Eliminating financial disincentives to living kidney donation - a call to action.

The incidence of end stage renal disease (ESRD) in the United States (US) is increasing each year. The lone curative treatment for ESRD remains kidney transplantation. Despite the demonstrated medical and economic benefits, living donor kidney transplantation (LDKT) only accounts for a small number of kidney transplantations each year. Direct and indirect costs exist that disincentivize potential living kidney donors from coming forward, such as the cost of travel and lodging, risk of death, potential loss of income due to an extended recovery time, and the inability to donate to a relative in the future if needed. Herein, we advocate for policy changes that make living kidney donation (LKD) a financially neutral process thereby incentivizing increased LDKT and mitigating the kidney donor shortage.

Selective CD28 blockade impacts T cell differentiation during homeostatic reconstitution following lymphodepletion.

Introduction: Costimulation blockade targeting the CD28 pathway provides improved long-term renal allograft survival compared to calcineurin inhibitors but may be limited as CTLA-4-Ig (abatacept, belatacept) blocks both CD28 costimulation and CTLA-4 coinhibition. Directly targeting CD28 while leaving CTLA-4 intact may provide a mechanistic advantage. Fc-silent non-crosslinking CD28 antagonizing domain antibodies (dAb) are currently in clinical trials for renal transplantation. Given the current standard of care in renal transplantation at most US centers, it is likely that lymphodepletion via thymoglobulin induction therapy could be used in patients treated with CD28 antagonists. Thus, we investigated the impact of T cell depletion (TCD) on T cell phenotype following homeostatic reconstitution in a murine model of skin transplantation treated with anti-CD28dAb. Methods: Skin from BALB/cJ donors was grafted onto C56BL/6 recipients which were treated with or without 0.2mg anti-CD4 and 10µg anti-CD8 one day prior to transplant and with or without 100µg anti-CD28dAb on days 0, 2, 4, 6, and weekly thereafter. Mice were euthanized six weeks post-transplant and lymphoid cells were analyzed by flow cytometry. Results: Anti-CD28dAb reversed lymphopenia-induced differentiation of memory CD4+ T cells in the spleen and lymph node compared to TCD alone. Mice treated with TCD+anti-CD28dAb exhibited significantly improved skin graft survival compared to anti-CD28dAb alone, which was also improved compared to no treatment. In addition, the expression of CD69 was reduced on CD4+ and CD8+ T cells in the spleen and lymph node from mice that received TCD+anti-CD28dAb compared to TCD alone. While a reduced frequency of CD4+FoxP3+ T cells was observed in anti-CD28dAb treated mice relative to untreated controls, this was balanced by an increased frequency of CD8+Foxp3+ T cells that was observed in the blood and kidney of mice given TCD+anti-CD28dAb compared to TCD alone. Discussion: These data demonstrate that CD28 signaling impacts the differentiation of both CD4+ and CD8+ T cells during homeostatic reconstitution following lymphodepletion, resulting in a shift towards fewer activated memory T cells and more CD8+FoxP3+ T cells, a profile that may underpin the observed prolongation in allograft survival.

Large Animal Models of Vascularized Composite Allotransplantation: A Review of Immune Strategies to Improve Allograft Outcomes.

Over the past twenty years, significant technical strides have been made in the area of vascularized composite tissue allotransplantation (VCA). As in solid organ transplantation, the allogeneic immune response remains a significant barrier to long-term VCA survival and function. Strategies to overcome acute and chronic rejection, minimize immunosuppression and prolong VCA survival have important clinical implications. Historically, large animals have provided a valuable model for testing the clinical translatability of immune modulating approaches in transplantation, including tolerance induction, co-stimulation blockade, cellular therapies, and ex vivo perfusion. Recently, significant advancements have been made in these arenas utilizing large animal VCA models. In this comprehensive review, we highlight recent immune strategies undertaken to improve VCA outcomes with a focus on relevant preclinical large animal models.

Non-invasive imaging for the diagnosis of acute rejection in transplantation: The next frontier.

