Complement-dependent Cytotoxicity Assay Research Articles

Uric acid increases cellular and humoral alloimmunity in primary human peripheral blood mononuclear cells.

Hyperuricaemia is common among kidney transplant recipients and has been associated with worse graft outcome. Since episodes of acute cellular rejection and chronic humoral rejection contribute to decreased graft survival, in this study the effect of uric acid on cellular and humoral alloimmunity was evaluated. Cellular alloimmunity was assessed by cell proliferation in two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity we developed a method in which humoral alloimmunity was induced in one-way MLR. Then the de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MRLs were used against resting PBMC similar to the stimulator cells of the above MLRs. Uric acid at a concentration above its crystallization threshold increased cellular proliferation in two-way MLRs. Supernatants from one-way MLRs performed in the presence of uric acid were more cytotoxic against PBMC from individuals that had conferred the stimulator cells for the above MLRs. Uric acid increases both cellular and humoral alloimmunity in human PBMC. These results offer a possible pathogenetic mechanism for the observed relation between hyperuricaemia and worse kidney allograft survival.

Class II Human Antigen in Iraqi patients with Hodgkin’s Lymphoma

Objective: To detect the association between human leukocyte antigen) HLA) class II and Iraqi patients with Hodgkin’s lymphoma (hl). Subject and methods: Study groups include 80 newly diagnosed Hodgkin’s lymphoma patients and two control group patients control which include 50 patients who were newly diagnosed to be affected by non Hodgkin’s lymphoma (NHL) and healthy individuals. Antibody mediated complement dependent cytotoxicity assay was done by treating sample of patients lymphocytes with a panel of anti-HLA antisera and complement. Results: The frequency expression of hla-dr3 and DQ1 was significantly greater in Hodgkin’s lymphoma patients than healthy control group with (p<0.001) for HLA-DR3 and (p<0.005) for DQ1. Conclusion: Hla-dr3 and dq1 are more related with Hodgkin’s lymphoma in Iraqi Arab population that reflect HLA alleles have immunogenetic factors of predisposition to Hodgkin lymphoma

Immunization of mice with egG1Y162-1/2 provides protection against Echinococcus granulosus infection in BALB/c mice

AimsThis study is to predict and purify the T-B combined epitopes of egG1Y162 antigen in Echinococcus granulosus, and to evaluate their immunogenicity in mice. MethodsThe bioinformatics software was used to predict the T-B combined epitopes of egG1Y162 antigen. Recombinant egG1Y161/2 peptides were constructed, expressed and purified. Mice were immunized with egG1Y161/2 peptides. The serum and spleen cells were isolated. The isolated spleen cells were stimulated with egG1Y161/2 peptides in vitro and the culture supernatant was collected. The levels of IgG in serum and levels of IL-4 and IFN-γ in the culture supernatant were measured by ELISA. The weight and number of the fresh hydatid cysts were evaluated. The serum ptotoscolicidal activity was measured by the complement dependent cytotoxicity assay. ResultsPeptides of 6–19aa, 64–82aa, 106–119aa were predicted as T-B combined epitopes of egG1Y162 antigen. And, recombinant protein egG1Y162-1 or egG1Y162-2, which contained T-B combined epitope(s) of the 6–19aa, or the 64–82aa and the 106–119aa in egG1Y162 antigen, respectively, was successfully expressed and purified. Serum IgG levels of mice immunized with egG1Y162-1/2 were significantly increased during the immune response to Echinococcus granulosus. The levels of IFN-γ, IL-4 and the ratio of IFN-γ/IL-4 after egG1Y162-1/2 immunization were significantly higher. Weight and number of the fresh hydatid cysts in egG1Y162-1/2 immunized mice was significantly decreased. And, the serum protoscolicidal activity after egG1Y162-1/2 immunization was enhanced. ConclusionsThe egG1Y162-1/2 induces production of serum IgG levels and Th1 cell immune response, which enhances the protective immunity in Echinococcus granulosus challenged mice and thus may be used as a potential vaccine candidate.

