Exploration of complement-dependent cytotoxicity technology for GTKO/hCD55 genetically engineered pig-to-human xenotransplantation / 中华器官移植杂志

Objective:To compare the inhibitory effect of human CD55(hCD55)expressed on porcine peripheral blood mononuclear cells(PBMC)on rabbit complement-and human complement-dependent cytotoxicity(CDC).Methods:Three α1, 3-galactosyltransferase gene knockout(GTKO)pigs from the same strain were selected.Two were transferred with hCD55 gene.According to the expression level of hCD55, the animals were divided into three groups of GTKO, GTKO/hCD55 Low(low-expression of hCD55)and GTKO/hCD55 High(high-expression of hCD55). After PBMC from these pigs were incubated with complement-inactivated pooled human serum(20 cases), rabbit complement-or human complement-dependent cytotoxicity and binding of antibodies(IgM/G)to pig PBMC were detected by flow cytometry. Results:No significant difference existed in binding of human serum xenoreactive antibodies to PBMC from three groups.The cytotoxicity to GTKO pig PBMC mediated by rabbit complement or human complement were 98.97%±0.50% and 82.73%±3.20% respectively.Both values were quite high.Compared with GTKO group, a low expression of hCD55 had no significant inhibitory effect on rabbit complement-dependent cytotoxicity(97.07%±2.25% vs. 98.9%±0.50%, P=0.2 267) while a high expression of hCD55 exerted a mild inhibitory effect on rabbit complement-dependent cytotoxicity(81.70%±5.86% vs. 98.9%±0.50%, P=0.0 355). Differently, a low expression of hCD55 had a potent inhibitory effect on human complement-dependent cytotoxicity(23.83%±3.53% vs. 82.73%±3.20%, P<0.0 001). Compared with hCD55 low-expression group, a high expression of hCD55 had a further inhibitory effect on human complement-dependent cytotoxicity(2.79%±0.45% vs. 82.73%±3.20%, P=0.009), attaining the level of negative control group.The inhibitory effect of low/high expression of hCD55 on human complement-mediated CDC was significantly better than that on rabbit complement-mediated CDC. Conclusions:Compared with traditional CDC counterpart using rabbit complement, modified CDC technology of commercial standard human complement is recommended for evaluating the regulatory effect of hCD55 expressed on cell surface from GTKO/hCD55 genetically engineered pigs.It thus provides experimental rationales for establishing a novel CDC experimental system of effectively evaluate the function of hCD55 after xenotransplantation.

Myelin Oligodendrocyte Glycoprotein-IgG Contributes to Oligodendrocytopathy in the Presence of Complement, Distinct from Astrocytopathy Induced by AQP4-IgG / 神经科学通报·英文版

Immunoglobulin G against myelin oligodendrocyte glycoprotein (MOG-IgG) is detectable in neuromyelitis optica spectrum disorder (NMOSD) without aquaporin-4 IgG (AQP4-IgG), but its pathogenicity remains unclear. In this study, we explored the pathogenic mechanisms of MOG-IgG in vitro and in vivo and compared them with those of AQP4-IgG. MOG-IgG-positive serum induced complement activation and cell death in human embryonic kidney (HEK)-293T cells transfected with human MOG. In C57BL/6 mice and Sprague-Dawley rats, MOG-IgG only caused lesions in the presence of complement. Interestingly, AQP4-IgG induced astroglial damage, while MOG-IgG mainly caused myelin loss. MOG-IgG also induced astrocyte damage in mouse brains in the presence of complement. Importantly, we also observed ultrastructural changes induced by MOG-IgG and AQP4-IgG. These findings suggest that MOG-IgG directly mediates cell death by activating complement in vitro and producing NMOSD-like lesions in vivo. AQP4-IgG directly targets astrocytes, while MOG-IgG mainly damages oligodendrocytes.

IgG Fc engineering to modulate antibody effector functions

Therapeutic monoclonal antibodies are among the most effective biotherapeutics to date. An important aspect of antibodies is their ability to bind antigen while at the same time recruit immune effector functions. The majority of approved recombinant monoclonal antibody therapies are of the human IgG1 subclass, which can engage both humoral and cellular components of the immune system. The wealth of information generated about antibodies has afforded investigators the ability to molecularly engineer antibodies to modulate effector functions. Here, we review various antibody engineering efforts intended to improve efficacy and safety relative to the human IgG isotype. Further, we will discuss proposed mechanisms by which engineering approaches led to modified interactions with immune components and provide examples of clinical studies using next generation antibodies.

