Antibody-dependent Complement-mediated Cytotoxicity Research Articles

Enhancement of complement-dependent cytotoxicity by linking factor-H derived short consensus repeats 19-20 to CD20 antibodies.

Antibody-mediated complement-dependent cytotoxicity (CDC) on malignant cells is regulated by several complement control proteins, including the inhibitory complement factor H (fH). fH consists of 20 short consensus repeat elements (SCRs) with specific functional domains. Previous research revealed that the fH-derived SCRs 19-20 (SCR1920) can displace full-length fH on the surface of chronic lymphocytic leukemia (CLL) cells, which sensitizes CLL cells for e.g. CD20-targeting therapeutic monoclonal antibody (mAb) induced CDC. Therefore, we constructed lentiviral vectors for the generation of cell lines that stably produce mAb-SCR-fusion variants starting from the clinically approved parental mAbs rituximab, obinutuzumab and ofatumumab, respectively. Flow-cytometry revealed that the modification of the mAbs by the SCRs does not impair the binding to CD20. Increased in vitro lysis potency compared to their parental mAbs was corroborated by showing specific and dose dependent target cell elimination by CDC when compared to their parental mAbs. Lysis of CLL cells was not affected by the depletion of NK cells, suggesting that antibody-dependent cellular cytotoxicity plays a minor role in this context. Overall, this study emphasizes the crucial role of CDC in the elimination of CLL cells by mAbs and introduces a novel approach for enhancing CDC by directly fusing fH SCR1920 with mAbs.

The Effect of Simvastatin on Thyroid Function in Experimental Autoimmune Thyroiditis Induced in Rats

Autoimmune thyroiditis or Hashimoto’s thyroiditis (HT): is the most common autoimmune thyroid disease. is characterized by marked lymphocytic infiltration and the presence of serum autoantibodies against Thyroid peroxidase (TPO) and Thyroglobulin (TG). HT is a prevalent cause of hypothyroidism in areas of the world where dietary iodine is sufficient. Genetic predisposition and cellular and humoral immunity play a role in thyroid autoimmune pathogenesis. Antithyroid antibodies are produced by interaction between APC, T cells, and B cells to fix complement. Thyroid tissue is more severely damaged by complement-dependent antibody-mediated cytotoxicity. Statins, the powerful inhibitors of HMG-CoA reductase have pleotropic effects including anti-inflammatory and immunomodulatory effects. In this current study, Experimental autoimmune thyroiditis was induced in twenty-four female Wistar rat. Simvastatin was given at a dose of 4mg/kg for 30 days. Thyroid autoantibodies and Thyroid hormones were measured by ELISA technique Results showed that Simvastatin reduced autoantibodies against TPO and TG. It also lowered serum level of TSH and increased serum level of T4. Due to the undesirable side effects associated with the current treatment options used in Autoimmune thyroiditis. Simvastatin might be a promising approach in the treatment of this disease.

Abstract 4990: Genotoxic chemotherapy impedes antibody-mediated complement dependent cytotoxicity (CDC), via a Chk1-CD59 axis

Abstract Monoclonal antibodies such as rituximab exert targeted tumor cell lysis via complement dependent cytotoxicity (CDC). These antibodies are often combined with chemotherapy in clinical treatment protocols. However, the impact of chemotherapy on cell-intrinsic pathways that regulate antibody-mediated CDC is not known. Through a phage display screen, we identified that genotoxic stress caused upregulation of a membrane complement regulatory protein (mCRP), CD46, prompting further investigation into its implications and mechanism. Using cell lines from diffuse large B cell lymphoma (DLBCL) as our model, we confirmed that genotoxic chemotherapy upregulates CD46, as well as other mCRPs such as CD55 and CD59. We hypothesized that upregulated mCRPs could impede Rituximab-mediated CDC. We assessed this in-vitro using rituximab and complement human serum. Aligned with our hypothesis, chemotherapy treatment of DLBCL cells significantly impeded rituximab-induced CDC. Using blocking antibodies to each mCRP, reversal of the effect of chemotherapy on CDC only occurred after co-treatment with a CD59 blocking antibody, suggesting that CD59 is a key player for chemotherapy-invoked resistance to CDC. To further elucidate the underlying cellular pathway, we performed a high-throughput small molecule screen using rituximab-induced CDC as a readout, with or without cotreatment of a genotoxic chemotherapeutic (etoposide). We identified Chk1 inhibitors as consistent top rescuers. qPCR and flow cytometry revealed that Chk1 inhibition prevented the upregulation of mCRPs transcriptionally. Sp1 is a known transcription factor driving constitutive CD59 expression. Inhibiting Sp1 similarly prevented chemotherapy-induced upregulation of CD59 and resistance to CDC. However, CHIP-qPCR showed no increased binding of total Sp1 protein to the CD59 promoter after chemotherapy. Instead, positive co-immunoprecipitation of pATM and p300 with Sp1 only with etoposide and not with co-treatment of Chk1 inhibitor suggests Chk1-mediated activation of Sp1. Mechanistically, we have found that chemotherapy-activated Chk1 modulates Sp1-mediated CD59 transcription and that the induced upregulation of CD59 is able to prevent antibody-mediated CDC. Overall, these results indicate novel immune regulation by DNA damage response, particularly in the context of complement regulatory proteins. Our results advocate consideration when using simultaneous administration of chemotherapy with CDC-inducing monoclonal antibodies, and suggest Chk1 blockade as a possible therapeutic addition. Citation Format: Allison S. Chan, Patrick W. Jaynes, Nurulhuda B. Mustafa, Akshaya A. Anbuselvan, Charmaine Z. Ong, Anand D. Jeyasekharan. Genotoxic chemotherapy impedes antibody-mediated complement dependent cytotoxicity (CDC), via a Chk1-CD59 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4990.

