Characterisation of anifrolumab, a fully human anti-interferon receptor antagonist antibody for the treatment of systemic lupus erythematosus.

OBJECTIVE: We investigated the mechanistic and pharmacological properties of anifrolumab, a fully human, effector-null, anti-type I interferon (IFN) alpha receptor 1 (IFNAR1) monoclonal antibody in development for SLE. METHODS: IFNAR1 surface expression and internalisation on human monocytes before and after exposure to anifrolumab were assessed using confocal microscopy and flow cytometry. The effects of anifrolumab on type I IFN pathway activation were assessed using signal transducer and activator of transcription 1 (STAT1) phosphorylation, IFN-stimulated response element-luciferase reporter cell assays and type I IFN gene signature induction. The ability of anifrolumab to inhibit plasmacytoid dendritic cell (pDC) function and plasma cell differentiation was assessed by flow cytometry and ELISA. Effector-null properties of anifrolumab were assessed in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays with B cells. RESULTS: Anifrolumab reduced cell surface IFNAR1 by eliciting IFNAR1 internalisation. Anifrolumab blocked type I IFN-dependent STAT1 phosphorylation and IFN-dependent signalling induced by recombinant and pDC-derived type I IFNs and serum of patients with SLE. Anifrolumab suppressed type I IFN production by blocking the type I IFN autoamplification loop and inhibited proinflammatory cytokine induction and the upregulation of costimulatory molecules on stimulated pDCs. Blockade of IFNAR1 suppressed plasma cell differentiation in pDC/B cell co-cultures. Anifrolumab did not exhibit CDC or ADCC activity. CONCLUSIONS: Anifrolumab potently inhibits type I IFN-dependent signalling, including the type I IFN autoamplification loop, and is a promising therapeutic for patients with SLE and other diseases that exhibit chronic dysfunctional type I IFN signalling.

Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging.

Regulatory authorities require that cell lines used in commercial production of recombinant proteins must be derived from a single cell progenitor or clone. The limiting dilution method of cell cloning required multiple rounds of low-density cell plating and microscopic observation of a single cell in order to provide evidence of monoclonality. Other cloning methods rely on calculating statistical probability of monoclonality rather than visual microscopic observation of cells. We have combined the single cell deposition capability of the Becton Dickinson Influx™ cell sorter with the microscopic imaging capability of the SynenTec Cellavista to create a system for producing clonal production cell lines. The efficiency of single cell deposition by the Influx™ was determined to be 98% using fluorescently labeled cells. The centrifugal force required to settle the deposited cells to the bottom of the microplate well was established to be 1,126g providing a 98.1% probability that all cells will be in the focal plane of the Cellavista imaging system. The probability that a single cell was deposited by the cell sorter combined with the probability of every cell settling into the focal plane of the imager yield a combined >99% probability of documented monoclonality.

The plasma cell signature in autoimmune disease.

OBJECTIVE: Production of pathogenic autoantibodies by self-reactive plasma cells (PCs) is a hallmark of autoimmune diseases. We undertook this study to investigate the prevalence of PCs and their relationship to known pathogenic pathways to increase our understanding of the role of PCs in disease progression and treatment response. METHODS: We developed a sensitive gene expression-based method to overcome the challenges of measuring PCs using flow cytometry. Whole-genome microarray analysis of sorted cellular fractions identified a panel of genes, IGHA1, IGJ, IGKC, IGKV4-1, and TNFRSF17, expressed predominantly in PCs. The sensitivity of the PC signature score created from the combined expression levels of these genes was assessed through ex vivo experiments with sorted cells. This PC gene expression signature was used for monitoring changes in PC levels following anti-CD19 therapy, for evaluating the relationship between PCs and other autoimmune disease-related genes, and for estimating PC levels in affected blood and tissue from patients with multiple autoimmune diseases. RESULTS: The PC signature was highly sensitive and capable of detecting a change in as few as 360 PCs. The PC signature was reduced more than 90% in scleroderma patients following anti-CD19 treatment, and this reduction was highly correlated (r = 0.80) with inhibition of collagen gene expression. Evaluation of multiple autoimmune diseases revealed that 30-35% of lupus and rheumatoid arthritis patients had increased levels of PCs. CONCLUSION: This newly developed PC signature provides a robust and accurate method of measuring PC levels in the clinic. Our results highlight subsets of patients across multiple autoimmune diseases who may benefit from PC-depleting therapy.

