Browsing by Author "Tector, Matthew"

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    Characterization of antibody binding to swine leukocyte antigen class II
    (2016-05-26) Ladowski, Joseph Matthew; Tector, A. Joseph; Tector, Matthew; Blum, Janice S.
    Though the elimination of carbohydrate xenoantigens has reduced the antibody barrier to clinical xenotransplantation, identification of additional targets of rejection could further increase the immunologic compatibility of pig tissues with humans. Many patients in need of organ transplantation have antibodies to proteins encoded by the human major histocompatibility complex (MHC) which have high similarity to their swine homologs. The goal of this thesis was to determine if the class II genes of the swine MHC can bind human antibodies. To characterize antibody binding effect to class II swine leukocyte antigens (SLA), a constitutively positive SLA class II cell was created through transfection with the human class II transactivator (CIITA). Cells expressing only SLA-DR or SLA-DQ were also created using the CRISPR/Cas9 gene knockout tools. These various lines were incubated with human sera and tested for binding to IgM and IgG in a flow cytometry crossmatch (FCXM). The results demonstrate reliable antibody binding to each of the SLA class II –DR and –DQ derivatives. A two-way paired t-test revealed statistical difference in total sera binding between to the DR(+)DQ(+) and DR(-)DQ(-) clones for IgG (p = 0.0059) but not IgM (p = 0.2460). Looking at the subset of individuals with and without anti-HLA class II sensitization, statistical difference was noted for IgG (p = 0.0229) but not IgM (p = 0.3045). Examining further the role of DR(+) vs DQ(+), statistical analysis revealed difference in the DR(+)DQ(-) vs. the DR(-)DQ(+) FCXM (p = 0.0099), the DR(+)DQ(-) vs. the DR(+)DQ(+) FCXM (p = 0.0192), and the DR(-)DQ(-) parent vs. DR(+)DQ(+) FCXM (p = 0.0329). No difference was found in the DR(-)DQ(+) vs. DR(+)DQ(+) FCXM (p = 0.1601). The results of this project suggest that SLA class II, specifically SLA-DQ, could be a target of antibody binding and cross-reactive anti-HLA class II antibodies may be capable of binding SLA class II.
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    Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease
    (American Association of Immunologists, 2014-12-01) Reyes, Luz M.; Estrada, Jose L; Wang, Zheng Yu; Blosser, Rachel J.; Smith, Rashod F.; Sidner, Richard A.; Paris, Leela L.; Blankenship, Ross L.; Ray, Caitlin N.; Miner, Aaron C.; Tector, Matthew; Tector, A. Joseph; Surgery, School of Medicine
    Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.
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    Porcine iGb3s gene silencing provides minimal benefit for clinical xenotransplantation
    (Wiley, 2016-03) Butler, James R.; Skill, Nicholas J.; Priestman, David; Platt, Frances; Li, Ping; Estrada, Jose L.; Martens, Gregory R.; Ladowski, Joseph M.; Tector, Matthew; Tector, A. Joseph; Department of Surgery, IU School of Medicine
    Background The Galα(1,3)Gal epitope (α-GAL), created by α-1,3-glycosyltransferase-1 (GGTA1), is a major xenoantigen causing hyperacute rejection in pig-to-primate and pig-to-human xenotransplantation. In response, GGTA1 gene-deleted pigs have been generated. However, it is unclear whether there is a residual small amount of α-Gal epitope expressed in GGTA1−/− pigs. Isoglobotrihexosylceramide synthase (iGb3s), another member of the glycosyltransferase family, catalyzes the synthesis of isoglobo-series glycosphingolipids with an α-GAL-terminal disaccharide (iGb3), creating the possibility that iGb3s may be a source of α-GAL epitopes in GGTA1−/− animals. The objective of this study was to examine the impact of silencing the iGb3s gene (A3GalT2) on pig-to-primate and pig-to-human immune cross-reactivity by creating and comparing GGTA1−/− pigs to GGTA1−/−- and A3GalT2−/−-double-knockout pigs. Methods We used the CRISPR/Cas 9 system to target the GGTA1 and A3GalT2 genes in pigs. Both GGTA1 and A3GalT2 genes are functionally inactive in humans and baboons. CRISPR-treated cells used directly for somatic cell nuclear transfer produced single- and double-gene-knockout piglets in a single pregnancy. Once grown to maturity, the glycosphingolipid profile (including iGb3) was assayed in renal tissue by normal-phase liquid chromatography. In addition, peripheral blood mononuclear cells (PBMCs) were subjected to (i) comparative cross-match cytotoxicity analysis against human and baboon serum and (ii) IB4 staining for α-GAL/iGb3. Results Silencing of the iGb3s gene significantly modulated the renal glycosphingolipid profile and iGb3 was not detected. Moreover, the human and baboon serum PBMC cytotoxicity and α-GAL/iGb3 staining were unchanged by iGb3s silencing. Conclusions Our data suggest that iGb3s is not a contributor to antibody-mediated rejection in pig-to-primate or pig-to-human xenotransplantation. Although iGb3s gene silencing significantly changed the renal glycosphingolipid profile, the effect on Galα3Gal levels, antibody binding, and cytotoxic profiles of baboon and human sera on porcine PBMCs was neutral.