A bivalent form of nanoparticle-based dengue vaccine stimulated responses that potently eliminate both DENV-2 particles and DENV-2-infected cells.

Dengue is the most prevalent mosquito-borne viral disease and continues to be a global public health concern. Although a licensed dengue vaccine is available, its efficacy and safety profile are not satisfactory. Hence, there remains a need for a safe and effective dengue vaccine. We are currently developing a bivalent dengue vaccine candidate. This vaccine candidate is composed of a C-terminus truncated non-structural protein 1 (NS11-279) and envelope domain III (EDIII) of DENV-2 encapsidated in the nanocarriers, N, N, N-trimethyl chitosan nanoparticles (TMC NPs). The immunogenicity of this bivalent vaccine candidate was investigated in the present study using BALB/c mice. In this work, we demonstrate that NS1+EDIII TMC NP-immunized mice strongly elicited antigen-specific antibody responses (anti-NS1 and anti-EDIII IgG) and T-cell responses (NS1- and EDIII-specific-CD4+ and CD8+ T cells). Importantly, the antibody response induced by NS1+EDIII TMC NPs provided antiviral activities against DENV-2, including serotype-specific neutralization and antibody-mediated complement-dependent cytotoxicity. Moreover, the significant upregulation of Th1- and Th2-associated cytokines, as well as the increased levels of antigen-specific IgG2a and IgG1, indicated a balanced Th1/Th2 response. Collectively, our findings suggest that NS1+EDIII TMC NPs induced protective responses that can not only neutralize infectious DENV-2 but also eliminate DENV-2-infected cells.

MiR-132-3p regulates antibody-mediated complement-dependent cytotoxicity in colon cancer cells by directly targeting CD55.

The overexpression of membrane-bound complement regulatory proteins (mCRPs) on tumour cells helps them survive complement attacks by suppressing antibody-mediated complement-dependent cytotoxicity (CDC). Consequently, mCRP overexpression limits monoclonal antibody drug immune efficacy. CD55, an mCRP, plays an important role in inhibiting antibody-mediated CDC. However, the mechanisms regulating CD55 expression in tumour cells remain unclear. Here, the aim was to explore CD55-targeting miRNAs. We previously constructed an in vitro model comprising cancer cell lines expressing α-gal and serum containing natural antibodies against α-gal and complement. This was used to simulate antibody-mediated CDC in colon cancer cells. We screened microRNAs that directly target CD55 using LoVo and Ls-174T colon cell lines, which express CD55 at low and high levels, respectively. miR-132-3p expression was dramatically lower in Ls-174T cells than in LoVo cells. miR-132-3p overexpression or inhibition transcriptionally regulated CD55 expression by specifically targeting its mRNA 3'-untranslated regions. Further, miR-132-3p modulation regulated colon cancer cell sensitivity to antibody-mediated CDC through C5a release and C5b-9 deposition. Moreover, miR-132-3p expression was significantly reduced, whereas CD55 expression was increased, in colon cancer tissues compared to levels in adjacent normal tissues. CD55 protein levels were negatively correlated with miR-132-3p expression in colon cancer tissues. Our results indicate that miR-132-3p regulates colon cancer cell sensitivity to antibody-mediated CDC by directly targeting CD55. In addition, incubating the LoVo human tumour cell line, stably transfected with the xenoantigen α-gal, with human serum containing natural antibodies comprises a stable and cheap in vitro model to explore the mechanisms underlying antibody-mediated CDC.

