Metabolic reprogramming by immune-responsive gene 1 up-regulation improves donor heart preservation and function.

Preservation quality of donor hearts is a key determinant of transplant success. Preservation duration beyond 4 hours is associated with primary graft dysfunction (PGD). Given transport time constraints, geographical limitations exist for donor-recipient matching, leading to donor heart underutilization. Here, we showed that metabolic reprogramming through up-regulation of the enzyme immune response gene 1 (IRG1) and its product itaconate improved heart function after prolonged preservation. Irg1 transcript induction was achieved by adding the histone deacetylase (HDAC) inhibitor valproic acid (VPA) to a histidine-tryptophan-ketoglutarate solution used for donor heart preservation. VPA increased acetylated H3K27 occupancy at the IRG1 enhancer and IRG1 transcript expression in human donor hearts. IRG1 converts aconitate to itaconate, which has both anti-inflammatory and antioxidant properties. Accordingly, our studies showed that Irg1 transcript up-regulation by VPA treatment increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in mice, which was accompanied by increased antioxidant protein expression [hemeoxygenase 1 (HO1) and superoxide dismutase 1 (SOD1)]. Deletion of Irg1 in mice (Irg1-/-) negated the antioxidant and cardioprotective effects of VPA. Consistent with itaconate's ability to inhibit succinate dehydrogenase, VPA treatment of human hearts increased itaconate availability and reduced succinate accumulation during preservation. VPA similarly increased IRG1 expression in pig donor hearts and improved its function in an ex vivo cardiac perfusion system both at the clinical 4-hour preservation threshold and at 10 hours. These results suggest that augmentation of cardioprotective immune-metabolomic pathways may be a promising therapeutic strategy for improving donor heart function in transplantation.

Xenotransplantation in transition.

The application of xenotransplantation of porcine organs and tissues for treatment of disease, sought for more than a century, might soon be realized. Until now, the immune response of recipients against xenogeneic organs and tissues posed the main obstacle to clinical application. However, decades of research into this immune response and identification of other molecular barriers together with advances in genetic engineering and cloning of large animals and immune therapeutics coalesced to support prolonged survival and function of porcine organ grafts in nonhuman primates. This experimental progress in turn sparks consideration of clinical trials. The papers in this special section provide authoritative views concerning the immune hurdles that still limit and potentially still preclude clinical application of xenotransplantation. Xenoreactive antibodies elicited in T cell-dependent B cell-responses constitute the most important hurdle and control of these responses impels use of intense regimens of immunosuppression. These antibodies pose a danger to xenografts and potentially compromise subsequent allografts. However, new insights into the specificity of these antibodies, the pathways and kinetics of production and genetic determinants of pathogenicity offer novel opportunities for intervention. Likewise, the rapid ability to propose and test new strategies in nonhuman primate models hastens needed advances. However further progress will depend on development and validation of laboratory methods for identification and assay of pathogenic immune responses and evaluation of the response to therapy.

Accommodation in allogeneic and xenogeneic organ transplantation: Prevalence, impact, and implications for monitoring and for therapeutics.

Accommodation refers to acquired resistance of organs or tissues to immune or inflammatory reactions that might otherwise cause severe injury or rejection. As first observed in ABO-incompatible kidney transplants and heterotopic cardiac xenografts, accommodation was identified when organ transplants continued to function despite the presence of anti-graft antibodies and/or other reactants in the blood of recipients. Recent evidence suggests many and perhaps most organ transplants have accommodation, as most recipients mount B cell responses specific for the graft. Wide interest in the impact of graft-specific antibodies on the outcomes of transplants prompts questions about which mechanisms confer protection against such antibodies, how accommodation might be detected and whether and how rejection could be superimposed on accommodation. Xenotransplantation offers a unique opportunity to address these questions because immune responses to xenografts are easily detected and the pathogenic impact of immune responses is so severe. Xenotransplantation also provides a compelling need to apply these and other insights to decrease the intensity and toxicity of immunosuppression that otherwise could limit clinical application.

TNFRSF13B in B cell responses to organ transplantation.

