Christopher M. Hope

Interleukin-17A-Induced Human Mesenchymal Stem Cells Are Superior Modulators of Immunological Function.

Interferon‐γ (IFN‐γ)‐preactivated mesenchymal stem cells (MSC‐γ) are highly immunosuppressive but immunogenic in vivo due to their inherent expression of major histocompatibility (MHC) molecules. Here, we present an improved approach where we modified human bone marrow‐derived MSC with interleukin‐17A (MSC‐17) to enhance T cell immunosuppression but not their immunogenicity. MSC‐17, unlike MSC‐γ, showed no induction or upregulation of MHC class I, MHC class II, and T cell costimulatory molecule CD40, but maintained normal MSC morphology and phenotypic marker expression. When cocultured with phytohemagglutinin (PHA)‐activated human T cells, MSCs‐17 were potent suppressors of T cell proliferation. Furthermore, MSC‐17 inhibited surface CD25 expression and suppressed the elaboration of Th1 cytokines, IFN‐γ, tumor necrosis factor‐α (TNF‐α), and IL‐2 when compared with untreated MSCs (UT‐MSCs). T cell suppression by MSC‐17 correlated with increased IL‐6 but not with indoleamine 2,3‐dioxygenase 1, cyclooxygenase 1, and transforming growth factor β‐1. MSC‐17 but not MSC‐γ consistently induced CD4+CD25highCD127lowFoxP3+ regulatory T cells (iTregs) from PHA‐activated CD4+CD25− T cells. MSC‐induced iTregs expressed CD39, CD73, CD69, OX40, cytotoxic T‐lymphocyte associated antigen‐4 (CTLA‐4), and glucocorticoid‐induced TNFR‐related protein (GITR). These suppressive MSCs‐17 can engender Tregs to potently suppress T cell activation with minimal immunogenicity and thus represent a superior T cell immunomodulator for clinical application. Stem Cells 2015;33:2850–2863

The immune phenotype may relate to cancer development in kidney transplant recipients.

High regulatory T-cell (Treg) numbers predict recurrent cutaneous squamous cell carcinoma in kidney transplant recipients, and the Treg immune phenotype may identify kidney transplant recipients at risk of developing squamous cell carcinoma and/or solid-organ cancer. To investigate this, a total of 116 kidney transplant recipients, of whom 65 had current or past cancer, were immune-phenotyped and followed up prospectively for a median of 15 months. Higher Treg (CD3+CD4+FOXP3+CD25(Hi)CD127(Lo)) proportion and numbers significantly increased the odds of developing cancer (odds ratios (95% CI) 1.61 (1.17-2.20) and 1.03 (1.00-1.06), respectively) after adjusting for age, gender, and duration of immunosuppression. Class-switched memory B cells (CD19+CD27+IgD-) had a significant association to cancer, 1.04 (1.00-1.07). Receiver operator characteristic (ROC) curves for squamous cell carcinoma development within 100 days of immune phenotyping were significant for Tregs, memory B cells, and γδ T cells (AUC of 0.78, 0.68, and 0.65, respectively). After cancer resection, Treg, NK cell, and γδ T-cell numbers fell significantly. Immune-phenotype profiles associated with both squamous cell carcinoma and solid-organ cancer in kidney transplant recipients and depended on the presence of cancer tissue. Thus, immune profiling could be used to stratify kidney transplant recipients at risk of developing cancers to identify those who could qualify for prevention therapy.

C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies

Acute antibody‐mediated rejection can occur in absence of circulating donor‐specific antibodies. Agonistic antibodies targeting the anti‐angiotensin II type 1 receptor (anti‐AT1R) are emerging as important non‐human leucocyte antigen (HLA) antibodies. Elevated levels of anti‐angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure.

Peripheral natural killer cell and allo-stimulated T-cell function in kidney transplant recipients associate with cancer risk and immunosuppression-related complications

Reducing immunosuppression has been proposed as a means of preventing cancer in kidney transplant recipients but this can precipitate graft rejection. Here we tested whether anti-tumor natural killer (NK) cell and allo-responsive T-cell function in kidney transplant recipients may predict cancer risk and define risk of rejection. NK cell function was measured by the release of lactate dehydrogenase and T-cell allo-response by interferon-γ quantification using a panel of reactive T-cell enzyme-linked immunospot (ELISPOT) in 56 kidney transplant recipients with current or past cancer and 26 kidney transplant recipients without cancer. NK function was significantly impaired and the allo-response was significantly lower in kidney transplant recipients with cancer. With prospective follow-up, kidney transplant recipients with poor NK cell function had a hazard ratio of 2.1 (95% confidence interval 0.97-5.00) for the combined end point of metastatic cancer, cancer-related death, or septic death. Kidney transplant recipients with low interferon-γ release were also more likely to reach this combined end point. Thus, posttransplant monitoring of allo-immunity and NK cell function is useful for assessing the risk of over immunosuppression for the development of malignancy and/or death from cancer or sepsis.

Angiotensin II type-1 receptor antibody (AT1Rab) associated humoral rejection and the effect of peri operative plasma exchange and candesartan

Angiotensin II type 1 antibodies (AT1Rab) can mediate antibody mediated rejection (AMR). Pre transplant AT1Rab levels, and risk of rejection were assessed in Kidney Transplant Recipients (KTR) transplanted in our centre from 2013 to 2014 (n=145). 14/145 (9.7%) KTR experienced antibody mediated rejection (AMR). The Hazard Ratio for AMR=3.7 [95% CI 2-26] (p=0.009) for KTR with AT1Rab levels >17.5U/ml. 6/11 of KTR with levels >25U/ml experienced AMR. In 2015 (n=80) KTR were transplanted and 6/80 KTR experienced rejection (2 AMR and 4 TCMR with vascular lesions). 7/80 of KTR had AT1Rab 17.5-25U/ml and none experienced rejection and were induced with ATG and candesartan. 7/80 had AT1Rab 25-40U/ml and received pre and post-operative plasma exchange, ATG and candesartan and 1/7 experienced TCMR with a vascular lesion. This perioperative regimen may alter the risk of rejection in patients with high levels of AT1Ab and further studies are needed.