Human IL-17 and TNF-α Additively or Synergistically Regulate the Expression of Proinflammatory Genes, Coagulation-Related Genes, and Tight Junction Genes in Porcine Aortic Endothelial Cells.

Immune rejection is the major limitation for porcine xenograft survival in primate recipients. Proinflammatory cytokines play important roles in immune rejection and have been found to mediate the pathological effects in various clinical and experimental transplantation trials. IL-17 and TNF-α play critical pathological roles in immune disorders, such as psoriasis and rheumatoid arthritis. However, the pathological roles of human IL-17 (hIL-17) and human TNF-α (hTNF-α) in xenotransplantation remain unclear. Here we found that hIL-17 and hTNF-α additively or synergistically regulate the expression of 697 genes in porcine aortic endothelial cells (PAECs). Overall, 415 genes were found to be synergistically regulated, while 282 genes were found to be additively regulated. Among these, 315 genes were upregulated and 382 genes were downregulated in PAECs. Furthermore, we found that hIL-17 and hTNF-α additively or synergistically induced the expression of various proinflammatory cytokines and chemokines (e.g., IL1α, IL6, and CXCL8) and decreased the expression of certain anti-inflammatory genes (e.g., IL10). Moreover, hIL-17 plus hTNF-α increased the expression of IL1R1 and IL6ST, receptors for IL1 and IL6, respectively, and decreased anti-inflammatory gene receptor expression (IL10R). hIL-17 and hTNF-α synergistically or additively induced CXCL8 and CCL2 expression and consequently promoted primary human neutrophil and human leukemia monocytic cell migration, respectively. In addition, hIL-17 and hTNF-α induced pro-coagulation gene (SERPINB2 and F3) expression and decreased anti-coagulation gene (TFPI, THBS1, and THBD) expression. Additionally, hIL-17 and hTNF-α synergistically decreased occludin expression and consequently promoted human antibody-mediated complement-dependent cytotoxicity. Interestingly, hTNF-α increased swine leukocyte antigen (SLA) class I expression; however, hIL-17 decreased TNF-α-mediated SLA-I upregulation. We concluded that hIL-17 and hTNF-α likely promote the inflammatory response, coagulation cascade, and xenoantibody-mediated cell injury. Thus, blockade of hIL-17 and hTNF-α together might be beneficial for xenograft survival in recipients.

In Mixed Lymphocyte Reaction, the Hypoxia-Inducible Factor Prolyl-Hydroxylase Inhibitor Roxadustat Suppresses Cellular and Humoral Alloimmunity.

Hypoxia-inducible factor (HIF) prolyl-hydroxylase inhibitors are currently used for the treatment of renal anemia. Since HIF affects immune cells, we evaluated the effect of such a drug, the roxadustat, on adaptive immunity. Cell proliferation was assessed in a two-way mixed lymphocyte reaction (MLR) with BrdU assay. In CD4+ T cells isolated from the two-way MLRs, western blotting was performed to detect the impact of roxadustat on HIF-1α and HIF-2α, the apoptotic marker cleaved caspase-3, and the master transcription factors of CD4+ T cells differentiation towards Th1, Th2, Th17, Treg and Tfh subsets. The signature cytokines of the above CD4+ T-cell subsets IFN-γ, IL-4, IL-17, IL-10, and IL-21 were measured in the supernatants. For assessing humoral immunity, we developed a suitable antibody-mediated complement-dependent cytotoxicity assay. Roxadustat stabilized HIF-1α and HIF-2α, suppressed cell proliferation, inhibited CD4+ T-cell differentiation into Th1 and Th17 subsets, while it favored differentiation towards Th2, Treg and Tfh. Roxadustat suppressed humoral immunity too. These immunosuppressive properties of roxadustat indicate that the recently introduced HIF prolyl-hydroxylase inhibitors in medical therapeutics may render the patients vulnerable to infections. This possibility should be further evaluated in clinical trials.

