b genetically fused to human albumin, was evaluated in patients with chronic hepatitis C with a previous non-response to interferon-α-based therapy. B-lymphocyte stimulator (BLyS) is an essential in vivo regulator of B-lymphocyte homeostasis. This analysis examined the relationship between serum BLyS level and virologic response across a range of alb-IFN doses. In all, 115 patients were randomized initially to three alb-IFN treatment arms (900 and 1200 µg every two weeks [q2wk], and 1200 µg every four weeks) with weight-based ribavirin, followed by sequential enrollment in two higher dose arms (1500 and 1800 µg q2wk). Serum BLyS level was assessed by enzyme-linked immunosorbent assay. Serum BLyS levels at baseline were lower in African-Americans (P 0.001). Significant BLyS inductions were observed at weeks 12 and 24 versus pretreatment; in general, serum BLyS levels returned to pretreatment levels following treatment completion. Induction of BLyS was greater in the highest dose group; a significant dose-response trend was observed at weeks 12 (P = 0.002) and 24 (P 0.001), as well as a significant time trend, with further BLyS induction increases at week 24 versus 12 (P 0.001). Week 24 BLyS level change correlated with hepatitis C virus RNA reduction (r = -0.28; P = 0.006), driven primarily by patients with BLyS increases 400%, but did not correlate with sustained virologic response. Higher alb-IFN doses demonstrated dose-related BLyS increases, although the correlation with virologic response was modest.

B lymphocytes are required for the pathogenesis of autoimmune diabetes in NOD mice. Previous studies established that a lymphopenic transitional (TR) B cell compartment reduces the competitive constraint on the entry of newly emerging TR B cells into the splenic follicle (FO), thereby disrupting a peripheral negative selection checkpoint in NOD mice. Thus, development of clinically feasible immunotherapeutic approaches for restoration of appropriate negative selection is essential for the prevention of anti-islet autoimmunity. In this study we hypothesized that in vivo neutralization of the B lymphocyte stimulator (BLyS/BAFF) may enhance the stringency of TR→FO selection by increasing TR B cell competition for follicular entry in NOD mice. This study demonstrated that in vivo BLyS neutralization therapy leads to the depletion of follicular and marginal zone B lymphocytes. Long-term in vivo BLyS neutralization caused an increased TR:FO B cell ratio in the periphery indicating a relative resistance to follicular entry. Moreover, in vivo BLyS neutralization: 1) restored negative selection at the TR→FO checkpoint, 2) abrogated serum insulin autoantibodies, 3) reduced the severity of islet inflammation, 4) significantly reduced the incidence of spontaneous diabetes, 5) arrested the terminal stages of islet cell destruction, and 6) disrupted CD4 T cell activation in NOD mice. Overall, this study demonstrates the efficacy of B lymphocyte-directed therapy via in vivo BLyS neutralization for the prevention of autoimmune diabetes.

Objective: To assess the effects of tumour necrosis factor (TNF) antagonist therapy on B lymphocyte stimulator (BLyS) expression in patients with rheumatoid arthritis (RA). Methods: Blood from 38 patients with RA from a single centre was collected prior to and following initiation of TNF antagonist therapy. Plasma BLyS protein levels, blood leukocyte BLyS mRNA levels and disease activity were longitudinally monitored. Twelve patients with RA who either refused or were felt not to be candidates for TNF antagonist therapy and five normal healthy volunteers served as TNF antagonist-naive controls. Results: Baseline plasma BLyS protein levels, but not blood leukocyte BLyS mRNA levels, were elevated in patients with RA. Plasma BLyS protein levels declined following initiation of TNF antagonist therapy in good responders (GR) to TNF antagonist therapy but not in poor responders (PR). By contrast, the erythrocyte sedimentation rate (ESR) declined in response to TNF antagonist therapy in GR and PR. TNF antagonist therapy did not promote change in blood leukocyte BLyS mRNA levels in either GR or PR, suggesting that the TNF antagonist-associated changes in circulating BLyS protein levels reflected changes in local BLyS production in the affected joints rather than changes in systemic BLyS production. BLyS expression did not change over time in either the normal or RA control groups. Conclusions: A good clinical response to TNF antagonist therapy in patients with RA is associated with a decline in plasma BLyS protein levels. Increased BLyS expression in affected joints may contribute to ongoing disease activity, and reduction of such expression may help promote a favourable clinical response to TNF antagonist therapy.

