Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease.

Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

Increased ribonuclease expression reduces inflammation and prolongs survival in TLR7 transgenic mice.

TLR7 activation is implicated in the pathogenesis of systemic lupus erythematosus. Mice that overexpress TLR7 develop a lupus-like disease with autoantibodies and glomerulonephritis and early death. To determine whether degradation of the TLR7 ligand RNA would alter the course of disease, we created RNase A transgenic (Tg) mice. We then crossed the RNase Tg to TLR7 Tg mice to create TLR7 × RNase double Tg (DTg) mice. DTg mice had a significantly increased survival associated with reduced activation of T and B lymphocytes and reduced kidney deposition of IgG and C3. We observed massive hepatic inflammation and cell death in TLR7 Tg mice. In contrast, hepatic inflammation and necrosis were strikingly reduced in DTg mice. These findings indicate that high concentrations of serum RNase protect against immune activation and inflammation associated with TLR7 stimulation and that RNase may be a useful therapeutic strategy in the prevention or treatment of inflammation in systemic lupus erythematosus and, possibly, liver diseases.

Anti-CD180 (RP105) activates B cells to rapidly produce polyclonal Ig via a T cell and MyD88-independent pathway.

CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is unclear. We report that injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses, with the exception of IgA and IgG2b, with up to 50-fold increases in serum IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR knockout [KO]), CD40 KO, IL-4 KO, and MyD88 KO mice. Anti-CD180 rapidly increased both transitional and mature B cells, with especially robust increases in transitional B cell number, marginal zone B cell proliferation, and CD86, but not CD80, expression. In contrast, anti-CD40 induced primarily follicular B cell and myeloid expansion, with increases in expression of CD80 and CD95 but not CD86. The expansion of splenic B cells was due, in part, to proliferation and occurred in wild-type and TCR KO mice, whereas T cell expansion occurred in wild-type, but not in B cell-deficient, mice, indicating a direct role for B cells in CD180 stimulation in vivo. Combination of anti-CD180 with various MyD88-dependent TLR ligands biased B cell fate because coinjection diminished Ig production, but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells, but it increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus, CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.

Rapid activation of glutamate cysteine ligase following oxidative stress.

Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the formation of the cellular antioxidant glutathione (GSH). The GCL holoenzyme consists of two separately coded proteins, a catalytic subunit (GCLC) and a modifier subunit (GCLM). Both GCLC and GLCM are controlled transcriptionally by a variety of cellular stimuli, including oxidative stress. This study addresses post-translational control of GCL activity, which increased rapidly in human lymphocytes following oxidative stress. Activation of GCL occurred within minutes of treatment and without any change in GCL protein levels and coincided with an increase in the proportion of GCLC in the holoenzyme form. Likewise, GCLM shifted from the monomeric form to holoenzyme and higher molecular weight species. Normal rat tissues also showed a distribution of monomeric and higher molecular weight forms. Neither GCL activation, nor the formation of holoenzyme, required a covalent intermolecular disulfide bridge between GCLC and GCLM. However, in immunoprecipitation studies, a neutralizing epitope associated with enzymatic activity was protected following cellular oxidative stress. Thus, the N-terminal portion of GCLC may undergo a change that stabilizes the GCL holoenzyme. Our results suggest that a dynamic equilibrium exists between low and high activity forms of GCL and is altered by transient oxidative stress. This provides a mechanism for the rapid post-translational activation of GCL and maintenance of cellular GSH homeostasis.

CD20-directed small modular immunopharmaceutical, TRU-015, depletes normal and malignant B cells.

PURPOSE: CD20-directed therapy with rituximab is effective in many patients with malignant lymphoma or follicular lymphoma. However, relapse frequently occurs within 1 year, and patients become increasingly refractory to retreatment. Our purpose was to produce a compact, single-chain CD20-targeting immunotherapeutic that could offer therapeutic advantages in the treatment of B-cell lymphoma. EXPERIMENTAL DESIGN: Rituximab is a chimeric antibody containing two heavy chains and two light chains. Here, we describe the properties of TRU-015, a small modular immunopharmaceutical specific for CD20, encoded by a single-chain construct containing a single-chain Fv specific for CD20 linked to human IgG1 hinge, CH2, and CH3 domains but devoid of CH1 and CL domains. RESULTS: TRU-015 mediates potent direct signaling and antibody-dependent cellular cytotoxicity but has reduced size and complement-mediated cytotoxicity activity compared with rituximab. TRU-015 is a compact dimer of 104 kDa that comigrates with albumin in size exclusion chromatography and retains a long half-life in vivo. TRU-015 induced growth arrest in multiple B lymphoma cell lines in vitro and showed effective antitumor activity against large, established subcutaneous Ramos or Daudi xenograft tumors in nude mice. TRU-015 also showed rapid, dose-dependent, and durable depletion of peripheral blood B cells following single-dose administration to nonhuman primates. CONCLUSION: These results indicate that TRU-015 may improve CD20-directed therapy by effectively depleting embedded malignant B cells and nonmalignant pathogenic B cells and do so with reduced complement activation.