Nanodelivery system enhances the immunogenicity of dengue-2 nonstructural protein 1, DENV-2 NS1

Nonstructural protein 1 (NS1) of dengue virus (DENV) is currently recognized as a dengue vaccine candidate. Unfortunately, most of non-replicating immunogens typically stimulate unsatisfactory immune responses, thus, the additional adjuvant is required. In this study, C-terminal truncated DENV-2 NS1 loaded in N,N,N, trimethyl chitosan nanoparticles (NS11-279TMC NPs) was prepared through the ionic gelation method. The immunogenicity of NS11-279TMC NPs was investigated using human ex vivo as well as the murine model. Through a human ex vivo model, it was demonstrated in this study that not only can TMC particles effectively deliver NS11-279 protein into monocyte-derived dendritic cells (MoDCs), but also potently stimulate those cells, resulting in increased expression of maturation marker (CD83), costimulating molecules (CD80, CD86 and HLA-DR) and markedly secreted various types of innate immune cytokines/chemokines. Moreover, mice administered with NS11-279TMC NPs strongly elicited both antibody and T cell responses, produced higher levels of IgG, IgG1, IgG2a and potently activated CD8+ T cells, as compared to mice administered with soluble NS11-279. Importantly, we further demonstrated that anti-NS11-279 antibody induced by this platform of NS11-279 effectively eliminated DENV-2 infected cells through antibody dependent complement-mediated cytotoxicity. Significantly, anti-DENV2 NS11-279 antibody exerted cross-antiviral activity against DENV-1 and −4 but not against DENV-3 infected cells. These findings demonstrate that TMC exerts a desirable adjuvant for enhancing delivery and antigenicity of NS1 based dengue vaccine.

Mortalin peptides exert antitumor activities and act as adjuvants to antibody-mediated complement-dependent cytotoxicity.

Cancer cells have developed numerous strategies to maintain their proliferative capacity and to withstand different kinds of stress. The mitochondrial stress‑70protein named glucose regulated protein 75 (GRP75), also known as mortalin, is an intriguing cancer pro‑survival factor. It is constitutively expressed in normal tissues but is upregulated in many tumors, and was shown to be a cancer prognostic biomarker. Mortalin is an inhibitor of complement‑dependent cytotoxicity(CDC) and may therefore protect cells from antibody‑based immunotherapy. To target mortalin for cancer therapy, our laboratory designed several mortalin mimetic peptides with sequences predicted to be involved in mortalin binding to its client proteins. The peptides were synthesized with a C‑terminal transactivator of transcription sequence. By using cell death methodologies, the mechanism of action of the mortalin mimetic peptides on cancer cells was studied. Two peptides in particular, Mot‑P2 and Mot‑P7, were found to be highly toxic to lymphoma and ovarian, breast and prostate carcinoma cells. The analysis of their mode of action revealed that they may induce, within minutes, plasma membrane perturbations and mitochondrial stress. Furthermore, Mot‑P2 and Mot‑P7 activated necrotic cell death, leading to plasma membrane perforation, mitochondrial inner membrane depolarization and decrease in ATP level. In addition, Mot‑P7, but not Mot‑P2, required extracellular calcium ions to fully mediate cell death and was partially inhibited by plasma membrane cholesterol. At sub‑toxic concentrations, the two peptides moderately inhibited cancer cell proliferation and blocked cell cycle at G2/M. Both peptides may bind intracellularly to mortalin and/or a mortalin‑binding protein, hence knocking down mortalin expression reduced cell death. Combining treatment with Mot‑P2 or Mot‑P7 and CDC resulted in increased cell death. This study identified highly cytotoxic mortalin mimetic peptides that may be used as monotherapy or combined with complement‑activating antibody therapy to target mortalin for precision cancer therapy.

Enhanced Fc-mediated complement activity with broadly neutralizing antibody 10E8v4 does not improve viral outcomes following SHIV challenge in rhesus macaques

Abstract Fc modified broadly neutralizing antibodies (bNAbs) are being developed for both prophylactic and therapeutic treatment of HIV with some success in extending half-life, but in vivo benefits of enhanced FcγR interaction remain unclear. Here we sought to improve bNAb efficacy by increasing Fc-mediated complement activity. A panel of Fc variants was screened for complement enhancement of 10 bNAbs. Modified 10E8v4, which weakly neutralizes SHIVSF162p3 (IC80 30 mg/ml) and does not mediate ADCC, was selected for macaque studies. Compared to wildtype, the EFTAE modification resulted in greater affinity for multiple FcγRs, >2-fold enhanced complement deposition, viral lysis, and antibody-mediated complement dependent cytotoxicity, but reduced serum half-life. Subcutaneous infusion with a sub-protective 5 mg/kg dose of 10E8v4 followed by a single high dose intrarectal challenge with SHIVSF162p3 showed unmodified 10E8v4 treated animals displayed lower plasma viremia compared to the complement/FcgR dual knockout (LALA) and controls. Unexpectedly, the EFTAE group displayed higher plasma viremia than controls, and viral DNA was significantly enhanced in tissues at necropsy (9 wpi, p <0.0001). Viremia in EFTAE treated animals inversely correlated with plasma neutralization titer at time of challenge; however, elevated viremia was not seen in animals treated with 10 and 20 mg/kg Ab, suggesting that complement mediated enhanced infection was mitigated by greater neutralizing activity. These are the first in vivo results substantiating in vitro reports of complement dependent enhanced infection mediated by non-neutralizing HIV Abs and indicate that future studies modulating complement induction by therapeutic bNAbs are warranted.