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.

DR3 is a death domain-containing receptor that is upregulated during T cell activation and whose overexpression induces apoptosis and NF-kappaB activation in cell lines. Here we show that an endothelial cell-derived TNF-like factor, TL1A, is a ligand for DR3 and decoy receptor TR6/DcR3 and that its expression is inducible by TNF and IL-1alpha. TL1A induces NF-kappaB activation and apoptosis in DR3-expressing cell lines, while TR6-Fc protein antagonizes these signaling events. Interestingly, in T cells, TL1A acts as a costimulator that increases IL-2 responsiveness and secretion of proinflammatory cytokines both in vitro and in vivo. Our data suggest that interaction of TL1A with DR3 promotes T cell expansion during an immune response, whereas TR6 has an opposing effect.

B lymphocyte stimulator (BLyS) is a novel member of the TNF family of proteins expressed by myeloid cells as membrane-bound and soluble forms. BLyS was shown to act specifically on B cells, inducing proliferation and immunoglobulin production both in vitro and in vivo. The present study was undertaken to characterize binding of radiolabeled BLyS to its cognate receptor on human B lymphocytes and examine intracellular events initiated by BLyS binding. Similar to other TNF family members, BLyS is present in solution as a homotrimer as determined by gel filtration chromatography and light scattering analysis. BLyS binding to B cells is specific as other TNF family members tested did not compete for 125 I-BLyS binding. Analysis of equilibrium binding of 125 I-labeled BLyS to purified human tonsillar B cells demonstrated saturable binding. Scatchard analysis of the binding data revealed a single class of high-affinity binding on human B cells with approximately 2600 binding sites per cell and an apparent dissociation constant (K D ) of about 0.1 nM. In addition we report that BLyS binding to B cells results in the activation of NF-κB and the Ets family transcription factor, ELF-1, and in the induction of mRNA for Polo-like kinase (PLK).

B-lymphocyte stimulator (BLyS) is a recently identified novel member of the tumor necrosis factor ligand superfamily shown to exist in a membrane-bound and soluble form. BLyS was found to be specifically expressed on cells of myeloid lineage and to selectively stimulate B-lymphocyte proliferation and immunoglobulin production. The expression of a cytokine involved in potentiation of humoral immune responses, such as BLyS, is expected to be strictly controlled. The goal of the present study was to examine regulation of BLyS levels in monocytic cells in response to cytokines and during their differentiation to macrophages and dendritic cells. The presence of BLyS on the cell surface and in the culture medium of both normal blood monocytes and on tumor cells of myelomonocytic origin was demonstrated. BLyS gene expression and levels of membrane-associated and soluble BLyS were found to be regulated by cytokines, in particular interferon (IFN)-γ and to a lesser extent interleukin-10 (IL-10). The expression of BLyS on monocyte membranes was retained following differentiation into macrophages, but detection on the surface of monocyte-derived dendritic cells required stimulation with IFN-γ. Both IFN-γ and IL-10 enhanced the release of soluble BLyS that was active in B-cell proliferation assays. Cells transfected with BLyS complementary DNA mutated in a predicted cleavage site failed to release BLyS into the culture medium, thereby suggesting that soluble BLyS was derived from the membrane form. These results provide further support for an important role for BLyS expressed in myeloid cells in B-cell expansion and antibody responses.

An expression cloning approach was employed to identify the receptor for B-lymphocyte stimulator (BLyS) and identified the tumor necrosis factor receptor superfamily member TACI as a BLyS-binding protein. Expression of TACI in HEK293T cells confers on the cells the ability to bind BLyS with subnanomolar affinity. Furthermore, a TACI-Fc fusion protein recognizes both the cleaved, soluble form of BLyS as well as the membrane BLyS present on the cell surface of a recombinant cell line. TACI mRNA is found predominantly in B-cells and correlates with BLyS binding in a panel of B-cell lines. We also demonstrate that TACI interacts with nanomolar affinity with the BLyS-related tumor necrosis factor homologue APRIL for which no clear in vivo role has been described. BLyS and APRIL are capable of signaling through TACI to mediate NF-kappaB responses in HEK293 cells. We conclude that TACI is a receptor for BLyS and APRIL and discuss the implications for B-cell biology.

