Jennifer Laskowski

IgM exacerbates glomerular disease progression in complement-induced glomerulopathy

While glomerular IgM deposition occurs in a variety of glomerular diseases the mechanism of deposition and its clinical significance remain controversial. Some have theorized IgM becomes passively trapped in areas of glomerulosclerosis. However, recent studies found that IgM specifically binds damaged glomeruli. Therefore, we tested whether natural IgM binds to neo-epitopes exposed after insults to the glomerulus and exacerbate disease in mice deficient in the complement regulatory protein factor H; a model of non-sclerotic and nonimmune-complex glomerular disease. Immunofluorescence microscopy demonstrated mesangial and capillary loop deposition of IgM while ultrastructural analysis found IgM deposition on endothelial cells and subendothelial areas. Factor H deficient mice lacking B cells were protected from renal damage, as evidenced by milder histologic lesions on light and electron microscopy. IgM, but not IgG, from wild-type mice bound to cultured murine mesangial cells. Furthermore, injection of purified IgM into mice lacking B cells bound within the glomeruli and induced proteinuria. A monoclonal natural IgM recognizing phospholipids also bound to glomeruli in vivo and induced albuminuria. Thus, our results indicate specific IgM antibodies bind to glomerular epitopes and that IgM contributes to the progression of glomerular damage in this mouse model of non-sclerotic glomerular disease.

Distinct roles for the complement regulators factor H and Crry in protection of the kidney from injury

Mutations in the complement regulatory proteins are associated with several different diseases. Although these mutations cause dysregulated alternative pathway activation throughout the body, the kidneys are the most common site of injury. The susceptibility of the kidney to alternative pathway-mediated injury may be due to limited expression of complement regulatory proteins on several tissue surfaces within the kidney. To examine the roles of the complement regulatory proteins factor H and Crry in protecting distinct renal surfaces from alternative pathway mediated injury, we generated mice with targeted deletions of the genes for both proteins. Surprisingly, mice with combined genetic deletions of factor H and Crry developed significantly milder renal injury than mice deficient in only factor H. Deficiency of both factor H and Crry was associated with C3 deposition at multiple locations within the kidney, but glomerular C3 deposition was lower than that in factor H alone deficient mice. Thus, factor H and Crry are critical for regulating complement activation at distinct anatomic sites within the kidney. However, widespread activation of the alternative pathway reduces injury by depleting the pool of C3 available at any 1 location.

Mapping the complement factor H-related protein 1 (CFHR1): C3b/C3d interactions

Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation.

Complement Activation via a C3a Receptor Pathway Alters CD4+ T Lymphocytes and Mediates Lung Cancer Progression

The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3-/- mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ+/TNFα+/IL10+ CD4+ and CD8+ T cells. Immunodepletion of CD4+ but not CD8+ T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5-dependent pathway.Significance: This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. Cancer Res; 78(1); 143-56. ©2017 AACR.

Chapter 14 – Factor B

Factor B is a component of the alternative pathway of complement. It contains a serine protease (SP) domain, and when activated it provides the catalytic activity of the alternative pathway C3 and C5 convertases. Factor B circulates as an inactive proenzyme (i.e., a zymogen), and only becomes activated after cleavage by the protein factor D. However, factor D can only cleave factor B when it is bound to the active forms of C3: C3(H2O) and C3b. Factor B is generated as a single-chain protein, and cleavage by factor D generates two peptide fragments (Ba and Bb). The Bb region (which contains the SP domain) remains bound to C3(H2O) and C3b, forming the alternative pathway convertases [C3(H2O)Bb and C3bBb]. As part of the C3 convertases, the SP domain of Bb has specific catalytic activity for cleavage of C3 molecules. The addition of another C3b molecule to the alternative pathway C3 convertase generates the C5 convertase (C3bBbC3b). As part of the alternative pathway C5 convertase, the SP domain of Bb cleaves C5 molecules, enabling the assembly of C5–C9 and the resultant formation of the membrane attack complex.

Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM

Natural IgM binds to glomerular epitopes in several progressive kidney diseases. Previous work has shown that IgM also binds within the glomerulus after ischemia/reperfusion (I/R) but does not fully activate the complement system. Factor H is a circulating complement regulatory protein, and congenital or acquired deficiency of factor H is a strong risk factor for several types of kidney disease. We hypothesized that factor H controls complement activation by IgM in the kidney after I/R, and that heterozygous factor H deficiency would permit IgM‐mediated complement activation and injury at this location. We found that mice with targeted heterozygous deletion of the gene for factor H developed more severe kidney injury after I/R than wild‐type controls, as expected, but that complement activation within the glomeruli remained well controlled. Furthermore, mice that are unable to generate soluble IgM were not protected from renal I/R, even in the setting of heterozygous factor H deficiency. These results demonstrate that factor H is important for limiting injury in the kidney after I/R, but it is not critical for controlling complement activation by immunoglobulin within the glomerulus in this setting. IgM binds to glomerular epitopes after I/R, but it is not a significant source of injury.

Complement factor H–related proteins in IgA nephropathy—sometimes a gentle nudge does the trick

Complement activation probably contributes to glomerular inflammation and damage in IgA nephropathy. In this issue, 2 groups report that levels of factor H-related protein 1 are elevated in patients with IgA nephropathy and correlate with disease progression. These studies provide new evidence that the complement cascade is important to the pathogenesis of this disease. These results also suggest that factor H-related protein 1 levels may be useful for identifying those patients at high risk of disease progression.

Abstract A121: Complement activation is critical for tumor progression in an orthotopic immunocompetent model of lung cancer

Background: The complement system is an important arm of the innate immune system, and complement proteins are produced by both cancer cells and cells of the TME. A primary function of the complement system is to remove pathogens and injured cells, and it had been assumed that complement activation would result in elimination of cancer cells. However, recent work has revealed new biologic effects of the complement activation fragments C3a and C5a. For example, C3a and C5a directly stimulate the growth of some tissues. Furthermore, inflammatory cells as well as CD4 and CD8 T cells express receptors for both of these activation fragments (C3aR and C5aR), and a recent study reported that C5a reduces the anti-tumor function of CD8+ T cells. Nevertheless, the role of complement activation in the growth and spread of non-small cell lung cancer is incompletely understood. While complement activation can proceed by multiple pathways, a central event is the deposition of C3 on the cell surface. The goal of this study was to examine the role of complement in lung cancer progression employing an immunocompetent orthotopic model which recapitulates many of the features of human lung cancer. Methods: Two murine lung cancer cell lines derived from C57BL/6 mice were used in an orthotopic model of tumor progression: Lewis Lung Carcinoma (LLC) cells, which are a de-differentiated cell line in which driver mutations have not been identified; and CMT167 cells, which are an epithelial lung cancer cell line that express oncogenic K-Ras. Tumor cells were implanted in the left lungs of syngeneic mice WT C57BL/6 mice, or mice which were globally knocked out for C3 (C3-KO). Primary tumor growth and formation of liver metastases were quantitated in the two groups of mice. Complement activation in the plasma was determined by ELISA. Single cell suspensions of the left lung of tumor-bearing mice were prepared and populations of myeloid cells and immune cells quantitated by flow cytometry. Results: Implantation of cancer lead to systemic complement activation as assessed by ELISA in plasma. Primary tumors and liver metastases were decreased in both cell lines implanted into C3-KO mice compared to controls. However, the degree of inhibition was much greater in CMT167 tumors. LLC cells expressed C3 in vitro, whereas CMT167 cells did not, suggesting that autonomous C3 production by cancer cells may contribute to tumor progression. Recruitment of inflammatory myeloid cells was not altered in tumors growing in C3-KO mice. At early time points, we observed equal increases in T cells (CD4, CD8) in the two groups of mice. However, at later time points (3 weeks), the number of T cells had decreased in WT mice, but remained elevated in C3-KO mice. Conclusions: Complement activation occurs in the setting of tumors. C3 plays a critical role in tumor progression, with strong inhibition observed in a subset of cancer cells. The differential response to complement inhibition may depend on the ability of the cancer cells to produce C3. Effects of complement activation may be mediated by inhibiting cytotoxic T cells. These data suggest that complement inhibitors may represent novel immunotherapeutic targets for lung cancer, and suggest interactions between the complement system and checkpoint inhibitors. Citation Format: Jeff Kwak, Howard Li, Jennifer Laskowski, Mary Weiser-Evans, Joshua Thurman, Raphael A. Nemenoff. Complement activation is critical for tumor progression in an orthotopic immunocompetent model of lung cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A121.