Antibodies directed against organ transplants are thought to pose the most vexing hurdle to enduring function and survival of the transplants, particularly organ xenotransplants, and accordingly basic and clinical investigation has focused on elucidating the specificity and pathogenicity of graft-specific antibodies. While much has been learned about these matters, far less is known about the B cells producing graft-specific antibodies and why these antibodies appear to injure some grafts but not others. With the goal of addressing those questions, we have investigated the properties of tumor necrosis factor receptor super family-13B (TNFRSF13B), which regulates various aspects of B cell responses. A full understanding of the functions of TNFRSF13B however is hindered by extreme polymorphism and by diversity of interactions of the protein. Nevertheless, TNFRSF13B variants have been found to exert distinct impact on natural and elicited antibody responses and host defense and mutations of TNFRSF13B have been found to influence the propensity for development of antibody-mediated rejection of organ transplants. Because B cell responses potentially limit application of xenotransplantation, understanding how TNFRSF13B diversity and TNFRSF13B variants govern immunity in xenotransplantation could inspire development of novel therapeutics that could in turn accelerate clinical implementation of xenotransplantation.

A non-human primate model of acute liver failure suitable for testing liver support systems.

Acute hepatic failure is associated with high morbidity and mortality for which the only definitive therapy is liver transplantation. Some fraction of those who undergo emergency transplantation have been shown to recover native liver function when transplanted with an auxiliary hepatic graft that leaves part of the native liver intact. Thus, transplantation could have been averted with the development and use of some form of hepatic support. The costs of developing and testing liver support systems could be dramatically reduced by the availability of a reliable large animal model of hepatic failure with a large therapeutic window that allows the assessment of efficacy and timing of intervention. Non-lethal forms of hepatic injury were examined in combination with liver-directed radiation in non-human primates (NHPs) to develop a model of acute hepatic failure that mimics the human condition. Porcine hepatocyte transplantation was then tested as a potential therapy for acute hepatic failure. After liver-directed radiation therapy, delivery of a non-lethal hepatic ischemia-reperfusion injury reliably and rapidly generated liver failure providing conditions that can enable pre-clinical testing of liver support or replacement therapies. Unfortunately, in preliminary studies, low hepatocyte engraftment and over-immune suppression interfered with the ability to assess the efficacy of transplanted porcine hepatocytes in the model. A model of acute liver failure in NHPs was created that recapitulates the pathophysiology and pathology of the clinical condition, does so with reasonably predictable kinetics, and results in 100% mortality. The model allowed preliminary testing of xenogeneic hepatocyte transplantation as a potential therapy.

Allogeneic and xenogeneic lymphoid reconstitution in a RAG2 -/- IL2RG y/- severe combined immunodeficient pig: A preclinical model for intrauterine hematopoietic transplantation.

Mice with severe combined immunodeficiency are commonly used as hosts of human cells. Size, longevity, and physiology, however, limit the extent to which immunodeficient mice can model human systems. To address these limitations, we generated RAG2 -/- IL2RG y/- immunodeficient pigs and demonstrate successful engraftment of SLA mismatched allogeneic D42 fetal liver cells, tagged with pH2B-eGFP, and human CD34+ hematopoietic stem cells after in utero cell transplantation. Following intrauterine injection at day 42-45 of gestation, fetuses were allowed to gestate to term and analyzed postnatally for the presence of pig (allogeneic) and human (xenogeneic) B cells, T-cells and NK cells in peripheral blood and other lymphoid tissues. Engraftment of allogeneic hematopoietic cells was detected based on co-expression of pH2B-eGFP and various markers of differentiation. Analysis of spleen revealed robust generation and engraftment of pH2B-eGFP mature B cells (and IgH recombination) and mature T-cells (and TCR-ß recombination), T helper (CD3+CD4+) and T cytotoxic (CD3+CD8+) cells. The thymus revealed engraftment of pH2B-eGFP double negative precursors (CD4-CD8-) as well as double positive (CD4+, CD8+) precursors and single positive T-cells. After intrauterine administration of human CD34+ hematopoietic stem cells, analysis of peripheral blood and lymphoid tissues revealed the presence of human T-cells (CD3+CD4+ and CD3+CD8+) but no detectable B cells or NK cells. The frequency of human CD45+ cells in the circulation decreased rapidly and were undetectable within 2 weeks of age. The frequency of human CD45+ cells in the spleen also decreased rapidly, becoming undetectable at 3 weeks. In contrast, human CD45+CD3+ T-cells comprised >70% of cells in the pig thymus at birth and persisted at the same frequency at 3 weeks. Most human CD3+ cells in the pig's thymus expressed CD4 or CD8, but few cells were double positive (CD4+ CD8+). In addition, human CD3+ cells in the pig thymus contained human T-cell excision circles (TREC), suggesting de novo development. Our data shows that the pig thymus provides a microenvironment conducive to engraftment, survival and development of human T-cells and provide evidence that the developing T-cell compartment can be populated to a significant extent by human cells in large animals.