Comparison of the effect of the aerobic glycolysis inhibitor dichloroacetate and of the Krebs cycle inhibitor LW6 on cellular and humoral alloimmunity.

Cell metabolism is altered during T-cell and B-cell activation and differentiation. Clarifying the exact metabolic shifts may lead to the development of new immunosuppressive medications. In this study, the effect of the aerobic glycolysis inhibitor dichloroacetate (DCA) and of the Krebs cycle enzyme malate dehydrogenase 2 (MDH2) inhibitor LW6 on T-cell alloimmune clonal expansion and on alloantibody production, was evaluated. T-cell clonal expansion was assessed in two-way mixed lymphocyte reaction (MLR). Humoral alloimmunity was evaluated by the alloantibody production in one-way MLR. For this purpose, an antibody-mediated complement-dependent cytotoxicity assay was developed in which the supernatants from one-way MLRs were used against resting peripheral blood mononuclear cells (PBMCs) derived from the same individual that contributed the stimulator cells for the respective MLR. DCA had a minimum effect on alloimmune T-cell clonal expansion, whereas it increased humoral immunity significantly. LW6 decreased both alloimmune T-cell proliferation and alloantibody production. The results indicate that MDH2 may be a perfect target for the development of new immunosuppressive medications, especially when inhibition of both cellular and humoral alloimmunity is desirable.

P045 Complement binding to single antigen beads is not directly related to antibody strength as determined in pairwise comparative studies

Aim Optical bead microarrays using Luminex have largely replaced anti-HLA antibody screening tests using cell based complement dependent cytotoxicity assays. One unintended consequence of this technologic development was the loss of the ability to distinguish between antibodies that fix complement from those that do not. This situation may have been addressed by the introduction of several assays that use the bead microarrays in conjunction with a reagent that detects whether components of the complement cascade are bound to the anti-HLA antibody – HLA complex on a reactive bead. One controversy centering on this technology has been the suggestion that the binding of complement in bead assays is dependent to a greater extend on the strength of the anti-HLA antibody rather than its complement binding activity. To address this issue we examined complement binding in single antigen bead (SAB) assays in sera that had multiple epitope specificities of different strengths. Methods Patient sera and sera from proficiency surveys were used. SAB assays of these sera were compared to C1q/C3d – SAB assays. The bead with the highest net MFI for each identified epitope in the microarray was recorded and compared to the same bead in C1q/C3d – SAB assays. Results We detected both strong and weak anti-HLA antibodies in SAB assay that were detected in C1q/C3d – SAB assays. In some sera complement bound to one set of epitope reactive SABs but not to another epitope set of SABs. Importantly, this was not dependent on the strength of the antibody. For example, in one patient’s sera, an epitope defined antigen bead, B∗42:01 (epitope 65GK), had an MFI of 12,989 (normalized score) in the SAB assay but only 99 MFI in the C1q – SAB assay. In the same serum another epitope defined bead (80 N), A∗23:01 with an MFI of 10,445 in the SAB assay exhibited an MFI of 9,162 in the C1q – SAB assay. We also observed the latter finding for HLA class 2 SABs and with the C3d assay. Conclusions Complement binding to SABs is not directly related to strength of the antibody as determined in pairwise comparative studies using sera that contained antibodies with different epitope specificities. C1q/C3d - SAB assays are therefore useful in evaluating the complement binding properties of pretransplant and post transplant anti-HLA antibodies.

P066 Structural aspects of HLA class I epitopes reacting with human alloantiserum

