Bruce Motyka

Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Is a Death Receptor for Granzyme B during Cytotoxic T Cell–Induced Apoptosis

The serine proteinase granzyme B is crucial for the rapid induction of target cell apoptosis by cytotoxic T cells. Granzyme B was recently demonstrated to enter cells in a perforin-independent manner, thus predicting the existence of a cell surface receptor(s). We now present evidence that this receptor is the cation-independent mannose 6-phosphate/insulin-like growth factor receptor (CI-MPR). Inhibition of the granzyme B-CI-MPR interaction prevented granzyme B cell surface binding, uptake, and the induction of apoptosis. Significantly, expression of the CI-MPR was essential for cytotoxic T cell-mediated apoptosis of target cells in vitro and for the rejection of allogeneic cells in vivo. These results suggest a novel target for immunotherapy and a potential mechanism used by tumors for immune evasion.

Granzyme B Induces Smooth Muscle Cell Apoptosis in the Absence of Perforin Involvement of Extracellular Matrix Degradation

Objective—T cell-induced cytotoxicity, of which granzyme B is a key mediator, is believed to contribute to the pathogenesis of inflammatory vascular diseases. In this report, we investigate the mechanism of granzyme B-induced smooth muscle cell (SMC) death. Methods and Results—The addition of purified granzyme B alone to cultured SMCs caused a significant reduction in cell viability. Chromatin condensation, phosphatidylserine externalization, and membrane blebbing were observed, indicating that the mechanism of granzyme B-induced SMC death was through apoptosis. Activated splenocytes from perforin-knockout mice induced SMC death through a granzyme B-mediated pathway. Inhibition of the proteolytic activities of caspases and granzyme B prevented granzyme B-induced SMC death, whereas attenuation of granzyme B internalization with mannose-6-phosphate (M6P) did not. Further, granzyme B induced the cleavage of several SMC extracellular proteins, including fibronectin, and reduced focal adhesion kinase phosphorylation. Conclusions—These results indicate that granzyme B can induce apoptosis of SMCs in the absence of perforin by cleaving extracellular proteins, such as fibronectin.

Identification of a Novel Human Granzyme B Inhibitor Secreted by Cultured Sertoli Cells

Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.

Modulation of the Tumor Cell Phenotype by IFN-γ Results in Resistance of Uveal Melanoma Cells to Granule-Mediated Lysis by Cytotoxic Lymphocytes

IFN-γ, a pleiotropic immune regulator, is implicated in both tumor immune surveillance and selection of tumor variants resistant to immune control, i.e., immunoediting. In uveal melanoma patients, elevated serum levels of IFN-γ correlate with the spread of metastasis and represent a negative prognostic marker. Treatment with IFN-γ boosted the MHC class I presentation machinery in uveal melanoma cells but suppressed their MHC class I-restricted CTL lysis. Tumor cells exposed to IFN-γ efficiently activated specific CTL but were less susceptible to permeabilization by perforin and exhibited a decreased capacity to bind and incorporate granzyme B. These results define a novel mechanism of resistance to granule-mediated CTL lysis in human tumors. Furthermore, the data suggest that immunoediting is not limited to genetic or epigenetic changes resulting in stable cellular phenotypes but also involves an inducible modulation of tumor cells in response to a microenvironment associated with immune activation.

Identification of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) isoforms in the Pekin duck.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD152) is an inhibitory T cell receptor predominately expressed on activated T cells. The duck CTLA-4 (DuCTLA-4) cDNA and a transcript lacking the predicted transmembrane encoding region (DuCTLA-4DeltaTM) were isolated from splenocytes using RT-PCR. The predicted DuCTLA-4 protein showed an identity of 92%, 49% and 47% with chicken, human and mouse homologues, respectively. Sequence comparison revealed conservation of residues implicated in the B7 ligand binding, disulfide linkages, glycosylation and intracellular signaling. DuCTLA-4 mRNA was predominately expressed in primary and secondary immune organs. DuCTLA-4 and DuCTLA-4DeltaTM transcripts were differentially regulated in PBMCs. Flow cytometric analysis showed constitutive expression of DuCTLA-4 protein on freshly isolated PBMCs and a modest increase upon mitogen stimulation. Our observations suggest that DuCTLA-4 and its isoform DuCTLA-4DeltaTM evolved before the divergence of birds and mammals. Both DuCTLA-4 isoforms have significant structural homology to mammalian CTLA-4 proteins but their individual roles in the regulation of duck immune responses remains to be elucidated.