Acute rejection is a leading cause of organ transplant failure and the most common indication for re-transplantation. Clinically, suspicion of acute rejection is often dependent upon serum laboratory values which may only manifest after organ injury. The gold standard for diagnosis requires an invasive biopsy which can carry serious clinical risks including bleeding and graft loss as well as the possibility of sampling error. The use of noninvasive imaging modalities to monitor transplanted organs is of great clinical value, particularly as a tool for early detection of graft dysfunction or acute rejection. Herein, we provide an overview of the existing literature evaluating noninvasive imaging modalities of solid organ and cellular allografts after transplantation, including both preclinical and clinical studies.

Cellular Immunotherapies in Preclinical Large Animal Models of Transplantation.

Hematopoietic stem cell (HSC) transplantation and solid organ transplantation remain the only curative options for many hematologic malignancies and end-stage organ diseases. Unfortunately, the sequelae of long-term immunosuppression, as well as acute and chronic rejection, carry significant morbidities, including infection, malignancy, and graft loss. Numerous murine models have demonstrated the efficacy of adjunctive cellular therapies using HSCs, regulatory T cells, mesenchymal stem cells, and regulatory dendritic cells in modulating the alloimmune response in favor of graft tolerance; however, translation of such murine approaches to other preclinical models and in the clinic has yielded mixed results. Large animals, including nonhuman primates, swine, and canines, provide a more immunologically rigorous model in which to test the clinical translatability of these cellular therapies. Here, we highlight the contributions of large animal models to the development and optimization of HSCs and additional cellular therapies to improve organ transplantation outcomes.

Programmed T cell differentiation: Implications for transplantation.

While T cells play a critical role in protective immunity against infection, they are also responsible for graft rejection in the setting of transplantation. T cell differentiation is regulated by both intrinsic transcriptional pathways as well as extrinsic factors such as antigen encounter and the cytokine milieu. Herein, we review recent discoveries in the transcriptional regulation of T cell differentiation and their impact on the field of transplantation. Recent studies uncovering context-dependent differentiation programs that differ in the setting of infection or transplantation will also be discussed. Understanding the key transcriptional pathways that underlie T cell responses in transplantation has important clinical implications, including development of novel therapeutic agents to mitigate graft rejection.

Impact of selective CD28 blockade on virus-specific immunity to a murine Epstein-Barr virus homolog.

CTLA-4Ig (belatacept) blocks the CD80/CD86 ligands for both CD28 and CTLA-4; thus, in addition to the intended effect of blocking CD28-mediated costimulation, belatacept also has the unintended effect of blocking CTLA-4-mediated coinhibition. Recently, anti-CD28 domain antibodies (dAb) that selectively target CD28 while leaving CTLA-4 intact were shown to more effectively inhibit alloimmune responses and prolong graft survival. However, the impact of selective CD28 blockade on protective immunity has not been extensively investigated. Here, we sought to compare the impact of CTLA-4Ig vs anti-CD28dAb on CD8+ T cell immunity to a transplant-relevant pathogen, a murine homolog of Epstein-Barr virus. Mice were infected with murine gammaherpesvirus-68 (MHV) and treated with vehicle, CTLA-4Ig, or anti-CD28dAb. Although anti-CD28dAb resulted in a decrease in virus-specific CD8+ T cell numbers as compared to CTLA-4Ig, cytolytic function and the expression of markers of high-quality effectors were not different from CTLA-4Ig treated animals. Importantly, MHV-68 viral load was not different between the treatment groups. These results suggest that preserved CTLA-4 coinhibition limits MHV-specific CD8+ T cell accumulation, but the population that remains retains cytolytic function and migratory capacity and is not inferior in its ability to control viral burden relative to T cell responses in CTLA-4Ig-treated animals.

Sepsis erodes CD8+ memory T cell-protective immunity against an EBV homolog in a 2B4-dependent manner.

