Intragraft memory-like CD127hiCD4+Foxp3+ Tregs maintain transplant tolerance.

CD4+Foxp3+ regulatory T cells (Tregs) play an essential role in suppressing transplant rejection, but their role within the graft and heterogeneity in tolerance are poorly understood. Here, we compared phenotypic and transcriptomic characteristics of Treg populations within lymphoid organs and grafts in an islet xenotransplant model of tolerance. We showed Tregs were essential for tolerance induction and maintenance. Tregs demonstrated heterogeneity within the graft and lymphoid organs of tolerant mice. A subpopulation of CD127hi Tregs with memory features were found in lymphoid organs, presented in high proportions within long-surviving islet grafts, and had a transcriptomic and phenotypic profile similar to tissue Tregs. Importantly, these memory-like CD127hi Tregs were better able to prevent rejection by effector T cells, after adoptive transfer into secondary Rag-/- hosts, than naive Tregs or unselected Tregs from tolerant mice. Administration of IL-7 to the CD127hi Treg subset was associated with a strong activation of phosphorylation of STAT5. We proposed that memory-like CD127hi Tregs developed within the draining lymph node and underwent further genetic reprogramming within the graft toward a phenotype that had shared characteristics with other tissue or tumor Tregs. These findings suggested that engineering Tregs with these characteristics either in vivo or for adoptive transfer could enhance transplant tolerance.

Antigen Specific Regulatory T Cells in Kidney Transplantation and Other Tolerance Settings.

Kidney transplantation is the most common solid organ transplant and the best current therapy for end-stage kidney failure. However, with standard immunosuppression, most transplants develop chronic dysfunction or fail, much of which is due to chronic immune injury. Tregs are a subset of T cells involved in limiting immune activation and preventing autoimmune disease. These cells offer the potential to provide tolerance or to allow reduction in immunosuppression in kidney transplants. The importance of Tregs in kidney transplantation has been shown in a number of seminal mouse and animal studies, including those with T cell receptors (TCRs) transgenic Tregs (TCR-Tregs) or Chimeric Antigen Receptor (CAR) Tregs (CAR-Tregs) showing that specificity increases the potency of Treg function. Here we outline the animal and human studies and clinical trials directed at using Tregs in kidney transplantation and other tolerance settings and the various modifications to enhance allo-specific Treg function in vivo and in vitro.

Regulatory T cells in kidney disease and transplantation.

Regulatory T cells (Tregs) have been shown to be important in maintaining immune homeostasis and preventing autoimmune disease, including autoimmune kidney disease. It is also likely that they play a role in limiting kidney transplant rejection and potentially in promoting transplant tolerance. Although other subsets of Tregs exist, the most potent and well-defined Tregs are the Foxp3 expressing CD4(+) Tregs derived from the thymus or generated peripherally. These CD4(+)Foxp3(+) Tregs limit autoimmune renal disease in animal models, especially chronic kidney disease, and kidney transplantation. Furthermore, other subsets of Tregs, including CD8 Tregs, may play a role in immunosuppression in kidney disease. The development and protective mechanisms of Tregs in kidney disease and kidney transplantation involve multiple mechanisms of suppression. Here we review the development and function of CD4(+)Foxp3(+) Tregs. We discuss the specific application of Tregs as a therapeutic strategy to prevent kidney disease and to limit kidney transplant rejection and detail clinical trials in this area of transplantation.

Methyl-Guanine-Methyl-Transferase Transgenic Bone Marrow Transplantation Allows N,N-bis(2-chloroethyl)-Nitrosourea Driven Donor Mixed-Chimerism Without Graft-Versus-Host Disease, and With Donor-Specific Allograft Tolerance.

BACKGROUND: Transplant tolerance has been achieved by mixed chimerism in animal models and in a limited number of kidney transplant patients. However, these mixed-chimerism strategies were limited either by loss of long-term mixed chimerism or risk of graft-versus-host disease (GVHD). Selective bone marrow (BM) engraftment using marrow protective strategies are currently reaching clinical use. In this study, we tested the utility of methyl-guanine-methyl-transferase (MGMT)-transgenic-C57BL/6 BM into a major histocompatibility complex mismatched-BALB/c model followed by N,N-bis(2-chloroethyl)-nitrosourea (BCNU) treatment to enhance donor-cell engraftment and then evaluated transplant tolerance induction. METHODS: A single-dose of anti-CD8 antibody and busulfan was administered into BALB/c-host-mice at day 1. The BALB/c-mice also received costimulatory blockade through multiple-doses of anti-CD40L antibody. 10 × 10(6) BM-cells from MGMT-transgenic-mice were transplanted into host BALB/c mice at day 0. The BCNU was administered at 4 time points after BM transplantation (BMT). Heterotopic donor C57BL/6 cardiac allografts were performed at day 243 after BMT. Skin transplantation with third-party CBA, host BALB/c and donor C57BL/6 grafts was performed at day 358 after BMT. RESULTS: The BALB/c-mice showed long-term stable and high-level donor-cell engraftment with MGMT transgenic C57BL/6 BMT after BCNU treatment, demonstrating full reconstitution and donor cardiac-allograft tolerance and no GVHD with expanded donor and host Foxp3 T regulatory cells. Further, skin grafts from donor, host, and third party showed good immune function with rejection of third-party grafts from all mice and benefit from enhanced chimerism after BCNU with less cell infiltrate and no chronic rejection in the donor skin grafts of BCNU treated mice compared no BCNU treated mice. CONCLUSIONS: High-level mixed chimerism without GVHD can be achieved using MGMT transgenic BM in a mixed-chimerism model receiving BCNU across a major histocompatibility complex mismatch. Enhanced mixed chimerism leads to long-term donor-specific allograft tolerance.