Aim Interaction between complementarity determining regions (CDR) of the antibody (Ab) and HLA (contact surface area of 700–900 A2) can be detected by mean fluorescence intensity (MFI) or lysis of cell membrane by complement dependent cytotoxicity (CDC). These biological functions of Ab depend on amino acid residue (AAR) composition of functional and structural epitopes (Ep) of the antigen. The aim of this study was to analyze Ep structure of HLA in order to interpret reactivity of Abs in the serum of kidney recipient (KR). Methods Ab analysis (IgG) and complement binding activity (C1q test) of alloantibodies were analyzed using solid phase single antigen (One Lambda, Inc.) (positive cut off MFI = 280–500) and cell based CDCx3 washes assays. Ep analysis was conducted by using HLAMatchmaker computer algorithm (MFI = 1000 was used as a positive cut off). Results Various combinations of four (63EE, 82LR, 127K, and 113YH) eplet-defined Eps were detected in HLAs Abs in KR serum reacted with, when SAA was applied. Consensus of AAR within 15 A radius of aforementioned eplets comprised sixteen positions. The results of reactivity of specific eplet carrying alleles in three assays are presented in Fig. 1. Our findings indicate that Ab reactivity is associated with AAR composition of structural Eps involved in contact with CDR of IgG. Binding to HLA likely depends on AARs at positions 44, 152, 156, and 158, whereas C1q reactivity is affected by AARs at 9H/24T and 163E/167S positions, and CDC reactivity is abolished by tryptophan (W) at 95 position. Conclusions Biological function of anti-HLA Ab requires not only the specific recognition of eplets but also depends on the interactions of their CDRs with structural Eps within the contact surface area. C1q + test does not always predict a positive CDC assay. The results of this study represent a new insight into organ allocation, immunomodulation therapy, and post-transplant Ab monitoring in solid organ recipients. Download : Download high-res image (603KB) Download : Download full-size image

Cytokine profiles in Tibetan macaques following α-1,3-galactosyltransferase-knockout pig liver xenotransplantation.

Pig-to-nonhuman primate orthotopic liver xenotransplantation is often accompanied by thrombocytopenia and coagulation disorders. Furthermore, the release of cytokines can trigger cascade reactions of coagulation and immune attacks within transplant recipients. To better elucidate the process of inflammation in liver xenograft recipients, we utilized a modified heterotopic auxiliary liver xenotransplantation model for xeno-immunological research. We studied the cytokine profiles and the relationship between cytokine levels and xenograft function after liver xenotransplantation. Appropriate donor and recipient matches were screened using complement-dependent cytotoxicity assays. Donor liver grafts from α1,3-galactosyltransferase gene-knockout (GTKO) pigs or GTKO pigs additionally transgenic for human CD47 (GTKO/CD47) were transplanted into Tibetan macaques via two different heterotrophic auxiliary liver xenotransplantation procedures. The cytokine profiles, hepatic function, and coagulation parameters were monitored during the clinical course of xenotransplantation. Xenograft blood flow was stable in recipients after heterotopic auxiliary transplantation. A Doppler examination indicated that the blood flow speed was faster in the hepatic artery (HA) and hepatic vein (HV) of xenografts subjected to the modified Sur II (HA-abdominal aorta+HV-inferior vena cava) procedure than in those subjected to our previously reported Sur I (HA-splenic artery+HV-left renal vein) procedure. Tibetan macaques receiving liver xenografts did not exhibit severe coagulation disorders or immune rejection. Although the recipients did suffer from a rapid loss of platelets, this loss was mild. In blood samples dynamically collected after xenotransplantation (post-Tx), dramatic increases in the levels of monocyte chemoattractant protein 1, interleukin (IL)-8, granulocyte-macrophage colony-stimulating factor, IL-6, and interferon gamma-induced protein 10 were observed at 1hour post-Tx, even under immunosuppression. We further confirmed that the elevation in individual cytokine levels was correlated with the onset of graft damage. Finally, the release of cytokines might contribute to leukocyte infiltration in the xenografts. Here, we established a modified auxiliary liver xenotransplantation model resulting in near-normal hepatic function. Inflammatory cytokines might contribute to early damage in liver xenografts. Controlling the systemic inflammatory response of recipients might prevent early post-Tx graft dysfunction.