Induction of Human Blood Group A Antigen Expression on Mouse Cells, Using Lentiviral Gene Transduction

The ABO histo-blood group system is the most important antigen system in transplantation medicine, yet no small animal model of the ABO system exists. To determine the feasibility of developing a murine model, we previously subcloned the human alpha-1,2-fucosyltransferase (H-transferase, EC 2.4.1.69) cDNA and the human alpha-1,3-N-acetylgalactosaminyltransferase (A-transferase, EC 2.4.1.40) cDNA into lentiviral vectors to study their ability to induce human histo-blood group A antigen expression on mouse cells. Herein we investigated the optimal conditions for human A and H antigen expression in murine cells. We determined that transduction of a bicistronic lentiviral vector (LvEF1-AH-trs) resulted in the expression of A antigen in a mouse endothelial cell line. We also studied the in vivo utility of this vector to induce human A antigen expression in mouse liver. After intrahepatic injection of LvEF1-AH-trs, A antigen expression was observed on hepatocytes as detected by immunohistochemistry and real-time RT-PCR. In human group A erythrocyte-sensitized mice, A antigen expression in the liver was associated with tissue damage, and deposition of antibody and complement. These results suggest that this gene transfer strategy can be used to simulate the human ABO blood group system in a murine model. This model will facilitate progress in the development of interventions for ABO-incompatible transplantation and transfusion scenarios, which are difficult to develop in clinical or large animal settings.

MHC-Matched A-Expressing Blood Cells Induce ABO Tolerance in Infant and Adult Mice

Purpose ABO-incompatible heart transplantation (ABOi HTx) is safe during infancy and allows increased donor access. Post-ABOi HTx B cell tolerance develops to donor blood group antigen(s) by mechanisms not fully defined. We developed A-transgenic mice (A-Tg) that express A-antigen on vascular endothelium and erythrocytes and demonstrated A-antigen specific tolerance induced by HTx into 4 wk-old, MHC-identical, wild-type (WT) mice. Herein, we explored intentional tolerance induction in infant and adult WT mice using A-Tg blood cells. Methods WT BALB/c mice were injected ip (weekly×3) with intact A-Tg BALB/c blood cells (±40Gy irradiated), beginning at 7 days (neonates) or 5 months of age (adults; see Table). Two weeks after treatment, all mice were injected ip (weekly×5) with human A-erythrocytes (‘A-sensitized’) in an attempt to elicit anti-A antibody (Ab) production. Serum anti-A and 3rd-party (non-A anti-human) Ab were assessed by hemagglutination assay. Results In response to A-sensitization, high levels of anti-A Ab were produced in untreated mice (group 1, Table). In contrast, anti-A remained undetectable in A-sensitized mice previously treated as neonates with A-Tg blood cells ±irradiation (groups 2&3). Treatment of adult mice (groups 4&5) with A-Tg blood cells resulted in reduced anti-A production in response to A-sensitization compared with untreated mice (group 1). Adult mice with undetectable natural anti-A (group 4) produced less anti-A vs those with pre-existing natural anti-A (group 5). Third-party antibody responses were high for all groups. Conclusions Our results suggest that the erythrocyte component of A-Tg blood cells can induce robust A-antigen-specific tolerance in WT mice. Importantly, our findings suggest that tolerance to A-antigen is not limited to the neonatal period but can also be induced in adults, especially in mice without previously detectable natural anti-A antibody. Intentional induction of tolerance to A/B-antigen(s) may allow subsequent ABOi HTx. Supported by Heart and Stroke Foundation of Canada; Women and Children’s Health Research Institute, University of Alberta; Alberta Innovates Health Solutions; and the Canadian Institutes of Health Research (CIHR) through the Canadian National Transplant Research Program (CNTRP). Table. No title available.