Limited Expansion of Human Hepatocytes in FAH/RAG2-Deficient Swine.

The mammalian liver's regenerative ability has led researchers to engineer animals as incubators for expansion of human hepatocytes. The expansion properties of human hepatocytes in immunodeficient mice are well known. However, little has been reported about larger animals that are more scalable and practical for clinical purposes. Therefore, we engineered immunodeficient swine to support expansion of human hepatocytes and identify barriers to their clinical application. Immunodeficient swine were engineered by knockout of the recombinase-activating gene 2 (RAG2) and fumarylacetoacetate hydrolase (FAH). Immature human hepatocytes (ihHCs) were injected into fetal swine by intrauterine cell transplantation (IUCT) at day 40 of gestation. Human albumin was measured as a marker of engraftment. Cytotoxicity against ihHCs was measured in transplanted piglets and control swine. We initially detected higher levels of human albumin in cord blood of newborn FAH/RAG2-deficient (FR) pigs compared with immunocompetent controls (196.26 ng/dL vs. 39.29 ng/dL, p = 0.008), indicating successful engraftment of ihHCs after IUCT and adaptive immunity in the fetus. Although rare hepatocytes staining positive for human albumin were observed, levels of human albumin did not rise after birth, but declined, suggesting rejection of xenografted ihHCs. Cytotoxicity against ihHCs increased after birth by 3.8% (95% CI: [2.1%-5.4%], p < 0.001) and inversely correlated with declining levels of human albumin (p = 2.1 × 10-5, R2 = 0.17). Circulating numbers of T cells and B cells were negligible in FR pigs. However, circulating natural killer (NK) cells exerted cytotoxicity against ihHCs. NK cell activity was lower in immunodeficient piglets after IUCT than in naive controls (30.4% vs. 40.1%, p = 0.011, 95% CI for difference [2.7%-16.7%]). In conclusion, ihHCs were successfully engrafted in FR swine after IUCT. NK cells were a significant barrier to expansion of hepatocytes. New approaches are needed to overcome this hurdle and allow large-scale expansion of human hepatocytes in immunodeficient swine. Impact statement There is currently a need for robust expansion of human hepatocytes. We describe an immunodeficient swine model into which we engrafted immature human hepatocytes (ihHCs). We identified the mechanism of the eventual graft rejection by the intact NK cell population, which has not been previously shown to have a significant role in xenograft rejection. By both improving engraftment and reducing NK cell-mediated cytotoxicity toward the graft through intrauterine cell transfer, we confirmed the presence of residual adaptive immunity in this model of immunodeficiency and the ability to induce hyposensitization in the NK cell population by taking advantage of the fetal microenvironment.

TNFRSF13B genotypes control immune-mediated pathology by regulating the functions of innate B cells.

Host genes define the severity of inflammation and immunity but specific loci doing so are unknown. Here we show that TNF receptor superfamily member 13B (TNFRSF13B) variants, which enhance defense against certain pathogens, also control immune-mediated injury of transplants, by regulating innate B cells' functions. Analysis of TNFRSF13B in human kidney transplant recipients revealed that 33% of those with antibody-mediated rejection (AMR) but fewer than 6% of those with stable graft function had TNFRSF13B missense mutations. To explore mechanisms underlying aggressive immune responses, we investigated alloimmunity and rejection in mice. Cardiac allografts in Tnfrsf13b-mutant mice underwent early and severe AMR. The dominance and precocity of AMR in Tnfrsf13b-deficient mice were not caused by increased alloantibodies. Rather, Tnfrsf13b mutations decreased "natural" IgM and compromised complement regulation, leading to complement deposition in allografted hearts and autogenous kidneys. Thus, WT TNFRSF13B and Tnfrsf13b support innate B cell functions that limit complement-associated inflammation; in contrast, common variants of these genes intensify inflammatory responses that help clear microbial infections but allow inadvertent tissue injury to ensue. The wide variation in inflammatory reactions associated with TNFRSF13B diversity suggests polymorphisms could underlie variation in host defense and explosive inflammatory responses that sometimes enhance morbidity associated with immune responses.