Generation of complement protein C3 deficient pigs by CRISPR/Cas9-mediated gene targeting

Complement protein C3 is the pivotal component of the complement system. Previous studies have demonstrated that C3 has implications in various human diseases and exerts profound functions under certain conditions. However, the delineation of pathological and physiological roles of C3 has been hampered by the insufficiency of suitable animal models. In the present study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the C3 gene in porcine fetal fibroblasts. Our results indicated that CRISPR/Cas9 targeting efficiency was as high as 84.7%, and the biallelic mutation efficiency reached at 45.7%. The biallelic modified colonies were used as donor for somatic cell nuclear transfer (SCNT) technology to generate C3 targeted piglets. A total of 19 C3 knockout (KO) piglets were produced and their plasma C3 protein was undetectable by western blot analysis and ELISA. The hemolytic complement activity and complement-dependent cytotoxicity assay further confirmed that C3 was disrupted in these piglets. These C3 KO pigs could be utilized as a valuable large animal model for the elucidation of the roles of C3.

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

A comparative analysis between proteasome and immunoproteasome inhibition in cellular and humoral alloimmunity

Triggered by the successful administration of the proteasome inhibitor bortezomib in kidney transplant recipients with acute or chronic antibody-mediated rejection, we evaluated the effect of the proteasome inhibitor CEP-18770 and of the selective immunoproteasome inhibitor ONX-0914 on cellular and humoral alloimmunity. Cellular alloimmunity was assessed by cell proliferation in a two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity we developed a method, where humoral alloimmunity was induced in one-way MLR. The de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MLRs were used against resting PBMC similar to the stimulator cells of the forementioned MLRs. In two-way MLRs ONX-0914 inhibited cell proliferation more than CEP-18770. In one-way MLRs CEP-18770 and ONX-0194 decreased alloantibody production to the same extent. Inhibition of the immunoproteasome is superior to inhibition of the proteasome in suppressing cellular alloimmunity, and equally effective as regards to humoral alloimmunity. Considering the selective expression of the immunoproteasome in immune cells and the expected restrictive toxicity of its inhibitors, these results render immunoproteasome an excellent target for the development of new immunosuppressive medications in the field of transplantation.

Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs.

As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.

A novel MAGE-A1-IgG antibody for lung adenocarcinoma.

e20085 Background: Melanoma-associated antigen A1 (MAGE-A1) has been reported to perform oncogenically and act as an ideal candidate in tumor immunotherapy. Methods: One-step quantitative real-time polymerase chain reaction (qPCR) test and immunohistochemistry (IHC) analysis were used to detect the relationship between MAGE-A1 expression and the clinicopathological characteristics of lung adenocarcinoma (LAC). Then a novel MAGE-A1 IgG antibody was constructed on the previous MAGE-A1 scFv antibody by eukaryotic expression vectors. CCK-8, wound healing, transwell, matrigel, apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC) and CDC (complement-dependent cytotoxicity) assays were performed to evaluate the inhibitory effects of MAGE-A1-IgG on LAC in vitro. Results: High MAGE-A1 expression was observed in LAC tissues and MAGE-A1 expression was associated with several malignant properties of LAC patients, including overall survival. MAGE-A1-IgG revealed significant roles in inhibiting proliferation, inducing apoptosis and activating ADCC and CDC of LAC in a dose- and time- dependent manner in vitro. Conclusions: MAGE-A1-IgG may provide a promising therapeutic strategy for the treatment of MAGE-A1-positive LAC.

First two bilateral hand transplantations in India (Part 4): Immediate post-operative care, immunosuppression protocol and monitoring.

Introduction:Being able to counter immune-mediated rejection has for decades been the single largest obstacle for the progress of vascular composite allotransplantation (VCA). The human immune system performs the key role of differentiating the 'self ' from the 'non-self '. This, although is quintessential to eliminate or resist infections, also resists the acceptance of an allograft which it promptly recognises as 'non-self'.Materials and Methods:Pre-operative evaluation of the recipient evaluation included immunological assessment in the form of panel reactive antibodies (PRA), human leucocyte antigen (HLA) typing, donor-specific antibody detection assays (DSA) and complement-dependent cytotoxicity assays (CDC). Induction immunosuppression was by thymoglobulin and the maintenance by the standard triple-drug therapy.Results:Both the recipients were managed by the standard triple drug therapy and have had only minor episodes of rejections thus far which have been managed appropriately.Discussion:Induction immunosuppression was by thymoglobulin and the maintenance by the standard triple-drug therapy. Various groups have tried various other formulations and regimes as well.Conclusion:A comprehensive plan has to be drawn up for immunological screening, selection and the post-operative immunosuppressant usage. The ultimate goal of these immunosuppression modalities is to achieve a state of donor-specific tolerance.