CIS is a cytokine-induced SH2-containing protein that was originally cloned as an interleukin (IL)-3-inducible gene. CIS is known to associate with the IL-3 receptor beta chain and erythropoietin receptor and to inhibit signaling mediated by IL-3 and erythropoietin. We now demonstrate that CIS also interacts with the IL-2 receptor beta chain (IL-2Rbeta). This interaction requires the A region of IL-2Rbeta (residues 313-382), which also mediates the association of IL-2Rbeta with Lck and Jak3. Correspondingly, CIS inhibits functions associated with both of these kinases: Lck-mediated phosphorylation of IL-2Rbeta and IL-2-mediated activation of Stat5. Thus, we demonstrate that CIS can negatively control at least two independent IL-2 signaling pathways. Although a functional SH2 binding domain of CIS was not required for its interaction with IL-2Rbeta in vitro, its phosphotyrosine binding capability was essential for the inhibitory action of CIS. On this basis, we have generated a mutant form of CIS protein with an altered SH2 domain that acts as a dominant negative and should prove useful in further understanding CIS action.

Phosphatidylinositol 3-kinase (PI 3-K) plays an important role in signaling via a wide range of receptors such as those for antigen, growth factors, and a number of cytokines, including interleukin-2 (IL-2). PI 3-K has been implicated in both IL-2-induced proliferation and prevention of apoptosis. A number of potential mechanisms for the recruitment of PI 3-K to the IL-2 receptor have been proposed. We now have found that tyrosine residues in the IL-2 receptor beta chain (IL-2Rbeta) are unexpectedly not required for the recruitment of the p85 component of PI 3-K. Instead, we find that Jak1, which associates with membrane-proximal regions of the IL-2Rbeta cytoplasmic domain, is essential for efficient IL-2Rbeta-p85 interaction, although some IL-2Rbeta-p85 association can be seen in the absence of Jak1. We also found that Jak1 interacts with p85 in the absence of IL-2Rbeta and that IL-2Rbeta and Jak1 cooperate for the efficient recruitment and tyrosine phosphorylation of p85. This is the first report of a PI 3-K-Jak1 interaction, and it implicates Jak1 in an essential IL-2 signaling pathway distinct from the activation of STAT proteins.

The interleukin-2 receptor alpha chain (IL-2Ralpha) is potently induced by antigens, mitogens, and certain cytokines that include IL-2 itself. This induction leads to the formation of high affinity IL-2 receptors when IL-2Ralpha is co-expressed with the beta (IL-2Rbeta) and gamma (gammac) chains of this receptor. We investigated the signaling pathways mediating IL-2-induced IL-2Ralpha mRNA expression using 32D myeloid progenitor cells stably transfected with either wild type IL-2Rbeta or mutants of IL-2Rbeta containing tyrosine to phenylalanine substitutions. Of the six cytoplasmic tyrosines in IL-2Rbeta, we have found that only the two tyrosines that mediate Stat5 activation (Tyr-392 and Tyr-510) contribute to IL-2-induced IL-2Ralpha gene expression and that either tyrosine alone is sufficient for this process. Interestingly, the IL-7 receptor contains a tyrosine (Tyr-429)-based sequence resembling the motifs encompassing Tyr-392 and Tyr-510 of IL-2Rbeta. Further paralleling the IL-2 system, IL-7 could activate Stat5 and drive expression of IL-2Ralpha mRNA in 32D cells transfected with the human IL-7R. However, IL-3 could not induce IL-2Ralpha mRNA in 32D cells, despite its ability to activate Stat5 via the endogenous IL-3 receptor. Moreover, the combination of IL-3 and IL-2 could not "rescue" IL-2Ralpha mRNA expression in cells containing an IL-2Rbeta mutant with phenylalanine substitutions at Tyr-392 and Tyr-510. These data suggest that Tyr-392 and Tyr-510 couple to an additional signaling pathway beyond STAT protein activation in IL-2-mediated induction of the IL-2Ralpha gene.