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    Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model
    (Wiley Blackwell (Blackwell Publishing), 2015-05) Higginbotham, Laura; Mathews, Dave; Breeden, Cynthia A.; Song, Mingqing; Farris, Alton Brad; Larsen, Christian P.; Ford, Mandy L.; Lutz, Andrew J.; Tector, Matthew; Newell, Kenneth A.; Tector, A. Joseph; Adams, Andrew B.; Department of Surgery, IU School of Medicine
    Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end-stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig-to-primate kidney xenotransplantation has met limited success. Preformed anti-pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti-pig antibody levels. We then selected animals with both low and high titers of anti-pig antibodies to proceed with kidney transplant from galactose-α1,3-galactose knockout/CD55 transgenic pig donors. All animals received T-cell depletion followed by maintenance therapy with costimulation blockade (either anti-CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti-pig antibody rejected the kidney xenograft within the first week. Low-titer animals treated with anti-CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21 days, respectively). Long-term surviving animals treated with the anti-CD154-based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig-to-non-human primate kidney xenotransplantation, now >125 days, provides promise for further study and potential clinical translation.
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    Reduced human platelet uptake by pig livers deficient in the asialoglycoprotein receptor 1 protein
    (Wiley, 2015-05) Paris, Leela L.; Estrada, Jose L.; Li, Ping; Blankenship, Ross L.; Sidner, Richard A.; Reyes, Luz M.; Montgomery, Jessica B.; Burlak, Christopher; Butler, James R.; Downey, Susan M.; Wang, Zheng-Yu; Tector, Matthew; Tector, A. Joseph; Surgery, School of Medicine
    BACKGROUND: The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia. METHODS: Transcription activator-like effector nucleases (TALENs) were targeted to the ASGR1 gene in pig liver-derived cells. ASGR1 deficient pig cells were used for somatic cell nuclear transfer (SCNT). ASGR1 knock out (ASGR1-/-) fetal fibroblasts were used to produce healthy ASGR1 knock out piglets. Human platelet uptake was measured in ASGR1+/+ and ASGR1-/- livers. RESULTS: Targeted disruption of the ASGR1 gene with TALENs eliminated expression of the receptor. ASGR1-/- livers phagocytosed fewer human platelets than domestic porcine livers during perfusion. CONCLUSIONS: The use of TALENs in liver-derived cells followed by SCNT enabled the production of healthy homozygous ASGR1 knock out pigs. Livers from ASGR1-/- pigs exhibit decreased human platelet uptake. Deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.
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    Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers
    (Wolters Kluwer, 2016-03) Butler, James R.; Paris, Leela L.; Blankenship, Ross L.; Sidner, Richard A.; Martens, Gregory R.; Ladowski, Joeseph M.; Li, Ping; Estrada, Jose L.; Tector, Matthew; Tector, A. Joseph; Department of Surgery, IU School of Medicine
    BACKGROUND: A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediated injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein, we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury. METHODS: Wild type (WT), ASGR1, GGTA1, and GGTA1CMAH knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1, GGTA1, and GGTA1 CMAH pigs. RESULTS: GGTA1, CMAH LSECs exhibited reduced levels of human platelet binding in vitro when compared with GGTA1 and WT LSECs. In a continuous perfusion model, GGTA1 CMAH livers consumed fewer human platelets than GGTA1 and WT livers. GGTA1 CMAH livers also consumed fewer human platelets than ASGR1 livers in a single-pass model. CONCLUSIONS: Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation.