Hiroyuki Tahara

Role for CD47-SIRPα signaling in xenograft rejection by macrophages

We have previously proven that human macrophages can phagocytose porcine cells even in the absence of Ab or complement opsonization, indicating that macrophages present a pivotal immunological obstacle to xenotransplantation. A recent report indicates that the signal regulatory protein (SIRP)α is a critical immune inhibitory receptor on macrophages, and its interaction with CD47, a ligand for SIRPα, prevents autologous phagocytosis. Considering the limited compatibility (73%) in amino acid sequences between pig and human CD47, we hypothesized that the interspecies incompatibility of CD47 may contribute to the rejection of xenogeneic cells by macrophages. In the present study, we have demonstrated that porcine CD47 does not induce SIRPα tyrosine phosphorylation in human macrophage-like cell line, and soluble human CD47-Fc fusion protein inhibits the phagocytic activity of human macrophages toward porcine cells. In addition, we have verified that manipulation of porcine cells for expression of human CD47 radically reduces the susceptibility of the cells to phagocytosis by human macrophages. These results indicate that the interspecies incompatibility of CD47 significantly contributes to the rejection of xenogeneic cells by macrophages. Genetic induction of human CD47 on porcine cells could provide inhibitory signaling to SIRPα on human macrophages, providing a novel approach to preventing macrophage-mediated xenograft rejection.

A model for personalized in vivo analysis of human immune responsiveness.

Personalized humanized mice can model intrinsic defects in human immune disease. Personalized Research From monogrammed towels to engraved wedding memorabilia, personalized gifts offer a way to give that special someone something truly unique. However, for all the talk of personalized medicine, translational research doesn’t always live up to this standard. For example, biomedical research is often performed in animal models that don’t recapitulate disease mechanisms as they manifest in any given human patient. Now, Kalscheuer et al. describe the development of what might be the perfect gift for some patients: “personalized immune” (PI) mice that allow individualized analysis of human immune responses. To generate these PI mice, the authors isolated hematopoietic stem cells (HSCs) from the bone marrow of human adults and injected them along with matched fetal thymic tissue (depleted of T cells to prevent graft rejection) into immunodeficient mice. The resulting T cells in these mice were functional, displayed a diverse repertoire, and didn’t attack healthy tissues in the recipient mouse. Kalscheuer et al. then examined PI mice derived with HSCs from both healthy and type 1 diabetic individuals. The researchers found that T cells that arose from HSCs donated by subjects with type 1 diabetes were more likely to have an activated and memory phenotype than were T cells from healthy subject–derived HSCs, even though the T cells developed in a similar environment. These data suggest that there are intrinsic differences in HSCs from type 1 diabetic patients that contribute to the autoimmune pathology. Extending these studies to other diseases should provide new insights into pathogenesis and suggest new treatment strategies. Studies of human immune diseases are generally limited to the analysis of peripheral blood lymphocytes of heterogeneous patient populations. Improved models are needed to allow analysis of fundamental immunologic abnormalities predisposing to disease and in which to assess immunotherapies. Immunodeficient mice receiving human fetal thymus grafts and fetal CD34+ cells intravenously produce robust human immune systems, allowing analysis of human T cell development and function. However, to use humanized mice to study human immune-mediated disorders, immune systems must be generated from adult hematopoietic cells. Here, we demonstrated robust immune reconstitution in mice with hematopoietic stem cells (HSCs) aspirated from bone marrow of adults with type 1 diabetes (T1D) and healthy control volunteers. In these humanized mice, cryopreservation of human leukocyte antigen allele–matched fetal thymic tissue prevented allogeneic adult HSC rejection. Newly generated T cells, which included regulatory T cells (Tregs), were functional and self-tolerant and had a diverse repertoire. The immune recognition of these mice mimicked that of the adult CD34+ cell donor, but the T cell phenotypes were more predominantly “naïve” than those of the adult donors. HSCs from T1D and control donors generated similar numbers of natural Tregs intrathymically; however, peripheral T cells from T1D subjects showed increased proportions of activated or memory cells compared to controls, suggesting possible HSC-intrinsic differences in T cell homeostasis that might underlie immune pathology in T1D. This “personalized immune” mouse provides a new model for individualized analysis of human immune responses that may provide new insights into not only T1D but also other forms of immune function and dysfunction as well.