Unexpected Positive Prospective Crossmatches in Organ Transplant.

Preformed donor-specific antibodies against human leukocyte antigen can induce antibody-mediated rejection after organ transplant. Hence, future transplant recipients undergo pretransplant screening for preformed antibodies (ie, virtual crossmatch). Subsequently, prospective (analytic) crossmatching is performed using conventional, complement-dependent cytotoxicity assays and/or flow cytometry-based methods. The present article reviews factors that must be considered when unexpected, positive, prospective crossmatches are observed. First, the prozone effect caused by the interference of complement or immunoglobulin M must be abrogated by treating the serum with moderate heat, dilution, hypotonic dialysis, EDTA, or dithiothreitol. Second, the physician must check for the presence of potentially interfering autoantibodies (in a context of autoimmune disease or human immunodeficiency virus infection) or therapeutic antibodies (such as rituximab and antithymocyte globulin). In conclusion, knowledge of each assay's technical characteristics will enable the physician to reliably interpret any discrepancies. The reasons for an unexpected, positive, prospective crossmatch must be elucidated before transplant to ensure efficient organ allocation and optimize patient outcomes.

Abstract B66: An integrated immuno-oncology platform using high-throughput cell based assays, gene editing and genomic screens in immune cells

Abstract Horizon is establishing a powerful in vitro immuno-oncology platform with the capability to identify combinations of agents that will synergise with checkpoint inhibitors, determine methods for making safer and more effective cell therapies and find novel targets for immuno-therapy. Horizon previously reported the establishment of a platform consisting of a suite of high-throughput cell-based assays modelling a variety of oncology relevant immune reactions. The platform includes primary human immune-cell based assays for T cell activation, Mixed lymphocyte reaction (MLR), Tumor cell lysis, Antibody-dependent cellular cytotoxicity (ADCC), Complement-dependent cytotoxicity (CDC) and Natural killer cell cytotoxicity assays that have been validated with appropriate clinically approved antibodies (e.g. nivolumab, blinotumamab, & rituximab). Miniaturized to a 384-well format and supported by automation at each experimental step the platform is highly customizable in terms of testing agents that either enhance or inhibit immune cell functions. We will present ongoing efforts to identify synergistic activities with other relevant therapeutics likely to bring benefits to patients. In addition to the continued development of our high throughput immuno-oncology cell based assays, we have expanded our cell engineering know how combined with CRISPR-Cas9 gene editing technology to build new models to understand better the immune response and its subversion in tumorigenesis. By modifying specific genes of interest in immune cells we aim to robustly identify and validate new targets for the clinic. For example, we have used high efficiency gene editing with CRISPR-Cas9 in human primary T cells to knock out and knock in genes using Neon transfection systems. Using this methodology we can rapidly generate primary T cell models lacking specific checkpoint proteins (such as PD-1) to better understand how T-cell signalling pathways interact and to nominate novel targets suitable for ex vivo gene editing. We have also employed pooled CRISPR-Cas9 screens to investigate the effects of metabolic changes on T cell biology. CD3+ T cells have been infected with a one vector lentiviral system that delivers both Cas9 and sgRNAs targeting genes involved in cellular metabolism. Results from these screens should identify potential targets involved in T cell metabolism that affect their capacity to respond to proliferative stimuli in the form of anti-CD3 and anti-CD28 antibodies. We anticipate that these and other data generated using libraries targeting essential genes will be invaluable for the design of more complex immuno-oncology screens. Overall, Horizon's integrated immuno-oncology platform will enable large scale interrogation of prospective immunotherapies, either alone or in combination, and could be useful for the discovery of novel checkpoint components, for deciphering the underlying mechanisms promoting an immunosuppressive tumor microenvironment and for the understanding of how pathways crucial for an anti-tumor immune response interact. Thus, this platform could contribute substantially to the discovery and development of future immunotherapies. Citation Format: An Frank, Sujatha Kumar, Christina Ghirelli, Kim Hoenderdos, Tabasum Huseni, Lauren Thibault, Lydia Kifle, Nava Almog, Felicia Zhao, Simon Scrace, Anatoly Myaskovsky, Chris Lowe, Janine Steiger, Nicola McCarthy, Jonathan Moore. An integrated immuno-oncology platform using high-throughput cell based assays, gene editing and genomic screens in immune cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B66.