The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma.

The WFDC2 (HE4) gene is amplified in ovarian carcinomas, whereas its expression in normal tissues, including ovary, is low. Although the function of the HE4 protein is unknown,it is a member of a family of stable 4-disulfide core proteins that are secreted at high levels. We therefore performed experiments to explore whether quantitation of HE4 protein levels in serum can be used as a biomarker for ovarian carcinoma. A fusion gene was constructed encoding the HE4 protein fused to a gene encoding the murine IgG2a Fc domain. Subsequently, protein produced in mammalian cells was purified by affinity chromatography and used to immunize mice to generate hybridomas specific for HE4. Hybridoma supernatants were screened for binding to a similar fusion protein that, instead, had a human immunoglobulin tail. Two hybridomas, 2H5 and 3D8, were selected that produce monoclonal antibodies to different HE4 epitopes, and a double determinant ("Sandwich") ELISA was constructed and shown to detect a signal at the 160-pg level. Blinded studies on sera from postmenopausal patients with ovarian carcinoma and controls indicate that the specificity and sensitivity of the HE4-based ELISA is equivalent to that of the CA125 assay. However, the HE4 assay may have an advantage over the CA125 assay in that it is less frequently positive in patients with nonmalignant disease.

Expression and characterization of recombinant soluble human CD3 molecules: presentation of antigenic epitopes defined on the native TCR-CD3 complex.

The TCR-CD3 complex consists of the clonotypic disulfide-linked TCRalphabeta or TCRdeltagamma heterodimers, and the invariant CD3delta, epsilon, gamma and zeta chains. We generated plasmid constructs expressing the extracellular domains of the CD3delta, epsilon or gamma subunits fused to human IgG1 Fc. Recombinant fusion proteins consisting of individual CD3delta, epsilon or gamma subunits reacted poorly with anti-CD3 mAb including G19-4, BC3, OKT3 and 64.1. Co-expression of the CD3epsilon-Ig with either the CD3delta-Ig (CD3epsilondelta-Ig) or the CD3gamma-Ig (CD3epsilongamma-Ig) resulted in fusion proteins with much increased binding to G19-4. A brief acid treatment of the purified CD3epsilondelta-Ig fusion protein substantially improved its binding to BC3, OKT3 and 64.1. Surface plasmon resonance analysis revealed that the dissociation constants for CD3epsilondelta-Ig and anti-CD3 mAb ranged from 10(-8) to 10(-9) M. Based on these results, a single-chain (sc) construct encoding the CD3delta chain linked to the CD3epsilon chain with a flexible linker followed by human IgG1 Fc was expressed. The sc CD3deltaepsilon-scIg reacted with anti-CD3 mAb without requiring acid treatment. Moreover, anti-CD3 mAb bound CD3epsilondelta-Ig at a higher affinity than CD3epsilongamma-Ig, suggesting potential structural differences between the CD3epsilondelta and CD3epsilongamma subunits. In summary, we report the expression of soluble recombinant CD3 proteins that demonstrate structural characteristics of the native CD3 complex expressed on the T cell surface. These CD3 fusion proteins can be used to further analyze the structure of the TCR-CD3 complex, and to identify molecules that can interfere with TCR-CD3-mediated signal transduction by disrupting the interaction between CD3 and TCR subunits.

Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB.

Monoclonal antibodies against the T-cell activation molecule 4-1BB have been effective in the treatment of established mouse tumors. To create a vaccine that stimulates the immune system similarly to the efficacious monoclonal anti-4-1BB antibody, 1D8, we constructed a vector encoding cell-bound single-chain Fv fragments from 1D8. We transfected the vector into cells from the K1735 melanoma, selected because of its low immunogenicity and very low expression of major histocompatibility complex class I. The transfected cells induced a strong type 1 T-helper cell response, for which CD4+ but not CD8+ T lymphocytes were necessary and that involved natural killer cells. Vaccinated mice rejected established wild-type K1735 tumors growing as subcutaneous nodules or in the lung. An analogous approach may be effective against micrometastases in human patients, including tumors whose expression of major histocompatibility complex class I is very low.

Cutting edge: CD83 regulates the development of cellular immunity.

We recently found that human CD83, a marker of mature dendritic cells, is an adhesion receptor that binds to resting monocytes and a subset of activated CD8(+) T cells. We injected CD83-Ig into mice transplanted with the immunogenic P815 mastocytoma and showed that it significantly enhanced the rate of tumor growth and inhibited the development of cytotoxic T cells. In contrast, mice immunized with CD83-transfected K1735 cells, a poorly immunogenic melanoma, could prevent the outgrowth of wild-type K1735 cells. Studies performed in vitro with human PBL showed that coimmobilized CD83-Ig and anti-CD3 enhanced T cell proliferation and increased the proportion of CD8(+) T cells. CD83-transfected B-lymphoblastoid T51 cells stimulated T cell proliferation more effectively than untransfected T51 cells in MLR cultures and increased the generation of cytolytic T cells. We conclude that CD83 is a functionally important receptor that can regulate the development of cellular immunity by interacting with its ligand(s).