Xenoantibodies and Complement Activity Determinations by Flow Cytometry in Pig-to-Primate Xenotransplantation.

In pig-to-primate xenotransplantation, flow cytometry assays allow the examination of antibody reactivity to intact antigens in their natural conformation and location on cell membranes. Here we describe in detail the procedures of two flow cytometry assays to measure the antibody-mediated complement-dependent cytotoxicity (CDC) response or serum levels of IgG and IgM xenoantibodies. This information is key for understanding the rejection process of vascularized xenografts and finding strategies to overcome it.

Hexabody-CD38, a Novel CD38 Antibody with a Hexamerization Enhancing Mutation, Demonstrates Enhanced Complement-Dependent Cytotoxicity and Shows Potent Anti-Tumor Activity in Preclinical Models of Hematological Malignancies

HexaBody-CD38 (GEN3014) is a novel, hexamerization-enhanced human IgG1 targeting CD38 with superior complement dependent cytotoxicity (CDC) activity, in addition to other effector mechanisms. HexaBody-CD38 carries the E430G mutation and binds a different epitope than the clinically validated CD38 monoclonal antibody daratumumab, which is currently being established as backbone therapy for the treatment of multiple myeloma. Introduction of the E430G mutationfacilitates the natural process of antibody hexamer formation through increased intermolecular Fc-Fc interactions after antigen binding at the cell surface (Diebolder et al., Science 2014; de Jong et al., PLoS Biol 2016). Improved IgG hexamer formation can increase binding of the hexavalent complement component C1q, thereby potentiating or unlocking antibody-mediated complement-dependent cytotoxicity (CDC). Preclinical data demonstrate highly potent CDC-mediated tumor cell kill in vitro in a panel of cell lines derived from hematological malignancies, including multiple myeloma (MM), B cell lymphoma and acute myeloid leukemia (AML). In these cell lines, at the highest dose tested (10 µg/mL), HexaBody-CD38 induced approximately 2-fold more CDC-mediated lysis compared to daratumumab. Of note, in those cell lines that were responsive to daratumumab in CDC assays (>50% tumor cell lysis), CDC activity of HexaBody-CD38 was superior to daratumumab, with IC50 values for HexaBody-CD38 2.4- to 13-fold lower than for daratumumab. Moreover, HexaBody-CD38 unlocked CDC activity in 17 out of 28 tumor cell lines that were not sensitive to daratumumab in CDC assays (<50% tumor cell lysis), including cell lines with lower expression of CD38 or higher expression of the complement inhibitory protein CD59. Importantly, in pilot experiments that are part of an ongoing larger study, HexaBody-CD38 was able to effectively kill MM cells from patients in CDC assays ex vivo, including in one patient that had relapsed from daratumumab (Figure 1). In addition to superior CDC, HexaBody-CD38 was shown to induce comparable antibody-dependent cell mediated cytotoxicity (ADCC) and antibody-dependent cell mediated phagocytosis (ADCP) as daratumumab. HexaBody-CD38 demonstrated more efficient inhibition of CD38 cyclase activity, which has been postulated to contribute to immune suppression in the tumor microenvironment. Importantly, in the presence of monocytes, HexaBody-CD38 treatment resulted in the removal of CD38 from the cell membrane of CD38 expressing cells, including T regulatory cells. This suggests downmodulation of CD38 as another potential mechanism to reduce CD38-generated metabolites and associated immune suppression. Finally, HexaBody-CD38 induced promising anti-tumor activity in vivo in PDX models of diffuse large B cell lymphoma in nude mice. Anti-tumor activity was associated with CD38 expression levels. In conclusion, HexaBody-CD38 is a novel CD38 antibody that shows superior capacity to induce CDC-mediated tumor cell kill compared to daratumumab, including in tumor samples from MM patients. Furthermore, HexaBody-CD38 induces FcγR-mediated effector functions and effectively inhibits CD38 enzymatic activity, either directly or indirectly by removal of CD38 from the cell membrane, thereby potentially contributing to immune activation. Targeting CD38 with HexaBody-CD38 could have therapeutic potential in daratumumab-naïve and -refractory MM patients, as well as in CD38-positive tumors in which daratumumab does not have single agent efficacy, such as DLBCL and AML. The promise of HexaBody-CD38 warrants further clinical investigation in CD38-positive hematological malignancies, including MM, B cell lymphoma and AML. Disclosures De Goeij: Genmab BV: Employment, Other: stock and/or warrants. Janmaat:Genmab BV: Employment, Other: stock and/or warrants. Andringa:Genmab BV: Employment, Other: stock and/or warrants. Kil:Genmab: Employment, Other: stock and/or warrants. Van Kessel:Genmab: Other: stock and/or warrants. Lingnau:Genmab: Employment, Other: stock and/or warrants. Freidig:Genmab BV: Employment, Other: stock and/or warrants. Mutis:Onkimmune: Research Funding; BMS: Research Funding; Janssen Pharmaceuticals: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Aduro: Research Funding. Sasser:Genmab: Employment, Other: stock and/or warrants. Breij:Genmab: Employment, Other: stock and/or warrants. Van De Donk:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Ahmadi:Genmab Inc: Employment, Other: stock and/or warrants. Satijn:Genmab BV: Employment, Other: stock and/or warrants.