Epstein-Barr virus (EBV) reactivation commonly occurs following sepsis, but the mechanisms underlying this are unknown. We utilized a murine EBV homolog (gHV) and the cecal ligation and puncture model of polymicrobial sepsis to study the impact of sepsis on gHV reactivation and CD8+ T cell immune surveillance following a septic insult. We observed a significant increase in the frequency of gHV-infected germinal center B cells on day 7 following sepsis. This increase in viral load was associated with a concomitant significant decrease in the frequencies of gHV-specific CD8+ T cells, as measured by class I MHC tetramers corresponding to the immunodominant viral epitopes. Phenotypic analysis revealed an increased frequency of gHV-specific CD8+ T cells expressing the 2B4 coinhibitory receptor in septic animals compared with sham controls. We sought to interrogate the role of 2B4 in modulating the gHV-specific CD8+ T cell response during sepsis. Results indicated that in the absence of 2B4, gHV-specific CD8+ T cell populations were maintained during sepsis, and gHV viral load was unchanged in 2B4-/- septic animals relative to 2B4-/- sham controls. WT CD8+ T cells upregulated PD-1 during sepsis, whereas 2B4-/- CD8+ T cells did not. Finally, adoptive transfer studies revealed a T cell-intrinsic effect of 2B4 coinhibition on virus-specific CD8+ T cells and gHV viral load during sepsis. These data demonstrate that sepsis-induced immune dysregulation erodes antigen-specific CD8+ responses against a latent viral infection and suggest that blockade of 2B4 may better maintain protective immunity against EBV in the context of sepsis.

Challenges and opportunities in targeting the CD28/CTLA-4 pathway in transplantation and autoimmunity.

INTRODUCTION: T cell activation is a complex process that requires multiple cell signaling pathways, including a primary recognition signal and additional costimulatory signals. One of the best-characterized costimulatory pathways includes the Ig superfamily members CD28 and CTLA-4 and their ligands CD80 and CD86. Areas covered: This review discusses past, current and future biological therapies that have been utilized to block the CD28/CTLA-4 cosignaling pathway in the settings of autoimmunity and transplantation, as well the challenges facing successful implementation of these therapies. Expert opinion: The development of CD28 blockers Abatacept and Belatacept provided a more targeted therapy approach for transplant rejection and autoimmune disease relative to calcineurin inhibitors and anti-proliferatives, but overall efficacy may be limited due to their collateral effect of simultaneously blocking CTLA-4 coinhibitory signals. As such, current investigations into the potential of selective CD28 blockade to block the costimulatory potential of CD28 while exploiting the coinhibitory effects of CTLA-4 are promising. However, as selective CD28 blockade inhibits the activity of both effector and regulatory T cells, an important goal for the future is the design of therapies that will maximize the attenuation of effector responses while preserving the suppressive function of T regulatory cells.

Donor Lymphocyte Infusion-Mediated Graft-versus-Host Responses in a Preclinical Swine Model of Haploidentical Hematopoietic Cell Transplantation.

We previously described successful hematopoietic stem cell engraftment across MHC barriers in miniature swine without graft-versus-host disease (GVHD) using novel reduced-intensity conditioning regimens consisting of partial transient recipient T cell-depletion, thymic or low-dose total body irradiation, and a short course of cyclosporine A. Here we report that stable chimeric animals generated with these protocols are strongly resistant to donor leukocyte infusion (DLI)-mediated GVH effects. Of 33 total DLIs in tolerant chimeras at clinical doses, 21 failed to induce conversion to full donor hematopoietic chimerism or cause GVHD. We attempted to overcome this resistance to conversion through several mechanisms, including using sensitized donor lymphocytes, increasing the DLI dose, removing chimeric host peripheral blood cells through extensive recipient leukapheresis before DLI, and using fully mismatched lymphocytes. Despite our attempts, the resistance to conversion in our model was robust, and when conversion was achieved, it was associated with GVHD in most animals. Our studies suggest that delivery of unmodified hematopoietic stem cell doses under reduced-intensity conditioning can induce a potent, GVHD-free, immune tolerant state that is strongly resistant to DLI.

MicroRNA miR-155 Is Necessary for Efficient Gammaherpesvirus Reactivation from Latency, but Not for Establishment of Latency.