Bilateral Wilms tumor and early presentation in pediatric patients is associated with the truncation of the Wilms tumor 1 protein.

OBJECTIVES: To investigate the frequency of constitutional Wilms tumor 1 gene (WT1) abnormalities in children with bilateral Wilms tumor (WT) and the age of tumor onset in patients with a mutation. STUDY DESIGN: Eight patients with bilateral WT were studied. High-resolution melting and direct sequencing were used to screen for the WT1 gene. Western blotting was performed to determine whether the identified mutations were associated with expressed truncated WT1 protein. RESULTS: The median age of tumor onset in patients with a mutation in the WT1 was lower (10 months) than in those without a mutation (39 months). Three novel heterozygous nonsense mutations were identified in exon 8 in peripheral blood from 3 individuals, whereas all 3 tumor tissues lacked the wild-type allele. All mutations led to a premature stop codon with truncation of the WT1 protein. In 1 patient, a truncated form of WT1 protein was identified, suggesting that development of the WT may have resulted from expression of an abnormal protein. Four distinct silent single-nucleotide polymorphisms (SNPs) were detected. All 3 patients with a pathogenic WT1 mutation had 2 synonymous SNPs, whereas only 1 of the remaining 5 patients had a single synonymous SNP (P < .05). CONCLUSIONS: Bilateral WT are associated with early presentation in pediatric patients and a high frequency of WT1 nonsense mutations in exon 8. Silent SNPs may also be involved in the development of WT.

Robust anti-tumor immunity and memory in Rag-1-deficient mice following adoptive transfer of cytokine-primed splenocytes and tumor CD80 expression.

Successful immunotherapy of solid tumors has proven difficult to achieve. The aim of the current study was to further investigate the effects of peripheral CD80-mediated co-stimulation on the efficacy of polyclonal anti-tumor effector CTL in an adoptive transfer model. Splenocytes obtained from wild-type mice immunized with CD80-transduced EL4 tumor cells were expanded in vitro in the presence of either IL-12 or IL-15 and irradiated CD80-transduced EL4 tumor cells. Polyclonal CD8 T cells were the major subset in the effector population. Primed effector cells were adoptively transferred into immuno-deficient Rag-1-deficient mice which were then challenged with syngeneic vector-control or CD80-transduced EL4 tumor cells. Expression of CD80 enhanced the elimination of EL4 tumors and mouse survival. Both IL-12 and IL-15 cultured cells had enhanced cytotoxicity. Importantly, anti-tumor memory was maintained without tumor evasion following re-challenge with either CD80-transduced and vector-control EL4 cells. We also show, using antibody-mediated depletion, that endogenous NK cells present in Rag-1-deficient mice exert anti-EL4 tumor activity that is enhanced by CD80 expression. Collectively these data show that peripheral co-stimulation by tumor expression of CD80 results in enhanced anti-tumor efficacy of NK and polyclonal effector T cells, and suggest that TCR repertoire diversity helps protect against tumor escape and provides memory with resultant robust immunity to subsequent tumor challenge irrespective of CD80 status.

Matching T-cell receptors identified in renal biopsies and urine at the time of acute rejection in pediatric renal transplant patients.

Urinary monitoring of kidney allograft function has been used for many years. More recently, molecular identification of cytotoxic T-cell products has been used as a diagnostic tool in acute rejection. Monitoring of T-cell infiltrates by analysis of the T-cell receptor (TcR) gene usage has been performed on biopsies with acute and chronic rejection, but not on urine samples. The aim of this study was to identify and compare TRBV gene usage assessing the CDR3 (Complementarity Determining Region 3) length distribution and sequence in urine and biopsies of pediatric renal allograft patients at the time of acute rejection and compare them with peripheral blood. We studied four pediatric renal transplant recipients with acute cellular rejection. We identified restricted and matched TRBV CDR3 spectratypes with overexpressed TRBV families and show identical, clonally expanded TRBV CDR3 sequences in all four patients present in the urine and renal allograft. We demonstrate that urinary monitoring can detect graft-infiltrating lymphocytes in acute rejection and may have a role in the monitoring of renal transplants.

Prophylactic bilateral nephrectomies in two paediatric patients with missense mutations in the WT1 gene.

BACKGROUND: Denys-Drash syndrome (DDS) is associated with mutations of the Wilms' tumour 1 (WT1) gene, and is characterized by pseudohermaphroditism, a progressive glomerulopathy, and the development of Wilms' tumour. More than 90% of patients with DDS who carry constitutional intragenic WT1 mutations are at high risk (90%) for the development of Wilms' tumour. WT1 is a signalling protein with 90% of WT1 mutations occurring in the WT1 zinc finger region as single nucleotide polymorphisms, the majority of which are missense mutations. METHODS: Constitutional DNA was extracted from peripheral blood. Direct sequencing and restriction enzymes were employed to analyse mutations. RESULTS: Two children, 46XY males who had evidence of pseudohermaphroditism, hypogonadism and renal failure with a glomerulopathy atypical for DDS, but no Wilms' tumour or nephroblastomatosis, on investigation, prior to transplant, were identified with missense mutations in the WT1 gene, in exons 8 and 9, respectively. The decision to do prophylactic nephrectomies was based on the genetic identification of WT1 mutations supporting a diagnosis of incomplete DDS, with the potential for increased risk of malignancy with the development of Wilms' tumour. The nephrectomy specimens demonstrated nephrogenic rests (nephroblastomatosis), which have a potential for malignant transformation. CONCLUSIONS: WT1 missense mutations in exons 8 and 9 can be regarded as having the potential for malignant change supporting prophylactic nephrectomy in apparent incomplete DDS patients with end-stage renal disease.