A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis.

Complement is an important component of the innate and adaptive immune response, yet complement split products generated through activation of each of the three complement pathways (classical, alternative, and lectin) can cause inflammation and tissue destruction. Previous studies have shown that complement activation through the alternative, but not classical, pathway is required to initiate antibody-induced arthritis in mice, but it is unclear if the alternative pathway (AP) plays a role in established disease. Previously, we have shown that human complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the AP of complement. Here, we present the crystal structure of murine CRIg and, using mutants, provide evidence that the structural requirements for inhibition of the AP are conserved in human and mouse. A soluble form of CRIg reversed inflammation and bone loss in two experimental models of arthritis by inhibiting the AP of complement in the joint. Our data indicate that the AP of complement is not only required for disease induction, but also disease progression. The extracellular domain of CRIg thus provides a novel tool to study the effects of inhibiting the AP of complement in established disease and constitutes a promising therapeutic with selectivity for a single complement pathway.

Structure of C3b in complex with CRIg gives insights into regulation of complement activation.

The complement system is a key part of the innate immune system, and is required for clearance of pathogens from the bloodstream. After exposure to pathogens, the third component of the complement system, C3, is cleaved to C3b which, after recruitment of factor B, initiates formation of the alternative pathway convertases. CRIg, a complement receptor expressed on macrophages, binds to C3b and iC3b mediating phagocytosis of the particles, but it is unknown how CRIg selectively recognizes proteolytic C3-fragments and whether binding of CRIg to C3b inhibits convertase activation. Here we present the crystal structure of C3b in complex with CRIg and, using CRIg mutants, provide evidence that CRIg acts as an inhibitor of the alternative pathway of complement. The structure shows that activation of C3 induces major structural rearrangements, including a dramatic movement (>80 A) of the thioester-bond-containing domain through which C3b attaches to pathogen surfaces. We show that CRIg is not only a phagocytic receptor, but also a potent inhibitor of the alternative pathway convertases. The structure provides insights into the complex macromolecular structural rearrangements that occur during complement activation and inhibition. Moreover, our structure-function studies relating the structural basis of complement activation and the means by which CRIg inhibits the convertases provide important clues to the development of therapeutics that target complement.

Complement receptor of the Ig superfamily enhances complement-mediated phagocytosis in a subpopulation of tissue resident macrophages.

An important function of the complement cascade is to coat self and foreign particles with C3-proteins that serve as ligands for phagocytic receptors. Although tissue resident macrophages play an important role in complement-mediated clearance, the receptors coordinating this process have not been well characterized. In the present study, we identified a subpopulation of resident peritoneal macrophages characterized by high expression of complement receptor of the Ig superfamily (CRIg), a recently discovered complement C3 receptor. Macrophages expressing CRIg showed significantly increased binding and subsequent internalization of complement-opsonized particles compared with CRIg negative macrophages. CRIg internalized monovalent ligands and was able to bind complement-opsonized targets in the absence of Ca(2+) and Mg(2+), which differs from the beta(2)-integrin CR3 that requires divalent cations and polyvalent ligands for activation of the receptor. Although CRIg dominated in immediate binding of complement-coated particles, CRIg and CR3 contributed independently to subsequent particle phagocytosis. CRIg thus identifies a subset of tissue resident macrophages capable of increased phagocytosis of complement C3-coated particles, a function critical for immune clearance.

Cancer immunotherapy: accomplishments to date and future promise.

Cancer remains a devastating disease as existing therapies are too often ineffective and toxicities remain unacceptably high. Immunotherapies for cancer offer the promise of the specificity and memory of the immune system against malignant cells to achieve durable cure with minimal toxicity. Beginning with the success of bone marrow transplantation for blood-borne cancers, and the more recent development of monoclonal antibody therapeutics for a variety of tumors, immunotherapies are already among the most successful class of treatments for cancer. Greater understanding of immunoregulatory mechanisms and improved techniques for immune cell manipulation and engineering have led to new immunomodulatory approaches and cell-based therapies for cancer that have generated great excitement within the biomedical community. As these technologies continue to improve, and as new approaches for harnessing the power and specificity of the immune system are developed, immunotherapies will play an increasingly important role in the treatment of cancer. Here, we review the history of immunotherapies for cancer and discuss existing and emerging immunotherapy technologies that hope to translate the promise of immunotherapy into clinical reality.