TNFRSF13B polymorphisms counter microbial adaptation to enteric IgA.

TNFRSF13B encodes the transmembrane activator and CAML interactor (TACI) receptor, which drives plasma cell differentiation. Although TNFRSF13B supports host defense, dominant-negative TNFRSF13B alleles are common in humans and other species and only rarely associate with disease. We reasoned that the high frequency of disruptive TNFRSF13B alleles reflects balancing selection, the loss of function conferring advantage in some settings. Testing that concept, we investigated how a common human dominant-negative variant, TNFRSF13B A181E, imparts resistance to enteric pathogens. Mice engineered to express mono- or biallelic A144E variants of tnrsf13B, corresponding to A181E, exhibited a striking resistance to pathogenicity and transmission of Citrobacter rodentium, a murine pathogen that models enterohemorrhagic Escherichia coli, and resistance was principally owed to natural IgA deficiency in the intestine. In WT mice with gut IgA and in mutant mice reconstituted with enteric IgA obtained from WT mice, IgA induces LEE expression of encoded virulence genes, which confer pathogenicity and transmission. Taken together, our results show that C. rodentium and most likely other enteric organisms appropriated binding of otherwise protective antibodies to signal induction of the virulence program. Additionally, the high prevalence of TNFRSF13B dominant-negative variants reflects balancing selection.

IgV somatic mutation of human anti-SARS-CoV-2 monoclonal antibodies governs neutralization and breadth of reactivity.

Abs that neutralize SARS-CoV-2 are thought to provide the most immediate and effective treatment for those severely afflicted by this virus. Because coronavirus potentially diversifies by mutation, broadly neutralizing Abs are especially sought. Here, we report a possibly novel approach to rapid generation of potent broadly neutralizing human anti-SARS-CoV-2 Abs. We isolated SARS-CoV-2 spike protein-specific memory B cells by panning from the blood of convalescent subjects after infection with SARS-CoV-2 and sequenced and expressed Ig genes from individual B cells as human mAbs. All of 43 human mAbs generated in this way neutralized SARS-CoV-2. Eighteen of the forty-three human mAbs exhibited half-maximal inhibitory concentrations (IC50) of 6.7 × 10-12 M to 6.7 × 10-15 M for spike-pseudotyped virus. Seven of the human mAbs also neutralized (with IC50 < 6.7 × 10-12 M) viruses pseudotyped with mutant spike proteins (including receptor-binding domain mutants and the S1 C-terminal D614G mutant). Neutralization of the Wuhan Hu-1 founder strain and of some variants decreased when coding sequences were reverted to germline, suggesting that potency of neutralization was acquired by somatic hypermutation and selection of B cells. These results indicate that infection with SARS-CoV-2 evokes high-affinity B cell responses, some products of which are broadly neutralizing and others highly strain specific. We also identify variants that would potentially resist immunity evoked by infection with the Wuhan Hu-1 founder strain or by vaccines developed with products of that strain, suggesting evolutionary courses that SARS-CoV-2 could take.

TNFRSF13B Diversification Fueled by B Cell Responses to Environmental Challenges-A Hypothesis.

B cell differentiation and memory are controlled by the transmembrane activator and CAML interactor (TACI), a receptor encoded by TNFRSF13B. TNFRSF13B mutations are frequently found in common variable immunodeficiency (CVID) and in IgA -deficiency; yet, ~98% of those with mutant TNFRSF13B are healthy. Indeed, TNFRSF13B is among the 5% most polymorphic genes in man. Other mammals evidence polymorphism at comparable loci. We hypothesize that TNFRSF13B diversity might promote rather than detract from well-being by controlling key elements of innate immunity. We shall discuss how extraordinary diversity of TNFRSF13B could have evolved and persisted across diverse species of mammals by controlling innate and adaptive B cell responses in apparently paradoxical ways.

The five dimensions of B cell tolerance.