Selective inhibition of cyclooxygenase-2 protects porcine aortic endothelial cells from human antibody-mediated complement-dependent cytotoxicity.

Cyclooxygenase-2 (COX-2) is an inducible enzyme with catalytic activity for biosynthesis of prostaglandins which are the key mediators of inflammation. COX-2 is also the therapeutic target for widely used non-steroidal anti-inflammatory drugs (NSAIDs). However, the involvement of COX-2 in xenotransplantation (eg, pig-to-non-human primate) remains poorly recognized. We investigatedthe mechanisms that regulate COX-2 expression and the effects of COX-2 on porcine aortic endothelial cell (PAEC) viability using in vitro pig-to-primate xenotransplantation modelandin vivopig-to-mouse cellular transplant model. Regulation of COX-2 expression was assessed by real-time quantitative polymerase chain reaction (qPCR) and Western blotting. The effects of inhibition or downregulation of COX-2 on PAEC viability were assessed by propidium iodide (PI)-Annexin V staining and Cell Counting Kit-8 assay. Human serum triggered robust COX-2 expression in PAECs in a dose- and time-dependent manner. Induction of COX-2 expression by human serum was partially through activation of both canonical and non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κb) signaling and increasing intracellular calcium. Cytokines like tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), IL-17, were able to induce COX-2 expression. Selective inhibition of COX-2 by celecoxib dramatically decreased PAEC death in vitro and in vivo as defined by propidium iodide (PI)-Annexin V staining. Consistently, downregulation of COX-2 expression by NF-κb inhibitors or calcium chelator BAPTA decreased human serum-induced PAEC death as well. Silencing of COX-2 expression by small interfering RNA (siRNA) protected PAEC viability when transplanted under kidney capsule of C57BL/6 mice. Taken together, our data suggest that COX-2 is highly induced in PAECs by xenogenic serum and associated with human antibody-mediated complement-dependent cytotoxicity. COX-2 might be a potential therapeutic target to improve xenotransplantation.

Antithyroid antibodies and autoimmune diseases of the thyroid

The significance and mechanisms of action of different antithyroid antibodies in diffuse toxic goiter (Graves' disease) and chronic lymphocytic thyroiditis (Hashimoto’s disease) arc analyzed. Antibodies immediately decreasing the level of cAMP in isolated thyrocytes were revealed in the sera of adolescents with juvenile struma, often resulting from lymphocytic thyroiditis. Complement-fixing cytotoxic antibodies are heterogeneous in patients with Graves' diseases and Hashimoto's thyroiditis. Thyrocytes from the tissue of diffuse toxic goiter are resistant to the cytolytic effect of such antibodies from patients with Graves' diseases but not from patients with Hashimoto's thyroiditis. The causes and mechanisms of development of resistance of thyrocytes from diffuse toxic goiter to antibody-dependent complement-mediated cytotoxicity of sera from patients with Graves' disease and the possibility of using this phenomenon as a differential diagnostic test are discussed.