Reconstitution of CD8+ T Cells by Retroviral Transfer of the TCR αβ-Chain Genes Isolated from a Clonally Expanded P815-Infiltrating Lymphocyte

Gene transfer of TCR αβ-chains into T cells may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated disorders. We report in this study the reconstitution of CD8+ T cells by transfer of TCR αβ-chain genes derived from an infiltrating T cell into P815. Analysis of the clonal expansion and Vβ subfamily usage of CD8+ TIL in the tumor sites demonstrated that T cells using Vβ10 efficiently infiltrated and expanded clonally. The TCR α- and β-chain sequences derived from a tumor-infiltrating CD8+/Vβ10+ single T cell clone (P09-2C clone) were simultaneously determined by the RT-PCR/single-strand conformational polymorphism method and the single-cell PCR method. When P09-2C TCR αβ-chain genes were retrovirally introduced into CD8+ T cells, the reconstituted T cells positively lysed the P815 tumor cells, but not the A20, EL4, or YAC-1 cells, in vitro. In addition, the CTL activity was blocked by the anti-H2Ld mAb. Furthermore, T cells containing both TCR α- and β-chains, but not TCR β-chain alone, accumulated at the tumor-inoculated site when the reconstituted CD8+ T cells were adoptively transferred to tumor-bearing nude mice. These findings suggest that it is possible to reconstitute functional tumor-specific CD8+ T cells by transfer of TCR αβ-chain genes derived from TIL, and that such T cells might be useful as cytotoxic effector cells or as a vehicle for delivering therapeutic agents.

Nucleosome-Specific Regulatory T Cells Engineered by Triple Gene Transfer Suppress a Systemic Autoimmune Disease

The mechanisms of systemic autoimmune disease are poorly understood and available therapies often lead to immunosuppressive conditions. We describe here a new model of autoantigen-specific immunotherapy based on the sites of autoantigen presentation in systemic autoimmune disease. Nucleosomes are one of the well-characterized autoantigens. We found relative splenic localization of the stimulative capacity for nucleosome-specific T cells in (NZB × NZW)F1 (NZB/W F1) lupus-prone mice. Splenic dendritic cells (DCs) from NZB/W F1 mice spontaneously stimulate nucleosome-specific T cells to a much greater degree than both DCs from normal mice and DCs from the lymph nodes of NZB/W F1 mice. This leads to a strategy for the local delivery of therapeutic molecules using autoantigen-specific T cells. Nucleosome-specific regulatory T cells engineered by triple gene transfer (TCR-α, TCR-β, and CTLA4Ig) accumulated in the spleen and suppressed the related pathogenic autoantibody production. Nephritis was drastically suppressed without impairing the T cell-dependent humoral immune responses. Thus, autoantigen-specific regulatory T cells engineered by multiple gene transfer is a promising strategy for treating autoimmune diseases.

Gene Therapy of Arthritis with TCR Isolated from the Inflamed Paw

In recent years, the treatment of autoimmune diseases has been significantly advanced by the use of biological agents. However, some biologics are accompanied with severe side effects, including tuberculosis and other types of infection. There is thus a critical need for nonsystemic and lesion-specific methods of delivering these therapeutic agents. We attempted to treat a mouse model of arthritis by using T cells that expressed a regulatory molecule and were specifically directed to the inflamed paw. To this end, we first identified the TCR αβ genes accumulating in the inflamed paw of mice with collagen-induced arthritis (CIA) by a combination of single-strand chain polymorphism analysis of TCR and single-cell sorting. We identified an expanded clone B47 which is autoreactive but is not specific to type II collagen. In vivo, TCR genes from B47-transduced T cells accumulated in the inflamed paw. Injection of cells cotransduced with the B47 and soluble TNFRIg genes resulted in a significant suppression of CIA. The suppression was correlated with the amount of TNFRIg transcripts in the hind paw, not with the serum concentrations of TNFRIg. Moreover, T cells cotransduced with the B47 and intracellular Foxp3 genes significantly suppressed CIA with reductions in TNF-α, IL-17A, and IL-1β expression and bone destruction. T cells cotransduced with B47 and Foxp3 genes also suppressed the progression of established CIA. Therefore, immunosuppressive therapy with autoreactive TCR is a promising therapeutic strategy for arthritis whether the TCRs are used to deliver either soluble or intracellular suppressive molecules.

Deficiency of N-glycolylneuraminic acid and Galα1-3Galβ1-4GlcNAc epitopes in xenogeneic cells attenuates cytotoxicity of human natural antibodies

Basnet NB, Ide K, Tahara H, Tanaka Y, Ohdan H. Deficiency of N‐glycolylneuraminic acid and Galα1‐3Galβ1‐4GlcNAc epitopes in xenogeneic cells attenuates cytotoxicity of human natural antibodies. Xenotransplantation 2010; 17: 440–448.

Immunological Property of Antibodies against N-Glycolylneuraminic Acid Epitopes in Cytidine Monophospho–N-Acetylneuraminic Acid Hydroxylase-Deficient Mice

The generation of pigs devoid of Galα1,3Galβ1,4GlcNAc (Gal) residues has stimulated interest in non-Gal Ags as potentially important targets for Ab binding leading to rejection of pig organ xenografts in humans. Although N-glycolylneuraminic acid (NeuGc) epitopes, which are widely expressed on the endothelial cells of all mammals except humans, are likely targets of anti–non-Gal Abs, this aspect has not been investigated intensively owing to the absence of an appropriate animal model. In this study, we used CMAH−/− mice, which are completely deficient in NeuGc and thus produce anti-NeuGc Abs. Sera obtained from CMAH−/− mice and healthy human volunteers having anti-NeuGc Abs initiated complement-mediated lysis against CMAH+/+ cells in vitro. The cytotoxic activity of anti-NeuGc Abs was also determined in vivo (i.e., NeuGc-expressing CMAH+/+ mouse splenocytes that had been i.v. injected were completely eliminated in syngeneic CMAH−/− mice). CMAH−/− mice rejected the islets transplanted from syngeneic CMAH+/+ mice. Thus, the anti-NeuGc Ab-mediated response may be crucially involved in xenograft loss. This is the first direct demonstration of the immunogenic property of NeuGc determinants as targets of the corresponding Abs in CMAH+/+-to-CMAH−/− transplantation setting.