The development of murine plasma cell tumors induced by raf/myc containing retroviruses is facilitated by T cells and completely dependent on IL-6. To determine whether kinases with differing specificities reflect alternative biochemical pathways in B cell tumorigenesis, we have employed an abl/myc containing retrovirus to assess neoplastic development. In contrast with raf/myc, abl/myc disease is T cell and IL-6 independent. An examination of the IL-6 signal transduction pathway reveals that this pathway, as defined by activation of Stat3, is inducible by IL-6 in raf/myc tumors but constitutively activated in abl/myc tumors. These findings provide a mechanism for the derivation of cytokine-independent plasma cell tumors and suggest that both IL-6-dependent and independent tumors may arise in vivo depending on the particular mutational events incurred during tumorigenesis.

One of the earliest events induced by interleukin 2 (IL-2) is tyrosine phosphorylation of cellular proteins, including the IL-2 receptor beta chain (IL-2Rbeta). Simultaneous mutation of three tyrosines (Y338, Y392, and Y510) in the IL-2Rbeta cytoplasmic domain abrogated IL-2-induced proliferation, whereas mutation of only Y338 or of Y392 and Y510 inhibited proliferation only partially. While Y392 and Y510 were critical for IL-2-induced activation of signal transducers and activators of transcription (STAT proteins), Y338 was required for Shc-IL-2Rbeta association and for IL-2-induced tyrosine phosphorylation of Shc. Thus, activation of both Jak-STAT and Shc-coupled signaling pathways requires specific IL-2Rbeta tyrosines that together act in concert to mediate maximal proliferation. In COS-7 cells, overexpression of Jak1 augmented phosphorylation of Y338 as well as Y392 and Y510, suggesting that the role for this Jak kinase may extend beyond the Jak-STAT pathway.

To understand the molecular bases for cytokine redundancy and plelotropy, we have compared the Stat proteins activated in peripheral blood lymphocytes (PBLs) by cytokines with shared and distinct actions. Interleukin-2 (IL-2) rapidly activated Stat5 in fresh PBL, and Stat3 and StatS in preactivated PBL. IL-7 and IL-15 induced the same complexes as IL-2, a feature explained by the existence of similar tyrosine-phosphorylated motifs in the cytoplasmic domains of IL-2RS and IL-7R that can serve as docking sites for Stat proteins. IL-13 induced the same complexes as IL-4, a finding explained by our studies implicating IL-4R as a shared component of the receptors. These studies demonstrate that a single cytokine can activate different combinations of Stat proteins under different physiological conditions, and also indicate two mechanisms by which distinct cytokines can activate the same Stat protein.

Objective.In patients with systemic lupus erythematosus (SLE), evidence suggests that most vaccines (except live-virus vaccines) are safe, although antibody response may be reduced. This substudy from the phase III, randomized, double-blind, placebo-controlled BLISS-76 trial evaluated the effects of belimumab on preexisting antibody levels against pneumococcal, tetanus, and influenza antigens in patients with SLE.Methods.In BLISS-76, patients with autoantibody-positive, active SLE were treated with placebo or belimumab 1 or 10 mg/kg every 2 weeks for 28 days and every 28 days thereafter, plus standard SLE therapy, for 76 weeks. This analysis included a subset of patients who had received pneumococcal or tetanus vaccine within 5 years or influenza vaccine within 1 year of study participation. Antibodies to vaccine antigens were tested at baseline and Week 52, and percentage changes in antibody levels from baseline and proportions of patients maintaining levels at Week 52 were assessed. Antibody titers were also assessed in a small number of patients vaccinated during the study.Results.Consistent with preservation of the memory B cell compartment with belimumab treatment, the proportions of patients maintaining antibody responses to pneumococcal, tetanus, and influenza antigens were not reduced. In a small group receiving influenza vaccine on study, antibody responses were frequently lower with belimumab, although titer levels were > 1:10 in all patients treated with 10 mg/kg and in the majority treated with 1 mg/kg.Conclusion.Treatment with belimumab did not affect the ability of patients with SLE to maintain antibody titers to previous pneumococcal, tetanus, and influenza immunizations. [ClinicalTrials.gov registration number NCT 00410384]