AT1R Antibody Assessment: How Should We Interpret Positive Results?

Introduction The angiotensin II type 1 receptor (AT1R) is a G-protein-coupled receptor that is expressed on vascular endothelium. It can signal cell growth and proliferation, fibrosis, and vascular remodeling. AT1R antibodies are being increasingly studied to examine their relevance in transplantation. The frequency and impact of AT1R antibodies in pediatric heart transplantation has not been well studied. Our aim is to measure the frequency of AT1R antibodies in this patient population as well as pediatric controls and to assess for non-specific reactivity in this assay. Methods We included 42 patients (n=154 samples) for whom pre- and post-transplant sera and HLA antibody data were available. AT1R antibody levels were measured by a commercially available ELISA test (distributed by One Lambda ThermoFisher). Samples were tested/interpreted as per product insert. Results were negative under 10 U/mL and positive over 17 U/mL. Age-matched, sex-balanced, non-transplant controls (n=27) were collected from the local cardiac-catheterization laboratory. A subset of samples with positive results (n=52 sera from 20 patients) as well as the assay’s positive control were re-tested following adsorption with Adsorb Out (One Lambda ThermoFisher.). Figure 1 details the samples and testing methodology. Analysis was performed using GraphPad. Figure. No caption available. Results No significant difference was observed between patient and control AT1R antibody levels (Figure 2). Values over the test method upper threshold (40 U/mL) were detected in 38% of patient sera. Adsorbed sera had significantly decreased AT1R values vs non adsorbed sera (Figure 3A). There was no significant change from pre- to post-transplant AT1R antibody detected overall (Figures 3B and 3C). In the UN-adsorbed sera, 57% of the pre-transplant sera and 64% of the post-transplant sera tested positive for AT1R antibody; 58% and 78% were positive above the 40U/mL threshold, respectively. In contrast, In the ADSORBED sera, 21% of the pre-transplant sera and 15% of the post-transplant sera tested positive for AT1R antibody; 29% and 20% were positive above the 40U/mL threshold, respectively. Figure. No caption available. Figure. No caption available. Conclusion Pediatric heart transplant patients appear to yield false positive results in this AT1R assay. This problem may be overcome by adsorbing for non-specific reactivity. Non-specific binding is a known concern in immunoassays such as ELISA. Typically, controls are included in these assays such as a ‘no antigen’ well and/or absorption steps are performed. This assay is lacking an essential control that is required by clinical laboratory standards. The pediatric heart transplant population may have especially high levels of non-specific reactivity in immunoassays due to the high frequency of thymectomy and mechanical circulatory support. We are now investigating the association of adsorbed and non-adsorbed AT1R antibody to transplant outcomes. Canadian National Transplant Research Program (CNTRP). Edmonton Civic Employee’s Research Award.

Antibody Response to Non-Self Blood Group A-Antigen depends on CD4 T Cells, Forigen Protein and CD22 Interaction