UNLABELLED: MicroRNA-155 (miR-155) has been shown to play significant roles in the immune response, including in the formation of germinal centers (GC) and the development and maturation of T follicular helper (Tfh) cells. There is in vitro evidence to support a critical role for cellular miR-155 and viral miR-155 homologs in the establishment of gammaherpesvirus latency in B cells. We sought to determine the contribution of miR-155 to the establishment and maintenance of latency in vivo using murine gammaherpesvirus (MHV-68) infection. MHV-68-infected mice deficient in miR-155 exhibited decreases in GC B cells and Tfh cells. However, the frequencies of spleen cells harboring latent MHV-68 genomes were the same in both miR-155-deficient and wild-type (WT) mice. Similar latent loads were also observed in mixed bone marrow chimeric mice, where B cell-extrinsic effects of miR-155 deficiency were normalized. Interestingly, we observed markedly lower efficiency of reactivation from latency in miR-155-deficient cells, indicating an important role for miR-155 in this process. These in vivo data complement previous in vitro studies and lead to the conclusion that miR-155 is not necessary for the establishment or maintenance of gammaherpesvirus latency but that it does affect reactivation efficiency. IMPORTANCE: Gammaherpesvirus infection leads to severe disease in immunosuppressed populations. miR-155 has been shown to play important roles in many pathological processes, including tumorigenesis and diseases caused by an overly aggressive immune response. Our work provides valuable in vivo data showing that miR-155 is dispensable for gammaherpesvirus latency but that it is critical for reactivation from latency, which is a crucial step in the viral life cycle.

CD4 and CD8 T cells directly recognize murine gammaherpesvirus 68-immortalized cells and prevent tumor outgrowth.

There has been extensive research regarding T cell recognition of Epstein-Barr virus-transformed cells; however, less is known regarding the recognition of B cells immortalized by gamma-2 herpesviruses. Here we show that B cells immortalized by murine gammaherpesvirus 68 (MHV-68, γHV-68) can be controlled by either CD4 or CD8 T cells in vivo. We present evidence for the direct recognition of infected B cells by CD4 and CD8 T cells. These data will help in the development of immunotherapeutic approaches combating gamma-2 herpesvirus-related disease.

Edema and tetraparesis in a miniature pig after allogeneic hematopoietic cell transplantation.

A 3-mo-old, 12-kg, intact, miniature pig presented with severe neurologic signs on day 8 after hematopoietic cell transplantation. This pig had received an immunosuppressive regimen before transplantation that included an antiCD3 immunotoxin for T-cell depletion, 100 cGy of total-body irradiation, and cyclosporine for 45 d. The pig began exhibiting erythematous lesions on posttransplantation day 7. He also demonstrated increased conscious proprioceptive deficits and recumbency but normal mentation. Neurologic signs worsened over several days; the pig became lethargic but remained afebrile. Conjunctival swelling developed on posttransplantation day 9, which subsequently spread to the animal's head, ears and hocks by day 10. Analgesics were given for pain, and cyclosporine levels were decreased. Despite the measures taken, neurologic signs progressed. Given the worsening subcutaneous edema and neurologic status, Escherichia coli infection was suspected, and treatment with a third-generation cephalosporin was instituted. The clinical signs resolved within 12 h after the start of antibiotics. 'Shiga-like' toxin from E. coli can cause peracute toxemia and induce ataxia, paralysis, and recumbency. Other common and pathognomonic findings include periocular edema and variable edema in other subcutaneous regions. A fecal sample demonstrated an overgrowth of gram-negative, lactose-fermenting colonies. On the basis of the clinical presentation, exclusion of other potential conditions compatible with edema and neurologic diseases, physical exam findings, microbiology and the resolution of signs after therapy, the pig was diagnosed with edema disease.

Lack of antidonor alloantibody does not indicate lack of immune sensitization: studies of graft loss in a haploidentical hematopoietic cell transplantation swine model.

Loss of chimerism is an undesirable outcome of allogeneic hematopoietic cell transplantation (HCT) after reduced-intensity conditioning. Understanding the nature of cellular and humoral immune responses to HCT after graft loss could lead to improved retransplantation strategies. We investigated the immunologic responses after graft loss in miniature swine recipients of haploidentical HCT that received reduced-intensity conditioning. After the loss of peripheral blood chimerism, antidonor cellular responses were present without detectable antidonor antibody. Reexposure to donor hematopoietic cells after graft loss induced a sensitized antidonor cellular response. No induced antidonor antibody response could be detected despite evidence of cellular sensitization to donor cells. In contrast, unconditioned animals exposed repeatedly to similar doses of haploidentical donor cells developed antidonor antibody responses. These results could have important implications for the design of treatment strategies to overcome antidonor responses in HCT and improve the outcome of retransplantation after graft loss.