A Questionnaire Survey of HLA Crossmatch Tests in Korea (2015)

BACKGROUND: We carried out a questionnaire survey for laboratories performing human leukocyte antigen-crossmatch (HLA-XM) to provide a basis for laboratory standardization of HLA-XM tests in Korea. METHODS: The questionnaires were distributed to 51 HLA laboratories participating in the HLA-XM part of the HLA proficiency survey program organized by the Korean Society for Laboratory Medicine and replies from 50 laboratories were analyzed. The questionnaires included following items: 1) HLA-XM methods performed and annual number of tests, 2) types of the specimen and lymphocyte separation methods, 3) test procedures and reagents for complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FCXM). RESULTS: The number of laboratories performing anti-human globulin (AHG) CDC-XM (47/49, 96%) and FCXM (30/50, 60%) was considerably increased compared to the 2005 survey (AHG CDC-XM, 35/43, 81%; FCXM, 7/44, 16%). As for the annual number of XM tests, more than 50% of the laboratories were low volume laboratories performing ≤50 tests, and only 10% of the laboratories were performing >500 tests. For cell isolation methods, negative selection was used by 43% (21/49) of laboratories performing CDC-XM. Number of cells reacted per 1 µL of serum varied among different laboratories in both CDC-XM (1,000–8,000) and FCXM tests (1,300-20,000). For the interpretation of FCXM, log fluorescence ratio (26/30, 87%) was more commonly used than channel shift values (5/30, 17%). CONCLUSIONS: Considerable variation is noted in both CDC-XM and FCXM methods performed by different laboratories. A continuous effort for laboratory standardization is needed to reduce inter-laboratory variation in the HLA-XM test results.

False-Positive T-Cell Cytotoxicity Crossmatch Results Due to Autoantibodies in Korean Network for Organ Sharing Crossmatch Tests / 대한이식학회지

BACKGROUND: Basic National Institute of Health (NIH) and sensitive antihuman globulin (AHG) methods are widely used for T-cell complement-dependent cytotoxicity crossmatch (XM) tests. Whereas NIH-negative, AHG-positive (NIH⁻/AHG⁺) results are caused by weak antibodies, NIH⁺/AHG⁻ results are usually due to autoantibodies. We found that solid organ transplantation candidates with NIH⁺/AHG⁻ XM results are repeatedly excluded from allocation of deceased donor organs by the Korean Network for Organ Sharing (KONOS) allocation system. Here, we attempted to demonstrate that these patients do not have donor-specific HLA antibodies (DSAs). METHODS: Sera showing NIH⁺/AHG⁻ results in the analysis of 1,668 KONOS T-cell XM tests were screened for panel reactive antibody (PRA) using a Luminex test. For screen-positive samples, antibody identification was conducted using a Luminex single antigen assay and the presence or absence of class I DSAs was determined. For positive controls, 42 KONOS XM tests showing probable true-positive (NIH⁻/AHG⁺ or NIH⁺/AHG⁺) results were reviewed for PRA results based on electronic medical records and the presence or absence of DSAs was determined. RESULTS: NIH⁺/AHG⁻ results were observed in 1.3% (21/1,668) of KONOS XM tests analyzed. Most of these (18/21, 85.7%) were negative for PRA or DSAs. All probable true-positive cases were either positive for DSAs (24/42, 57.1%) or had high PRA (mean, 92% [range; 42%~100%]), complicating accurate identification of antibody specificities. CONCLUSIONS: NIH⁺/AHG⁻ results are not rare (1.3%) in KONOS XM tests. Most of these results are not due to DSAs, and these patients should not be excluded from organ allocation.

Influence of serum complement and IgG on rituximab-dependent NK cell-mediated cytotoxicity to Raji cells / 白血病·淋巴瘤

