Post-transplant cyclophosphamide limits reactive donor T cells and delays the development of graft-versus-host disease in a humanized mouse model.

Graft-versus-host disease (GVHD) is a major complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT) that develops when donor T cells in the graft become reactive against the host. Post-transplant cyclophosphamide (PTCy) is increasingly used in mismatched allo-HSCT, but how PTCy impacts donor T cells and reduces GVHD is unclear. This study aimed to determine the effect of PTCy on reactive human donor T cells and GVHD development in a preclinical humanized mouse model. Immunodeficient NOD-scid-IL2Rγnull mice were injected intraperitoneally (i.p.) with 20 × 106 human peripheral blood mononuclear cells stained with carboxyfluorescein succinimidyl ester (CFSE) (day 0). Mice were subsequently injected (i.p.) with PTCy (33 mg kg-1 ) (PTCy-mice) or saline (saline-mice) (days 3 and 4). Mice were assessed for T-cell depletion on day 6 and monitored for GVHD for up to 10 weeks. Flow cytometric analysis of livers at day 6 revealed lower proportions of reactive (CFSElow ) human (h) CD3+ T cells in PTCy-mice compared with saline-mice. Over 10 weeks, PTCy-mice showed reduced weight loss and clinical GVHD, with prolonged survival and reduced histological liver GVHD compared with saline-mice. PTCy-mice also demonstrated increased splenic hCD4+ :hCD8+ T-cell ratios and reduced splenic Tregs (hCD4+  hCD25+  hCD127lo ) compared with saline-mice. This study demonstrates that PTCy reduces GVHD in a preclinical humanized mouse model. This corresponded to depletion of reactive human donor T cells, but fewer human Tregs.

α-Parvin and ß-parvin in the rat uterus during decidualisation and uterine receptivity.

During early pregnancy, the uterine luminal epithelial cells (UECs) and endometrial stromal cells (ESCs) undergo morphological changes to enable blastocyst implantation. The present study investigates, for the first time, the cytoskeletal-associated proteins and α-actinin superfamily members, α-parvin and ß-parvin, during early pregnancy in the rat uterus. These two PARVA proteins are involved in cell adhesion, morphological changes and regulation of other cytoskeletal proteins, through binding with proteins such as actin and integrin-linked kinase. α-parvin is present in UECs at fertilisation and significantly decreases by the time of implantation. ß-parvin acts in opposition; significantly increasing in both UECs and ESCs at the time of implantation, suggesting a role in the process of decidualisation. Additionally, the presence of a serine-8 residue-phosphorylated α-parvin, which is associated with cell morphology changes, was found in the nuclear region of both UECs and ESCs during implantation and decidualisation. We also show that the presence of both ß-parvin and phosphorylated α-parvin in ESCs is dependent on decidualisation occurring. This study demonstrates that the changing balance and localisation of the two PARVA proteins are dependent on the time of uterine receptivity, suggesting a co-dependent role in the cytoskeletal re-organisation crucial to the changing conditions necessary for implantation and decidualisation.

Change in distribution of cytoskeleton-associated proteins, lasp-1 and palladin, during uterine receptivity in the rat endometrium.

The epithelium of the uterine lumen is the first point of contact with the blastocyst before implantation. To facilitate pregnancy, these uterine epithelial cells (UECs) undergo morphological changes specific to the receptive uterus. These changes include basal, lateral and apical alterations in the plasma membrane of UECs. This study looked at the cytoskeletal and focal adhesion-associated proteins, lasp-1 and palladin, in the uterus during early pregnancy in the rat. Two palladin isoforms, 140 kDa and 90 kDa, were analysed, with the migration-associated 140-kDa isoform increasing significantly at the time of implantation when compared with the time of fertilisation. Lasp-1 was similarly increased at this time, whilst also being located predominantly apically and laterally in the UECs, suggesting a role in the initial contact between the UECs and the blastocyst. This is the first study to investigate palladin and lasp-1 in the uterine luminal epithelium and suggests an importance for these cytoskeletal proteins in the morphological changes the UECs undergo for pregnancy to occur.

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.

Key driver genes as potential therapeutic targets in renal allograft rejection.