Candidate pathways for promoting differentiation or quiescence of oligodendrocyte progenitor-like cells in glioma.

Platelet-derived growth factor receptor alpha-positive oligodendrocyte progenitor cells (OPC) located within the mature central nervous system may remain quiescent, proliferate, or differentiate into oligodendrocytes. Human glioblastoma multiforme tumors often contain rapidly proliferating oligodendrocyte lineage transcription factor 2 (Olig2)-positive cells that resemble OPCs. In this study, we sought to identify candidate pathways that promote OPC differentiation or quiescence rather than proliferation. Gene expression profiling conducted in both normal murine OPCs and highly proliferative Olig2-positive glioma cells identified all the transcripts associated with the highly proliferative state of these cells and showed that among the various cell types found within the brain, Olig2-positive tumor cells are most similar to OPCs. We then subtracted OPC transcripts found in tumor samples from those found in normal brain samples and identified 28 OPC transcripts as candidates for promoting differentiation or quiescence. Systematic analysis of human glioma data revealed that these genes have similar expression profiles in human tumors and were significantly enriched in genomic deletions, suggesting an antiproliferative role. Treatment of primary murine glioblastoma cells with agonists of one candidate gene, Gpr17, resulted in a decreased number of neurospheres. Together, our findings show that comparison of the molecular phenotype of progenitor cells in tumors to the equivalent cells in the normal brain represents a novel approach for the identification of targeted therapies.

Metastatic breast cancer cells in the bone marrow microenvironment: novel insights into oncoprotection.

Among all cancers, malignancies of the breast are the second leading cause of cancer death in the United States after carcinoma of the lung. One of the major factors considered when assessing the prognosis of breast cancer patients is whether the tumor has metastasized to distant organs. Although the exact phenotype of the malignant cells responsible for metastasis and dormancy is still unknown, growing evidence has revealed that they may have stem cell-like properties that may account for resistance to chemotherapy and radiation. One process that has been attributed to primary tumor metastasis is the epithelial-to-mesenchymal transition. In this review, we specifically discuss breast cancer dissemination to the bone marrow and factors that ultimately serve to shelter and promote tumor growth, including the complex relationship between mesenchymal stem cells (MSCs) and various aspects of the immune system, carcinoma-associated fibroblasts, and the diverse components of the tumor microenvironment. A better understanding of the journey from the primary tumor site to the bone marrow and subsequently the oncoprotective role of MSCs and other factors within that microenvironment can potentially lead to development of novel therapeutic targets.

Recruited cells can become transformed and overtake PDGF-induced murine gliomas in vivo during tumor progression.

BACKGROUND: Gliomas are thought to form by clonal expansion from a single cell-of-origin, and progression-associated mutations to occur in its progeny cells. Glioma progression is associated with elevated growth factor signaling and loss of function of tumor suppressors Ink4a, Arf and Pten. Yet, gliomas are cellularly heterogeneous; they recruit and trap normal cells during infiltration. METHODOLOGY/PRINCIPAL FINDINGS: We performed lineage tracing in a retrovirally mediated, molecularly and histologically accurate mouse model of hPDGFb-driven gliomagenesis. We were able to distinguish cells in the tumor that were derived from the cell-of-origin from those that were not. Phenotypic, tumorigenic and expression analyses were performed on both populations of these cells. Here we show that during progression of hPDGFb-induced murine gliomas, tumor suppressor loss can expand the recruited cell population not derived from the cell-of-origin within glioma microenvironment to dominate regions of the tumor, with essentially no contribution from the progeny of glioma cell-of-origin. Moreover, the recruited cells can give rise to gliomas upon transplantation and passaging, acquire polysomal expression profiles and genetic aberrations typically present in glioma cells rather than normal progenitors, aid progeny cells in glioma initiation upon transplantation, and become independent of PDGFR signaling. CONCLUSIONS/SIGNIFICANCE: These results indicate that non-cell-of-origin derived cells within glioma environment in the mouse can be corrupted to become bona fide tumor, and deviate from the generally established view of gliomagenesis.