To assess the effects of the B lymphocyte stimulator (BLyS)-specific inhibitor belimumab on immunologic biomarkers, including B cell and T cell populations, and maintenance of antibody titers to prior vaccines in autoantibody-positive systemic lupus erythematosus (SLE) patients.Pooled data from 2 phase III trials, the Study of Belimumab in Subjects with SLE 52-week (BLISS-52) and 76-week (BLISS-76) trials, comparing belimumab 1 mg/kg or 10 mg/kg versus placebo (plus standard SLE therapy for each group) were analyzed for changes in autoantibody, immunoglobulin, and complement levels. BLISS-76 patients were also analyzed for changes in B cell and T cell populations and effects on prior vaccine-induced antibody levels.Belimumab-treated patients experienced significant sustained reductions in IgG and autoantibodies and improvement in C3/C4 levels, resulting in greater positive-to-negative conversion rates for IgG anti-double-stranded DNA (anti-dsDNA), anti-Sm, anticardiolipin, and anti-ribosomal P autoantibodies and normalization of hypergammaglobulinemia and low C3/C4 levels. Belimumab-treated patients experienced significant decreases in the numbers of naive and activated B cells, as well as plasma cells, whereas memory B cells and T cell populations did not decrease. Belimumab did not substantially affect preexisting antipneumococcal or anti-tetanus toxoid antibody levels. Post hoc analysis showed greater reductions in SLE disease activity and the risk of severe flares in patients treated with belimumab 10 mg/kg (P≤0.01) who were anti-dsDNA positive and had low C3/C4 levels at baseline. Normalization of the C3 or anti-dsDNA level by 8 weeks, irrespective of therapy, was predictive of a reduced risk of severe flare over 52 weeks.Belimumab appears to promote normalization of serologic activity and reduce BLyS-dependent B cell subsets in serologically and clinically active SLE. Greater serologic activity may predict a better treatment response to belimumab.

Background. Immunologic rejection is a major barrier to successful long-term outcomes in clinical transplantation. The importance of B lymphocytes—and their secretory products, alloantibodies—in the pathogenesis of allograft rejection is accepted. Furthermore, it is now clear that the dominant regulator of peripheral B-cell homeostasis and tolerance is the B-Lymphocyte Stimulator (BLyS), also referred to as the B-cell activating factor (BAFF). Recently, a novel class of clinical immunotherapeutic agents specific for BLyS, and its family of cytokines, has emerged for the treatment of B-cell-mediated diseases. In this study, we demonstrate the potential utility of BLyS-directed immunotherapy in preventing allograft rejection using a murine islet transplantation model. Methods. A transient period of mature peripheral B-cell depletion was induced by means of in vivo BLyS neutralization using a murine analog of the monoclonal antibody, Benlysta. Subsequently, fully major histocompatibility complex-mismatched islets were transplanted into naive diabetic mice followed by a short course of rapamycin. Results. After BLyS neutralization, indefinite islet allograft survival was achieved. Induction therapy with rapamycin was necessary, but not sufficient, for the achievement of this long-term graft survival. The tolerant state was associated with (1) abrogation of the donor-specific antibody response, (2) transient preponderance of immature/transitional B cells in all lymphoid organs, (3) impaired CD4 T-cell activation during the period of B-cell depletion, and (4) presence of a “regulatory” cytokine milieu. Conclusions. In vivo BLyS neutralization effectively induces humoral tolerance and promotes long-term islet allograft survival in mice. Therefore, B-lymphocyte-directed immunotherapy targeting the homeostatic regulator, BLyS, may be effective in promoting transplantation tolerance.