Mycophenolic acid differentially impacts B cell function depending on the stage of differentiation.

Production of pathogenic Abs contributes to disease progression in many autoimmune disorders. The immunosuppressant agent mycophenolic acid (MPA) has shown clinical efficacy for patients with autoimmunity. The goal of these studies was to elucidate the mechanisms of action of MPA on B cells isolated from healthy individuals and autoimmune patients. In this study, we show that MPA significantly inhibited both proliferation and differentiation of primary human B cells stimulated under various conditions. Importantly, MPA did not globally suppress B cell responsiveness or simply induce cell death, but rather selectively inhibited early activation events and arrested cells in the G0/G1 phase of the cell cycle. Furthermore, MPA blocked expansion of both naive and memory B cells and prevented plasma cell (PC) differentiation and Ab production from healthy controls and individuals with rheumatoid arthritis. Finally, whereas MPA potently suppressed Ig secretion from activated primary B cells, terminally differentiated PCs were not susceptible to inhibition by MPA. The target of MPA, IMPDH2, was found to be downregulated in PCs, likely explaining the resistance of these cells to MPA. These results suggest that MPA provides benefit in settings of autoimmunity by directly preventing activation and PC differentiation of B cells; however, MPA is unlikely to impact autoantibody production by preexisting, long-lived PCs.

A novel role of HLA class I in the pathology of medulloblastoma.

BACKGROUND: MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. METHODS: We investigated expression of four essential components of MHC class I (heavy chain, beta2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. RESULTS: The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of beta2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. CONCLUSION: MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or beta2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility.

Critical role for IgM in host protection in experimental filarial infection.

We have previously shown that B cells (in particular B1 cells) are important in host protection against brugian infections in a murine i.p. model. In this study, we show that mice deficient in circulating IgM (secIgM-/-), but otherwise normal in their humoral responses, manifest a significant impairment in worm elimination, suggesting that one critical B cell function is the production of Ag-specific IgM. Efficient elimination of larvae is IgM dependent for both primary and challenge infections. The ability to eliminate worms is restored in secIgM-/- mice by administering sera from primed mice. We corroborated these in vivo studies with in vitro observations which show that IgM is the only isotype that reacts strongly with the surface of Brugia L3. Furthermore, activated peritoneal exudate cells adhere to L3 only in the presence of filaria-specific sera or IgM purified from them. This attachment is not reduced by heat inactivation of the serum, suggesting complement independent activity. Peritoneal exudate cells from primed mice, especially activated macrophages, carry high levels of IgM on their surfaces. Our observations suggest that an IgM-mediated reaction initiates the formation of host-protective granulomas.

Kinetics of cellular responses to intraperitoneal Brugia pahangi infections in normal and immunodeficient mice.

Filarial infections evoke exuberant inflammatory responses in the peritoneal cavities of immunocompetent mice. Clearance of infection appears to be dependent on complex interactions between B1 and B2 B lymphocytes, T cells, eosinophils, macrophages, and the products of these cells. In an earlier communication, we described the course of infection in normal immunocompetent mice. In this study, we utilize mice with well-characterized mutations that disable one or more effector components of adaptive immunity in order to determine their roles in host protection. We characterize peritoneal exudate cells by flow cytometry and determine the kinetics of accumulation of each of the different cell types following infection with Brugia pahangi. We find that (i) four-color flow-cytometric analysis of peritoneal exudate cells using anti-CD3, -CD11b, -CD19, and -Gr1 can distinguish up to six different populations of cells; (ii) an initial influx of neutrophils occurs within 24 h of infection, independent of the adaptive immune status of mice, and these cells disappear by day 3; (iii) an early influx of eosinophils is seen at the site of infection in all strains studied, but a larger, second wave occurs only in mice with T cells; (iv) the presence of T cells and eosinophils is important in causing an increase in macrophage size during the course of infection; and (v) most unexpectedly, T-cell recruitment appears to be optimal only if B cells are present, since JHD mice recruit significantly fewer T cells to the site of infection.