Stem cells and regenerative medicine: accomplishments to date and future promise.

More than 50 years have passed since the first allogeneic hematopoietic stem cell transplant in patients; however, the promise of other stem cell populations for tissue replacement and repair remains unachieved. When considering cell-based interventions for personalized medicine, the factors influencing therapeutic success and safety are more complicated than for traditional small-molecule pharmacological agents and protein biologics. Failure to progress personalized stem cell therapies to the clinic has resulted from complications that include an incomplete understanding of developmental programs and the diversity of host-donor interactions. In order to more rapidly extend the use of stem cells to the clinic, a better understanding of the different stem cell sources and the implications of their host interactions is required. In this review, we introduce the currently available sources and highlight recent literature that instructs the potential and limitations of their use.

Negative regulation of autoimmune demyelination by the inhibitory receptor CLM-1.

Multiple sclerosis and its preclinical model, experimental autoimmune encephalomyelitis, are marked by perivascular inflammation and demyelination. Myeloid cells, derived from circulating progenitors, are a prominent component of the inflammatory infiltrate and are believed to directly contribute to demyelination and axonal damage. How the cytotoxic activity of these myeloid cells is regulated is poorly understood. We identify CMRF-35-like molecule-1 (CLM-1) as a negative regulator of autoimmune demyelination. CLM-1 is expressed on inflammatory myeloid cells present in demyelinating areas of the spinal cord after immunization of mice with MOG35-55 (myelin oligodendrocyte glycoprotein) peptide. Absence of CLM-1 resulted in significantly increased nitric oxide and proinflammatory cytokine production, along with increased demyelination and worsened clinical scores, whereas T cell responses in the periphery or in the spinal cord remained unaffected. This study thus identifies CLM-1 as a negative regulator of myeloid effector cells in autoimmune demyelination.

Modeling Adult Gliomas Using RCAS/t-va Technology.

Malignant gliomas remain the most devastating childhood and adult tumors of the central nervous system. Although adult and pediatric gliomas are histologically indistinguishable, they differ in location, behavior, and molecular characteristics. This implies that the molecular pathways and pathophysiology of malignant gliomagenesis in these two populations are distinct. Such differences between adult and pediatric gliomas may predict different therapeutic responses. Therefore, accurate genetically engineered models of adult and pediatric gliomas may help understand the biology of these tumors and evaluate therapeutic agents in preclinical studies. It has been proposed that gliomas arise from the subventricular zone in mice during development. Here, we demonstrate that, in adult mice, gliomas may arise not only when injected in the subventricular zone but also when injected in the cortex and cerebellum. Our work demonstrates a versatile and highly reproducible adult mouse model of glioma, which can be easily incorporated into preclinical studies.

CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens.

The complement system serves an important role in clearance of pathogens, immune complexes, and apoptotic cells present in the circulation. Complement fragments deposited on the particle surface serve as targets for complement receptors present on phagocytic cells. Although Kupffer cells, the liver resident macrophages, play a dominant role in clearing particles in circulation, complement receptors involved in this process have yet to be identified. Here we report the identification and characterization of a Complement Receptor of the Immunoglobulin superfamily, CRIg, that binds complement fragments C3b and iC3b. CRIg expression on Kupffer cells is required for efficient binding and phagocytosis of complement C3-opsonized particles. In turn, Kupffer cells from CRIg-deficient mice are unable to efficiently clear C3-opsonized pathogens in the circulation, resulting in increased infection and mortality of the host. CRIg therefore represents a dominant component of the phagocytic system responsible for rapid clearance of C3-opsonized particles from the circulation.