Successful hepatitis B vaccination in liver transplant recipients with donor-specific hyporesponsiveness.

Currently, patients are prescribed lifelong treatment with hepatitis B immunoglobulin (HBIg) after liver transplantation (LT) for hepatitis B virus (HBV)‐related diseases in order to prevent reinfection with HBV. Active immunization with an HBV vaccine would be a preferable alternative; however, the immunosuppressive environment in LT recipients is believed to elicit a poor response to vaccination. Minimizing the exposure of the HBV‐infected LT recipients to immunosuppressants would be beneficial in inducing adaptive immunity against HBV by vaccination. In this study, in addition to efforts to minimize immunosuppression, prophylaxis with HBV vaccination combined with continuous HBIg administration was performed in 17 LT recipients who had undergone transplantation attributable to HBV‐related diseases. During the observation period, the overall response rate to HBV vaccination was 64.7%. The immune status of the recipients was evaluated by a mixed lymphocyte reaction assay in response to allostimulation. Patients showing a donor‐specific hyporesponse with a well‐maintained response to the third‐party stimulus always achieved a sustained immune response to the vaccine, whereas patients showing a hyporesponse to both the donor and the third‐party stimulus were unable to do so. Thus, inducing an anti‐donor‐specific immunosuppressive status by minimizing immunosuppression should enable post‐transplant HBV vaccination to be a promising prophylactic strategy.

Microsurgical hepatic artery reconstruction during living‐donor liver transplantation by using head‐mounted surgical binocular system

We have described our experience with arterial reconstruction during living‐donor liver transplantation by using Varioscope® AF3 – a head‐mounted surgical binocular system with automatic focusing and continuous zoom magnification from 3.6× to 7.2×. From July 1996 to December 2006, 91 grafts were implanted in 89 living‐donor liver transplantation recipients, including two that required retransplantation. For microsurgical reconstruction of the graft hepatic artery, a conventional operating microscope was used in the first 10 transplants and Varioscope, in the subsequent 81. The time required to complete arterial reconstruction while using a conventional operating microscope and Varioscope was 78.6 ± 44.6 min and 35.5 ± 15.5 min, respectively. No arterial complications, including hepatic artery thrombosis, occurred in any of the 89 patients during the observation period. In living‐donor liver transplantation, successful hepatic artery reconstruction can be safely carried out using Varioscope.

Expression of MRE11 complex (MRE11, RAD50, NBS1) and hRap1 and its relation with telomere regulation, telomerase activity in human gastric carcinomas.

The MRE11 complex (MRE11, RAD50, NBS1) are required for the repair of DNA double-strand breaks and have another important function in regulating telomere length. The silent information regulator (Sir) proteins required for telomere position effect also bind telomeres. hRap1 protein is a human ortholog of yeast Rap1 which regulates telomere length by interacting with TRF2 and is recruited to telomeres by TRF2. We examined the expression of the MRE11 complex (MRE11, RAD50, NBS1), Sir2 and hRAP1 in 20 gastric carcinomas by reverse transcription polymerase chain reaction and then analyzed the relation between telomerase activity and other telomerase components such as human telomerase reverse transcriptase (TERT), human telomerase RNA component (hTR), human telomerase-associated protein (TEP1), telomeric repeat binding factor 1 (TRF1), TRF2- and TRF1-interacting, ankyrin-related ADP-ribose polymerase (tankyrase) as well as TRF1-interacting nuclear protein 2 (TIN2). Of twenty gastric carcinomas examined, 13 (65%), 14 (70%), 16 (80%), 12 (60%) and 13 (65%) expressed MRE11, RAD50, NBS1, Sir2 and hRap1 at higher levels than corresponding nonneoplastic gastric mucosa, respectively. No obvious correlation was observed between MRE11 complex expression and telomerase activity or expression of TERT, hTR, TEP1, tankyrase and TIN2. Carcinomas with high TRF1 expression expressed significantly higher levels of MRE11 and RAD50 than those with low TRF1 expression (p < 0.05). On the other hand, carcinomas with high TRF2 expression expressed significantly higher levels of MRE11, NBS1 and hRap1 than those with low TRF2 expression (p < 0.05). These results suggest that gastric carcinomas with high TRF1 and TRF2 expression may need a large quantity of the MRE11 complex. Moreover, gastric carcinomas with high TRF1 expression may require a large quantity of hRap1.