B cell tolerance has been generally understood to be an acquired property of the immune system that governs antibody specificity in ways that avoid auto-toxicity. As useful as this understanding has proved, it fails to fully explain the existence of auto-reactive specificities in healthy individuals and contribution these may have to health. Mechanisms underlying B cell tolerance are considered to select a clonal repertoire that generates a collection of antibodies that do not bind self, ie tolerance operates more or less in three dimensions that largely spare autologous cells and antigens. Yet, most B lymphocytes in humans and probably in other vertebrates are auto-reactive and absence of these auto-reactive B cells is associated with disease. We suggest that auto-reactivity can be embodied by extending the concept of tolerance by two further dimensions, one of time and circumstance and one that allows healthy cells to actively resist injury. In this novel concept, macromolecular recognition by the B cell receptor leading to deletion, anergy, receptor editing or B cell activation is extended by taking account of the time of development of normal immune responses (4th dimension) and the accommodation (or tolerance) of normal cells to bound antibody, activation of complement, and interaction with inflammatory cells (fifth dimension). We discuss how these dimensions contribute to understanding B cell biology in health or disease.

Cell Fusion in Malignancy: A Cause or Consequence? A Provocateur or Cure?

Cell fusion has been observed in malignancy, and cancer cells have been found especially apt to fuse with other cells. Investigation of human and experimental malignancies suggests spontaneous fusion of normal cells can induce manifold genetic changes and manifestations of malignant transformation. Fusion of transformed cells with other cells can promote the progression of cancer to more malignant forms. However, observations in various fields suggest cell fusion also potentially contributes to natural defenses against cancer. Thus, cell fusion potentially corrects genetic and/or phenotypic changes underlying malignant transformation. Cell fusion also might help nonmalignant cells in tumors thwart tumor growth. Perhaps most importantly, cell fusion may generate genetic changes that lead to the expression of neoantigens, provide the mass of neoantigen expression needed to elicit immunity, and promote the function of antigen-presenting cells in a way that favors protective immunity as a defense against malignancy. To the extent that cell fusion promotes cellular, tissue, and/or systemic resistance to malignancy, the propensity of tumor cells to fuse with other cells might constitute a natural defense against malignancy.

Non-canonical B cell functions in transplantation.

B cells are differentiated to recognize antigen and respond by producing antibodies. These activities, governed by recognition of ancillary signals, defend the individual against microorganisms and the products of microorganisms and constitute the canonical function of B cells. Despite the unique differentiation (e.g. recombination and mutation of immunoglobulin gene segments) toward this canonical function, B cells can provide other, "non-canonical" functions, such as facilitating of lymphoid organogenesis and remodeling and fashioning T cell repertoires and modifying T cell responses. Some non-canonical functions are exerted by antibodies, but most are mediated by other products and/or direct actions of B cells. The diverse set of non-canonical functions makes the B cell as much as any cell a central organizer of innate and adaptive immunity. However, the diverse products and actions also confound efforts to weigh the importance of individual non-canonical B cell functions. Here we shall describe the non-canonical functions of B cells and offer our perspective on how those functions converge in the development and governance of immunity, particularly immunity to transplants, and hurdles to advancing understanding of B cell functions in transplantation.

The role of B cell immunity in VCA graft rejection and acceptance.

Vascularized composite allotransplantation (VCA) has emerged as the most recent field of transplantation to offer an alternative treatment for those patients that have failed or are not suitable candidates for conventional therapy. Most of the current clinical experience in this field is with recipients of skin containing grafts such as the face, upper extremity and abdominal wall transplants. Like solid organ recipients, VCA recipients require lifelong systematic immunosuppression to maintain their grafts. To date, the most successful immunosuppressant regimens are calcineurin inhibitor based and have been targeted to the control of T cells. While these regimens have resulted in excellent short term graft survival in solid organ transplantation, achieving significant improvements in long term survival has been more challenging. The reasons are multi-factorial, but a role for B cells and humoral immunity has been proposed. Antibody mediated rejection leading to chronic rejection has been cited as the leading cause of renal graft loss. While the number of VCA transplants performed is still small, evidence to date suggests that antibody mediated rejection may occur less frequently than seen in solid organ transplants. Here we will discuss the role of B cell immunity in solid organ transplantation as it pertains and contrasts to the field of VCA and present some examples of possible sequela of B cell immunity in a series of hand transplant recipients.