Effect of pre-existing anti-diphtheria toxin antibodies on T cell depletion levels following diphtheria toxin-based recombinant anti-monkey CD3 immunotoxin treatment.

Diphtheria toxin (DT)-based anti-CD3 immunotoxins have clinical relevance in numerous applications including autoimmune disease therapies and organ transplantation tolerance protocols. Pre-existing anti-DT antibodies acquired either by vaccination against diphtheria toxin or infections with C. diphtheriae may interfere or inhibit the function of these anti-CD3 immunotoxins. Previously, a full-length anti-rhesus monkey CD3 immunotoxin, FN18-CRM9, was shown to be less effective at depleting circulating T cells in animals with pre-existing anti-DT antibody titers than in animals without antibodies, and subsequent doses were ineffective. In this study, the T cell depletion function of a truncated DT based recombinant anti-monkey CD3 immunotoxin, A-dmDT390-scfbDb (C207), as part of a reduced intensity conditioning regimen prior to hematopoietic cell transplantation, was compared between two groups of monkeys: those with and without pre-existing anti-diphtheria titers. T cell depletion was comparable in both groups of monkeys, and therefore appeared to be unaffected by the presence of moderate levels of pre-existing anti-diphtheria antibodies.

Effects of mobilization regimens in donors on outcomes of hematopoietic cell transplantation in miniature Swine.

Toxicities and complications associated with hematopoietic cell transplantation currently limit this potentially curative therapy for malignant and nonmalignant blood disorders. Miniature swine provide a clinically relevant model for studies to improve posttransplantation outcomes. Miniature swine recipients of high-dose haploidentical hepatopoietic cell transplantation after reduced-intensity conditioning consisting of low-dose (100 cGy) total-body irradiation, partial T-cell depletion by using a CD3 immunotoxin, and a 45-d course of cyclosporine A typically successfully engraft without graft-versus-host disease. We recently observed broad variability in engraftment outcomes that correlates with the occurrence of adverse reactions in donors after cytokine treatment to mobilize hematopoietic progenitor cells from the bone marrow to the peripheral blood for collection. Haploidentical recipients (n = 16) of cells from donors remaining healthy during cytokine treatment engrafted with multilineage chimerism, did not develop graft-versus-host disease, and did not require any blood products. In comparison, identically conditioned recipients of cells from donors that had severe reactions during cytokine treatment had adverse outcomes, including the development of clinically significant thrombocytopenia requiring blood product support in 8 of 11 swine. Furthermore, all 11 recipients lost peripheral blood myeloid chimerism (indicating lack of engraftment of donor stem cells). These data suggest that posttransplantation complications in swine are influenced by the health status of the donor before and during the collection of hematopoietic cells by leukapheresis.

Expression and purification of non-N-glycosylated porcine interleukin 3 in yeast Pichia pastoris.

Yeast Pichia pastoris has been widely utilized to express heterologous recombinant proteins. P. pastoris expressed recombinant porcine interleukin 3 (IL3) has been used for porcine stem cell mobilization in allo-hematopoietic cell transplantation models and pig-to-primate xeno-hematopoietic cell transplantation models in our lab for many years. Since the yeast glycosylation mechanism is not exactly the same as those of other mammalian cells, P. pastoris expressed high-mannose glycoprotein porcine IL3 has been shown to result in a decreased serum half-life. Previously this was avoided by separation of the non-glycosylated porcine IL3 from the mixture of expressed glycosylated and non-glycosylated porcine IL3. However, this process was very inefficient and lead to a poor yield following purification. To overcome this problem, we engineered a non-N-glycosylated version of porcine IL3 by replacing the four potential N-glycosylation sites with four alanines. The codon-optimized non-N-glycosylated porcine IL3 gene was synthesized and expressed in P. pastoris. The expressed non-N-glycosylated porcine IL3 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50 HQ. In vivo mobilization studies performed in our research facility demonstrated that the non-N-glycosylated porcine IL3 still keeps the original stem cell mobilization function.

Development of a diphtheria toxin based antiporcine CD3 recombinant immunotoxin.

Anti-CD3 immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.