Marked central nervous system pathology in CD59 knockout rats following passive transfer of Neuromyelitis optica immunoglobulin G

Neuromyelitis optica spectrum disorders (herein called NMO) is an inflammatory demyelinating disease of the central nervous system in which pathogenesis involves complement-dependent cytotoxicity (CDC) produced by immunoglobulin G autoantibodies targeting aquaporin-4 (AQP4-IgG) on astrocytes. We reported evidence previously, using CD59−/− mice, that the membrane-associated complement inhibitor CD59 modulates CDC in NMO (Zhang and Verkman, J. Autoimmun. 53:67–77, 2014). Motivated by the observation that rats, unlike mice, have human-like complement activity, here we generated CD59−/− rats to investigate the role of CD59 in NMO and to create NMO pathology by passive transfer of AQP4-IgG under conditions in which minimal pathology is produced in normal rats. CD59−/− rats generated by CRISPR/Cas9 technology showed no overt phenotype at baseline except for mild hemolysis. CDC assays in astrocyte cultures and cerebellar slices from CD59−/− rats showed much greater sensitivity to AQP4-IgG and complement than those from CD59+/+ rats. Intracerebral administration of AQP4-IgG in CD59−/− rats produced marked NMO pathology, with astrocytopathy, inflammation, deposition of activated complement, and demyelination, whereas identically treated CD59+/+ rats showed minimal pathology. A single, intracisternal injection of AQP4-IgG in CD59−/− rats produced hindlimb paralysis by 3 days, with inflammation and deposition of activated complement in spinal cord, optic nerves and brain periventricular and surface matter, with most marked astrocyte injury in cervical spinal cord. These results implicate an important role of CD59 in modulating NMO pathology in rats and demonstrate amplification of AQP4-IgG-induced NMO disease with CD59 knockout.

Assignment of C1q-binding HLA antibodies as unacceptable HLA antigens avoids positive CDC-crossmatches prior to transplantation of deceased donor organs

Soon, a virtual crossmatch shall replace the complement-dependent cytotoxicity (CDC) allocation crossmatch in the Eurotransplant region. To prevent positive CDC-crossmatches in the recipient centre, careful definition of unacceptable antigens is necessary. For highly sensitized patients, this is difficult by CDC alone. Assignment of all antibodies detected by sensitive assays, however, could prevent organ allocation. To assess the usefulness of the Luminex C1q-assay to prevent positive CDC-crossmatches, all CDC-crossmatches performed prior to deceased kidney transplantation in a 16-month-period were reviewed. Sera causing positive crossmatches were investigated by the C1q-assay. 31 out of 1432 crossmatches (2.2%) were positive. Sera involved in 26 positive crossmatches were available. C1q-binding donor-specific antibodies were detected in 19 sera (73.1%). The other sera were from recipients without any HLA antibodies detectable by CDC or common solid phase assays. Three patients had known Non-HLA antibodies causing positive CDC-results. Four crossmatches were only weak positive. Therefore, avoidance of donors with HLA antigens against whom C1q-binding antibodies were detected would have prevented all positive crossmatches due to HLA antibodies. Provided that all HLA specificities against which antibodies are detected by the Luminex C1q-assay are considered as unacceptable antigens, CDC-crossmatches prior to transplantation might safely be omitted in many patients. They should be maintained in highly immunized patients, however, for whom assignment of all C1q-positive antibodies as unacceptable antigens could lead to a significant delay or even prevention of transplantation.