Background ABO-incompatible heart transplantation (ABOi-HTx) is safe during infancy and allows increased donor access. B-cell tolerance develops to donor A/B-antigen(s) (Ag) after ABOi-HTx by mechanisms remaining unclear. We developed transgenic mice (A-Tg) constitutively expressing human A-Ag on vascular endothelium and erythrocytes (RBC) to study anti-A antibody responses. CD22 participates in B-cell tolerance and we found that B cells express high-levels of CD22 in human B cells, decreasing with age. Here we used a mouse model to study the anti-A response in the context of MHC syngeneic, allogeneic and xenogeneic stimulation, and the impact of CD22 expression. Methods Part I: Adult wild-type (WT) C57BL/6 (B6/H-2b), BALB/c (BALB/H-2d), C3H/He (C3H/H-2k), or CD22-deficient B6 (CD22KO) mice received intraperitoneal injections of B6 or BALB A-Tg blood cells or human-RBC membranes (100ul/10%v/v) from blood group-A (hu-A) or O (hu-O); or A-incompatible heart allografts. Serum anti-A Ab was measured by hemagglutination and ELISA (IgG and IgM); graft survival was assessed by palpation. Part II: a) To assess requirement of foreign protein to stimulate anti-A, hu-O RBC/syngeneic A-Tg cells or allogeneic A-Tg blood were co-injected in WT mice; b) to assess T cell dependence of anti-A response, CD4+ T cells were depleted from WT B6 mice before hu-A RBC injection. Part III: To assess the role of CD22, A-Tg or hu-A-RBC, were injected into CD22KO mice with or without CD4+ T-cell depletion. Results Part I: Exposure to allogeneic A-Tg blood cells/heart graft or xenogeneic hu-RBC induced anti-A production (Table), whereas syngeneic A-Tg blood cells did not. Part II: a) mixture of syngeneic A-Tg/hu-O RBC did not induce anti-A; b) after CD4+ T-cell depletion, hu A-RBC failed to elicit anti-A. Part III: Hu A-RBC induced a very high anti-A in CD22KO mice compared to WT B6. In contrast to WT B6 mice, anti-A Ab was elicited in CD22KO mice following injection with A-Tg blood cells or hu A-RBC with CD4+ T cell depletion. Figure. No caption available. Conclusions Our results show that in WT mice, anti-A antibody production depends not only on exposure to A-antigen but also co-engagement with foreign protein and a requirement for CD4+ cells; consistent with a T-dependent anti-A response. Conversely, in CD22KO mice there was no requirement for foreign protein or CD4+ cells to elicit an anti-A antibody response; consistent with a T-independent anti-A response. These findings suggest an important role for the regulatory CD22 receptor in the B cell response to ABH antigens.

Antibody-Mediated Rejection in a Blood Group A-Transgenic Mouse Model of ABO-Incompatible Heart Transplantation.

Background ABO-incompatible (ABOi) organ transplantation is performed owing to unremitting donor shortages. Defining mechanisms of antibody-mediated rejection, accommodation, and tolerance of ABOi grafts is limited by lack of a suitable animal model. We report generation and characterization of a murine model to enable study of immunobiology in the setting of ABOi transplantation. Methods Transgenesis of a construct containing human A1- and H-transferases under control of the ICAM-2 promoter was performed in C57BL/6 (B6) mice. A-transgenic (A-Tg) mice were assessed for A-antigen expression by histology and flow cytometry. B6 wild-type (WT) mice were sensitized with blood group A-human erythrocytes; others received passive anti-A monoclonal antibody and complement after heart transplant. Serum anti-A antibodies were assessed by hemagglutination. “A-into-O” transplantation (major histocompatibility complex syngeneic) was modeled by transplanting hearts from A-Tg mice into sensitized or nonsensitized WT mice. Antibody-mediated rejection was assessed by morphology/immunohistochemistry. Results A-Tg mice expressed A-antigen on vascular endothelium and other cells including erythrocytes. Antibody-mediated rejection was evident in 15/17 A-Tg grafts in sensitized WT recipients (median titer, 1:512), with 2 showing hyperacute rejection and rapid cessation of graft pulsation. Hyperacute rejection was observed in 8/8 A-Tg grafts after passive transfer of anti-A antibody and complement into nonsensitized recipients. Antibody-mediated rejection was not observed in A-Tg grafts transplanted into nonsensitized mice. Conclusions A-Tg heart grafts transplanted into WT mice with abundant anti-A antibody manifests characteristic features of antibody-mediated rejection. These findings demonstrate an effective murine model to facilitate study of immunologic features of ABOi transplantation and to improve potential diagnostic and therapeutic strategies.