Severe Sepsis Caused by Listeria monocytogenes in A Patient Given Monoclonal Antibodies Against CD38 and Proteosome Inhibitor

Severe Sepsis Caused by Listeria monocytogenes in A Patient Given Monoclonal Antibodies Against CD38 and Proteosome Inhibitor

TNF-\u03b1 promotes human antibody-mediated complement-dependent cytotoxicity of porcine endothelial cells through downregulating P38-mediated Occludin expression

BackgroundThe major limitation of organ transplantation is the shortage of available organs. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection is a major hurdle for the successful survival of pig xenografts in primate recipients. Cytokines play important roles in inflammation and many diseases including allotransplantation, however, their roles in xenotransplantation have been less well investigated.MethodsWe assessed the role of several cytokines in xenotransplantation using an in vitro model of human antibody-mediated complement-dependent cytotoxicity (CDC). Porcine aortic endothelial cells (PAECs) and porcine iliac endothelial cells (PIECs) were selected as target cells. The complement regulators (CD46, CD55 and CD59) and junction protein genes were assessed by real-time PCR, flow cytometry, or western-blotting assay. Flow cytometry assay was also used to evaluate C3 and C5b-9 deposition, as well as the extent of human IgM and IgG binding to PIECs. Gene silencing was used to reduce genes expression in PIECs. Gene overexpression was mediated by adenovirus or retrovirus.ResultsRecombinant human TNF-α increased the cytotoxicity of PAECs and PIECs in a human antibody-mediated CDC model. Unexpectedly, we found that the expression of complement regulators (CD46, CD55 and CD59) increased in PIECs exposed to human TNF-α. Human TNF-α did not modify C3 or C5b-9 deposition on PIECs. The extent of human IgM and IgG binding to PIECs was not affected by human TNF-α. Human TNF-α decreased the expression of Occludin in PIECs. Gene silencing and overexpression assay suggested that Occludin was required for human TNF-α-mediated cytotoxicity of PIECs in this model. P38 gene silencing or inhibition of P38 signaling pathway with a specific inhibitor, SB203580, inhibited the reduction of Occludin expression induced by TNF-α, and suppressed TNF-α-augmented cytotoxicity of PIECs.ConclusionOur data suggest that human TNF-α increases the cytotoxicity of porcine endothelial cells in a human antibody-mediated CDC model by downregulating P38-dependent Occludin expression. Pharmacologic blockade of TNF-α is likely to increase xenograft survival in pig-to-primate organ xenotransplantation.Graphical abstract

Abstract 4831: Using reporter-based bioassays and engineered TNFalpha and VEGF+ target cells to measure Fc-mediated ADCC and CDC activity of anti-TNFá and anti-VEGF therapeutic antibodies

Abstract Fc receptor-mediated effects contribute to the therapeutic response in terms of efficacy and safety of anti-TNF antibodies. Measurement of Fc-mediated antibody-mediated cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) during antibody drug discovery and development is not only important for antibodies that harness ADCC and/or CDC as their primary mechanism of action (e.g. rituximab, trastuzumab), but also for antibodies designed to target and block soluble ligands such as TNFα and VEGF. We previously reported the development of a cell-based reporter bioassay platform which has been used to measure ADCC and ADCP mediated through FcgRI, FcgRIIa and FcgRIIIa. These reporter bioassays exhibit the specificity, accuracy, precision, and robustness necessary for qualification according to ICH guidelines and have been used extensively to characterize and measure the potency of antibody-based biologics drugs that target cell surface immune receptors. In the current study, we sought to evaluate Fc-mediated ADCC and CDC activities of therapeutic antibodies designed to target and block soluble ligands including TNFa and VEGF. To measure ADCC activity of anti-TNFa and anti-VEGF blocking antibodies, we developed engineered target cells that express either membrane-bound TNFα or VEGF. When used as target cells with reporter-based effector cells expressing a relevant FcgR, ADCC activity of adalimumab (anti-TNFa) and bevacizumab (anti-VEGF) was detected in a specific and dose-dependent manner. Similarly, when used in a luminescence-based CDC assay, the engineered target cells elicited an appropriate FcgR-mediated response. The assay signals demonstrated IgG isotype specificity as IgG4 variants showed minimal activity in both ADCC and CDC assays. Our results demonstrate that the combined use of cell-based reporter bioassays with target cells engineered to express membrane-bound soluble ligands can provide a simple, specific, and quantitative platform to measure Fc-mediated effector functions of therapeutic antibodies targeting soluble ligands. Citation Format: Denise Garvin, Jamison Grailer, Rich Moravec, Jim Hartnett, Chris Heid, Frank Fan, Mei Cong, Jey Cheng. Using reporter-based bioassays and engineered TNFalpha and VEGF+ target cells to measure Fc-mediated ADCC and CDC activity of anti-TNFá and anti-VEGF therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4831.

A potential role of TLR2 in xenograft rejection of porcine iliac endothelial cells: An in vitro study.

Porcine vascular endothelial cells are a major participant in xenograft rejection. The Toll-like receptor 2 (TLR2) pathway plays an important role in both innate and adaptive immunity. The specific role of TLR2 in the response to a xenograft has not been reported. Whether the TLR2 pathway in pig vascular endothelial cells is involved in acute rejection needs to be investigated, and the mechanism is explored. We used a modified antibody-dependent complement-mediated cytotoxicity (ADCC) assay to conduct in vitro experiments. In porcine iliac artery endothelial cells (PIECs), siRNA was used to knock down the expression of TLR2, CXCL8, and CCL2. The effect of human serum or inactivated human serum on the expression of TLR2 was analyzed by real-time PCR and Western blotting, and transwell assays were used to assess the chemotactic efficiency of PIECs on human monocyte-macrophages (THP-1 cells) and human neutrophils. The downstream signaling pathways activated by human serum were detected by Western blotting, and the regulation of proinflammatory chemokines and cytokines by TLR2 signaling was assessed by real-time PCR and ELISA. TLR2 was significantly upregulated in PIECs after exposure to human serum, and porcine proinflammatory chemokines, CXCL8 and CCL2, were induced, at least partially, in a TLR2-dependent pattern; the upregulated chemokines participated in the chemotaxis of human neutrophils and THP-1 cells across the species barrier. (i) TLR2 is significantly upregulated in PIECs by human serum, (ii) the elevated TLR2 participates in the chemotaxis of inflammatory cells through the secretion of chemokine CCL2 and CXCL8, and (iii) blockade of TLR2 would be beneficial for xenograft survival.

Indoleamine 2,3-dioxygenase suppresses humoral alloimmunity via pathways that different to those associated with its effects on T cells.

Chronic antibody-mediated rejection remains a major cause of late graft loss. Regarding cellular alloimmunity, the immunosuppressive properties of indoleamine 2,3-dioxygenase (IDO) have been well investigated; however, little is known of its effects on humoral alloimmunity. Therefore, the present study aimed to evaluate the effects of IDO on humoral alloimmunity. We developed a method for the induction of humoral alloimmunity in a one-way mixed lymphocyte reaction (MLR), which was measured with an antibody-mediated complement-dependent cytotoxicity assay using resting cells, which are similar to the stimulator cells of the aforementioned MLR. In parallel, cellular alloimmunity was assessed in two-way MLRs. The IDO inhibitor 1-methyl-DL-tryptophan was used for evaluating the role of IDO. In order to investigate whether the pathways known to serve a role in the effects of IDO on T cells are applied in humoral alloimmunity, the general control nonderepressible-2 (GCN-2) kinase activator tryptophanol and the aryl hydrocarbon receptor (AhR) inhibitor CH223191 were employed. The IDO inhibitor was revealed to increased cellular autoimmunity, but was decreased by the GCN-2 kinase activator. Unexpectedly, the AhR inhibitor decreased cellular alloimmunity. In addition, the IDO inhibitor was observed to suppress humoral alloimmunity, which may occur in manners independent of GCN-2 kinase AhR. The present study proposed that IDO may decrease humoral alloimmunity in primary human peripheral blood mononuclear cells via pathways that differ to those associated with its effect on T cells.

Upregulation of Complement Factor H by SOCS-1/3⁻STAT4 in Lung Cancer.

Complement factor H (CFH) is a fluid phase regulator of complement proteins and functions to prevent complement attack and immune surveillance. CFH is known to inactivate therapeutic antibody-dependent complement-mediated cellular cytotoxicity. We found that CFH was highly expressed in human lung cancer cells and tissues. To investigate mechanisms of CFH upregulation, we searched for a CFH transcription factor and its regulatory factors. First, signal transducer and activator of transcription 4 (STAT4) expression patterns coincided with CFH expression patterns in lung cancer tissues. Knockdown of STAT4 led to decreased CFH secretion from lung cancer cells. STAT4 bound directly to the CFH promoter, as demonstrated by luciferase reporter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay, suggesting that STAT4 is a transcription factor for CFH. In addition, a low level of suppressors of cytokine signaling (SOCS)-1/3, a Janus kinase (JAK) inhibitor, was observed in lung cancer cells and its transfection decreased CFH protein levels and promoter activity. Unexpectedly, the low level of SOCS-1/3 was not due to epigenetic silencing. Instead, differential methylation was found on the regulatory region of STAT4 between normal and lung cancer cells. In conclusion, our results demonstrated that CFH is upregulated by constitutive activation of STAT4, which is accounted for by SOCS silencing in lung cancer cells.