Objective To determine the influence of serum complement and IgG on rituximabdependent NK cell-mediated cytotoxicity to Raji cells in vitro.Methods FcγR Ⅲ a (CD16a) polymorphism of NK cells were detected by nest-PCR. Effects of serum IgG on FcγRⅢ a expression of NK cells in vitro were analyzed by flow cytometry.The target cells(Raji cells) were stained with DIO,cultured with effector cells(NK cells) and rituximab with or without serum IgG/complement,and finally stained with propidium iodide (PI),then these cells were tested by flow cytometry and the cytotoxic index was calculated as well. Results The cytotoxic indexes of the ADCC +CDC groups were higher than those of ADCC groups, but the serum IgG groups were lower than the ADCC groups. In FcγRⅢa-158Ⅴ/Ⅴ groups, the cytotoxic indexs of the ADCC+ CDC groups,the serum IgG groups and the ADCC groups were (94.25±1.79) ％,(59.79±0.66) ％ and(69.05± 2.38) ％,respectively,and the differences among the groups were statistically significant (P＜ 0.05).In FcγRⅢ a-158Ⅴ/F groups,the cytotoxic indexs of these three groups were (66.71±5.57) ％,(18.13±2.99) ％ and (39.63±3.86) ％, respectively, and the differences among the groups were also statistically significant (P＜ 0.05).Conclusions Complement may enhance the rituximab-mediated NK cell cytotoxicity to Raji cells, whereas,serum IgG may weaken the cytotoxicity against Raji cells. It is clued up that for patients treated by tumorspecific monolonal antibody (MAb), combined infusion of fresh frozen plasma could promote its anti-tumor effect,however,MAb combined with IVIG may impair its anti-tumor effect.

Generation of 1E8 Single Chain Fv-Fc Construct Against Human CD59

BACKGROUND: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). METHODS: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reverse-transcriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. RESULTS: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. CONCLUSION: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.

Clinical Implications of Pre-Transplantation Panel Reactive Antibody in Renal Transplantation in Koreans / 대한이식학회지

PURPOSE: Panel reactive antibody (PRA) is a screening test for HLA alloimmunization. The purpose of this study is to clarify the clinical implications of pre-transplantation PRA in renal transplantation in Koreans. METHODS: Study subjects included 99 renal transplant recipients whose HLA cross match tests were conducted between Jan. 1994 and Dec. 1995. Their sera were tested for PRA using 50 lymphocyte panels from Koreans by NIH and AHG methods. RESULTS: PRA was positive in 18 (18%) patients by NIH method, and in 19 (19%) patients by AHG method. NIH PRA positivity was higher in the female group (33% vs. 12% in males, P=0.01) while the age of AHG PRA (+) group was higher than the (-) group (37+/-16 vs. 28+/-13, P 45, donor age > 50 and AHG PRA (+) were associated with worse graft survival. In multivariate analysis, donor age > 50 along with AHG PRA (+), or donor age > 50 with recipient age > 45 were significant prognostic factors for graft survival. Recipient age >45, donor age > 50 and AHG PRA (+) were still prognostic of long-term graft fates in cadaveric transplants. CONCLUSION: AHG PRA correlates better with clinical data than NIH PRA and pre-transplant PRA is a significant prognostic factor for long-term graft fates in cadaveric renal recipients in Koreans.

Comparison of NIH-CDC and AHG-CDC methods for the Detection of Panel Reactive Antibodies / 대한임상병리학회지

BACKGROUND: Panel reactive antibody (PRA) test is used for anti-HLA antibody screening and characterization in patients awaiting organ transplantation. Complement-dependent cytotoxicity (CDC) is the most widely used standard procedure and addition of antihuman globulin (AHG) reagent to the basic (NIH) CDC method increases the sensitivity of detection of HLA antibodies. We compared NIH-CDC and AHG-CDC methods for the detection of HLA class I panel reactive antibodies. METHODS: A total of 314 sera from 253 patients were analysed for the detection of HLA class I antibodies by NIH-CDC and AHG-CDC methods using a panel of 50 lymphocytes. PRA% and reaction strength (mean score) were calculated and antibody specificities were identified with r value calculated for antibody specificity. RESULTS: A total of 46 (15%) out of 314 sera were PRA-positive (PRA%> or =10%) by either NIH-CDC (33 sera) or AHG-CDC (43 sera). Concordance of PRA test results between these two methods was 96% (301/314). AHG-CDC was more sensitive in the detection of HLA antibodies compared with NIH-CDC, showing significantly higher PRA% (44% vs 29%, P=0.0001) and reaction strength (mean score 7.3 vs 6.1, P=0.0015) for PRA-positive samples. Among 46 PRA-positive sera, HLA antibody specificities were identified in 21 samples (46%) by NIH-CDC and in 32 samples (70%) by AHG-CDC. AHG-CDC methods frequently detected a wider range of antibody specificities compared with NIH-CDC and provided a more accurate assessment of the antibody specificities. Follow up PRA tests were useful providing information on change of alloimmunization status and antibody specificities in prospective organ transplantation patients. CONCLUSIONS: Compared with NIH-CDC, AHG-CDC method is more sensitive in the detection of panel reactive antibodies and provides a more accurate assessment of the HLA antibody specificities.