Mortality in patients with shock during the 2001 US anthrax outbreak was high [1, 2]. Although the mechanisms and optimal management of this shock remain unclear, the risk of anthrax persists, as demonstrated by recent cases among injection drug abusers in Europe [3, 4]. Seventeen of 52 patients from this outbreak died, many with progressive shock despite standard intensive care unit (ICU) support. Anthrax bacilli produce 2 toxins: lethal toxin (LT) and edema toxin (ET) [5, 6]. Each is composed of a toxic moiety (lethal factor [LF] or edema factor [EF], respectively) and protective antigen (PA), a component necessary for LF or EF uptake by host cells. LF is a zinc endopeptidase that cleaves mitogen-activated protein kinase kinases (MAPKK), whereas EF is a calmodulin-dependent adenyl cyclase. Of the two, LT is the more lethal and thought to play a central role in the pathogenesis of anthrax shock [7–20]. Despite the contribution of LT-induced shock to the pathogenesis of anthrax, its treatment has received little study. Conventional therapy for septic shock (which would include hemodynamic support with fluid and vasopressor administration titrated to physiologic endpoints like mean arterial pressure) has not been investigated in models of anthrax. In rats, fixed (nontitrated) doses of either normal saline or norepinephrine (NE) administered as 24-hour infusions were beneficial against lipopolysaccharide (LPS) or Escherichia coli challenge, but not LT challenge [20, 21]. Notably, however, inability to titrate these therapies in the LT-challenged rodent model may have produced adverse effects that limited benefit. Besides hemodynamic support, toxin inhibition has also been proposed for the treatment of life-threatening anthrax infection. Both polyclonal (Anthrax Immune Globulin Intravenous [AIGIV], Cangene Corp, Winnipeg, Canada) and monoclonal (raxibacumab; Human Genome Sciences, Rockville, MD) antibody preparations, directed in part or solely against PA, have been added to the US Strategic National Stockpile. However, whether such preparations have additive benefit when combined with conventionally titrated hemodynamic support is not clear [21–27]. Defining the effects of titrated hemodynamic support, alone or together with protective antigen–directed monoclonal antibody (PA-mAb), for LT-associated shock is important. We previously developed a sedated, instrumented, and mechanically ventilated canine model in which LT, infused over 24 hours to simulate toxin release during infection, produced progressive hypotension and organ dysfunction [16]. We employed this model to investigate the effects of titrated hemodynamic support and PA-directed monoclonal antibody (raxibacumab) alone or together, for LT-induced shock. Although hemodynamic support was available (if indicated) from the start of toxin infusion, PA-mAb was administered either early at the initiation of toxin (0 hours), or later at 9 or 12 hours to investigate the effect of delayed treatment as might occur clinically.

Transplantation tolerance remains an elusive goal as B-cell-initiated chronic humoral rejection evades current immunosuppression. B-cell-directed therapy is thus emerging as a key component in achieving transplantation tolerance and long-term graft survival. Here, we propose strategies of B-cell repertoire remodeling to achieve humoral unresponsiveness to donor antigens with implementation of fundamental B-cell immunobiology and use of newly developed B-cell-directed agents.

Mortality among patients with shock during the 2001 US anthrax outbreak was high [1, 2]. Although the mechanisms and optimal management of this shock remain unclear, anthrax risk persists, as demonstrated by recent cases of highly fatal soft-tissue infection among intravenous drug users in the United Kingdom and Europe [3, 4]. Lethal (LeTx) and edema (ETx) toxins are important in the pathogenesis of anthrax [5, 6]. Both are binary-type toxins that, in addition to lethal factor and edema factor, include protective antigen (PA), the component necessary for binding and uptake by host cells. Lethal factor is a zinc endopeptidase that cleaves mitogenactivated protein kinase kinases, and edema factor is a potent calmodulin-dependent adenyl cyclase. Despite the importance of LeTx and ETx during anthrax infection, there has been limited characterization of their cardiovascular effects [6–9]. Most studies are in small animals, in which measurement of cardiac preload, afterload, and output are difficult and monitoring is of limited duration. The effect of cardiovascular changes with toxin on noncardiac organ dysfunction has also not been assessed. Incomplete understanding of the pathophysiologic effects of LeTx and ETx hampers the development of beneficial supportive strategies. Therefore, in the present investigation recombinant LeTx, ETx, or control challenge was infused over 24 h in sedated and instrumented canines receiving mechanical ventilation; canines are an anthrax-susceptible species [10]. Cardiopulmonary, renal, and hepatic measurements were performed up to 72 h after completion of toxin infusions. To define the influence of fluid administration on the hemodynamic effects of the toxins, measurements were repeated daily after volume loading. An initial study (study 1) investigated and compared the individual effects of similarly lethal dose ranges of each toxin, and 50% lethal doses (LD50) were determined. A second study (study 2) investigated the effects of an LD50 of LeTx and an equimolar dose of ETx administered alone and together.