Acute rejection (AR) in renal transplantation is an established risk factor for reduced allograft survival. Molecules with regulatory control among immune pathways of AR that are inadequately suppressed, despite standard-of-care immunosuppression, could serve as important targets for therapeutic manipulation to prevent rejection. Here, an integrative, network-based computational strategy incorporating gene expression and genotype data of human renal allograft biopsy tissue was applied, to identify the master regulators - the key driver genes (KDGs) - within dysregulated AR pathways. A 982-meta-gene signature with differential expression in AR versus non-AR was identified from a meta-analysis of microarray data from 735 human kidney allograft biopsy samples across 7 data sets. Fourteen KDGs were derived from this signature. Interrogation of 2 publicly available databases identified compounds with predicted efficacy against individual KDGs or a key driver-based gene set, respectively, which could be repurposed for AR prevention. Minocycline, a tetracycline antibiotic, was chosen for experimental validation in a murine cardiac allograft model of AR. Minocycline attenuated the inflammatory profile of AR compared with controls and when coadministered with immunosuppression prolonged graft survival. This study demonstrates that a network-based strategy, using expression and genotype data to predict KDGs, assists target prioritization for therapeutics in renal allograft rejection.

Low-Dose Interleukin-2 Combined With Rapamycin Led to an Expansion of CD4+CD25+FOXP3+ Regulatory T Cells and Prolonged Human Islet Allograft Survival in Humanized Mice.

Islet transplantation is an emerging therapy for type 1 diabetes and hypoglycemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study, we established a diabetic humanized NOD-scidIL2Rγnull (NSG) mouse model of T-cell-mediated human islet allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin-2 (IL-2) combined with low-dose rapamycin to prolong graft survival. NSG mice that had received renal subcapsular human islet allografts and were transfused with 1 × 107 of human spleen mononuclear cells reconstituted human CD45+ cells that were predominantly CD3+ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3 × 106 IU/m2 or 1 × 106 IU/m2) or rapamycin alone (0.5-1 mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet allograft survival. Graft survival was associated with expansion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) and enhanced transforming growth factor-ß production by CD4+ T cells. CD8+ T cells showed reduced interferon-γ production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet allograft rejection by expanding CD4+FOXP3+ Tregs in vivo and suppressing effector cell function and could be the basis of effective tolerance-based regimens.

Standardisation of flow cytometry for whole blood immunophenotyping of islet transplant and transplant clinical trial recipients.

Understanding the immunological phenotype of transplant recipients is important to improve outcomes and develop new therapies. Immunophenotyping of whole peripheral blood (WPB) by flow cytometry is a rapid method to obtain large amounts of data relating to the outcomes of different transplant treatments with limited patient impact. Healthy individuals and patients with type 1 diabetes (T1D) enrolled in islet transplantation were recruited and WPB was collected. 46 fluorochrome-conjugated mouse-anti-human antibodies were used (43 of 46 antibodies were titrated). BD cytometer setup and tracking beads were used to characterize and adjust for cytometer performance. Antibody cocktails were pre-mixed <60 minutes before staining. Multicolour panels were designed based on fluorochrome brightness, antigen density, co-expression, and fluorochrome spillover into non-primary detectors in each panel on a 5 laser flow cytometer. WPB sample staining used 50-300 µl WPB for each panel and was performed within 2 hours of blood sample collection. Samples were acquired on a BD-LSRFortessa. The operating procedures, including specimen collection, antibody cocktails, staining protocol, flow-cytometer setup and data analysis, were standardized. The staining index of 43 antibodies and the spillover spreading matrix for each panel was calculated. The final concentrations for the 46 antibodies used was determined for staining of WPB samples. Absolute cell-count and 7 leukocyte profiling panels consisting of subsets and/or status of granulocytes, monocytes, dendritic, B, NK, and T cells including regulatory T cells (Tregs) and NKT were designed and established on a 5 laser BD-LSR Fortessa. 13 T1D patients, including 4 islet transplant recipients and 8 healthy controls, were evaluated. The ability to reproducibly measure immune subsets and immune-profiles of islet transplant patients up to 18 months post transplantation has been established as a tool to measure immune cell reconstitution after transplantation.