Case-based review of cross-match techniques in kidney transplantation

To avoid renal allograft rejection and loss, human leucocyte antigen compatibility and lymphocytotoxic cross-matching of renal donors against prospective renal allograft recipients are obligatory and fundamental steps in pretransplant evaluation. The complement-dependent cytotoxicity assay has been the mainstay technique for ascertaining the presence of donor-specific antibodies since the 1970s. Improvement in the performance of cross-matching has seen the introduction of more sensitive and specific techniques such as flow cytometry, and highly sensitive enzyme-linked immunosorbent assay, Luminex platforms and virtual cross-match. This review article is an in-depth comparison of the advantages and disadvantages of each of the cross-match techniques. We demonstrate the application of these techniques through a clinical case scenario. A step-by-step analysis and interpretation of cross-match modalities of varying levels of sensitivity and specificity is needed to arrive at a pragmatic decision, which will lead to better transplantation outcome. In some patients with immunologically complex issues, a spectrum of cross-match techniques may be required for a safe outcome of transplant.

Correlation of Pretransplant Donor-specific Antibody Assay Using Luminex Crossmatch with Graft Outcome in Renal Transplant Patients.

The significance of pretransplant anti-human leukocyte antigen antibody levels that are detectable by more sensitive platforms (including the Luminex platform) yet undetected by complement-dependent cytotoxicity (CDC) assay remains unclear. The aim of this study was to determine the clinical significance of the donor-specific antibody (DSA) assay Luminex crossmatch and its impact on short-term renal graft outcome such as acute rejections, graft survival, and graft function. The results of pretransplant DSA-lymphocyte crossmatching (LCXM) assay in 126 renal allograft recipients whose CDCs crossmatches were negative were retrospectively analyzed for correlation with posttransplant outcomes. Of the 126 recipients, 32 (25.4%) had pretransplant DSA positive. Statistically significant association was found between DSA-LCXM positivity with 14th day estimated glomerular filtration rate (eGFR) (P = 0.05), DSA Class I with 3rd (P = 0.014) and 6th month (P = 0.02) eGFR, DSA Class II with 14th day (P = 0.06) and 1st month (P = 0.10) eGFR, mean fluorescent intensity (MFI) DSA with 7th day (P = 0.08) and 14th day (P = 0.09) eGFR, and maximum MFI DSA with 7th day eGFR (P = 0.09). The posttransplant eGFR was higher at various time intervals in DSA-LCXM-negative patients as compared to DSA-positive patients. However, pretransplant DSA-LCXM results did not predict the rejection episodes, graft loss, and 1-year posttransplant 24 h urine protein. Pretransplant DSA detected by LCXM in patients with a negative CDC does not predict adverse short-term outcomes. However, the difference in posttransplant eGFR supports further investigation in long-term effects.

A neutralized human LMP1-IgG inhibits ENKTL growth by suppressing the JAK3/STAT3 signaling pathway.

Latent membrane protein 1 (LMP1), which is associated with the development of different types of Epstein-Barr virus (EBV) related lymphoma, has been suggested to be an important oncoprotein. In this study, a human anti-LMP1 IgG antibody (LMP1-IgG) was constructed and characterized by ELISA, western blotting (WB), affinity and immunohistochemistry (IHC) analyses. CCK-8, MTT, apoptosis assays, antibody-dependent cell-mediated cytotoxicity (ADCC) and CDC (complement-dependent cytotoxicity) assays were performed to evaluate the inhibitory effects of LMP1-IgG on extranodal nasal-type natural killer (NK)/T-cell lymphoma (ENKTL). Then, the influence of LMP1-IgG on the JAK/STAT signaling pathway was investigated. The results showed that the successfully constructed LMP1-IgG inhibited proliferation, induced apoptosis, and activated ADCC and CDC of ENKTL in a concentration- and time- dependent manner. Moreover, phosphorylation of JAK3 and STAT3 was inhibited by LMP1-IgG. Our data indicate that LMP1-IgG may provide a novel and promising therapeutic strategy for the treatment of LMP1-positive ENKTL.