Immune profiling and cancer post transplantation

Half of all long-term (> 10 year) australian kidney transplant recipients (KTR) will develop squamous cell carcinoma (SCC) or solid organ cancer (SOC), making cancer the leading cause of death with a functioning graft. At least 30% of KTR with a history of SCC or SOC will develop a subsequent SCC or SOC lesion. Pharmacological immunosuppression is a major contributor of the increased risk of cancer for KTR, with the cancer lesions themselves further adding to systemic immunosuppression and could explain, in part, these phenomena. Immune profiling includes; measuring immunosuppressive drug levels and pharmacokinetics, enumerating leucocytes and leucocyte subsets as well as testing leucocyte function in either an antigen specific or non-specific manner. Outputs can vary from assay to assay according to methods used. In this review we define the rationale behind post-transplant immune monitoring assays and focus on assays that associate and/or have the ability to predict cancer and rejection in the KTR. We find that immune monitoring can identify those KTR of developing multiple SCC lesions and provide evidence they may benefit from pharmacological immunosuppressive drug dose reductions. In these KTR risk of rejection needs to be assessed to determine if reduction of immunosuppression will not harm the graft.

Reductions in immunosuppression after haematological or solid organ cancer diagnosis in kidney transplant recipients

Few data exist on how immunosuppression is altered in kidney transplant recipients (KTR) following a diagnosis of cancer. This study investigated how immunosuppression was altered in KTR after cancer diagnosis and its effect on patient and graft survival. All KTR diagnosed with cancer at our centre from 1990 to 2012 were assessed. Drug regime and serum creatinine levels were recorded 1 year before, at time of, and 1 year after cancer diagnosis. Of 87 KTR who developed cancer (7.3% of transplanted population, n = 1189), 30 developed haematological malignancies and 57 developed solid organ cancers (SOC). In total, 38% of KTR presented with nodal or metastatic disease and 23 of 87 (26%) KTR died within 6 months of cancer diagnosis. Fifty‐five KTR had records of pre‐ and postcancer diagnosis drug regimes. Thirty‐six KTR had a (>50%) dose reduction or cessation of 1 or more immunosuppressive agents, and 19 no reduction in immunosuppression. In total, 2 of 36 (6%) of KTR who underwent a dose reduction suffered acute rejection that was reversed with methylprednisolone. Dose reduction/cessation of immunosuppression did not impair graft function, but also did not affect cancer free survival. Further larger prospective studies are needed to determine whether dose reduction alters relapse free cancer survival in KTR.

Characterization of 3D-Printed Human Regulatory T-Cells

Introduction 3D bioprinting is an innovative technology that allows for the rapid and precise fabrication of complex 3D architectures. Co-printing of regulatory T-cells (Tregs) and islets in a 3D-printed lattice may overcome current immunosuppressive obstacles in islet transplantation. This project aims to evaluate the effect of bioprinting on Treg viability and functionality and to evaluate what effect modifying the hydrogel ‘ink’ with IL-2 may have on these parameters. Methods CD4+ CD25hi CD127low natural Tregs (nTregs) and CD4+ CD45RA+ (naïve) T cells were isolated from human blood by FACS. Naïve CD4+ T-cells were induced to a Treg phenotype (iTreg) by culture with TGF-&bgr;, all-trans retinoic acid (ATRA) and rapamycin. nTregs and iTregs were expanded with IL-2 and anti-CD3/28-conjugated beads over 14 days. These cells were either suspended in media (‘non-printed’), or printed to a 6mm diameter disc structure (at 2x106 cells/mL) within an alginate-gelMA hydrogel, photo-crosslinked (at 400nm) and chemically crosslinked with CaCl2 (2% w/v). These ‘printed’ cells were recovered by dissolving the hydrogel with TrypLE Express. Viability and Treg functional markers were quantified by flow-cytometry using propidium iodide and anti-LAP, CD69, CD39 and CTLA-4 antibodies. Results and Discussion The viability of nTregs decreased from 92% (±1.6%) to 85% (±2.3%) with IL-2, and from 89% (±1.9%) to 80% (±4.6%) without IL-2, upon printing (p<0.0001 for both). iTreg viability decreased from 91% (±2.2%) to 87% (±3.4%) with IL-2, and from 91% (±2.4%) to 85% (±1.5%) without IL-2, upon printing (p=0.0042 and <0.0001, respectively). This demonstrated that the effect of bioprinting, on both nTreg and iTreg viability, is statistically significant but minimal. Hydrogel modification with IL-2 had no significant effect on viability at day 1. However, at day 3, IL-2 significantly increased printed nTreg viability by 15% (72±4.8%) compared without IL-2 (57±8.9%, p=0.0003). While this demonstrated the merit of hydrogel modification with IL-2, it did not mimic the 48% improvement shown under non-printed conditions (91±2.4% with IL-2 and 53±9.3% without IL-2, p<0.0001). This may be due to IL-2 leaching into the media, decreasing the concentration in the hydrogel. IL-2 retention within the hydrogel could be improved using a microsphere sustained-release system. Moreover, no decrease in LAP, CD69, CD39 or CTLA-4 expression was observed upon printing, suggesting functional markers are not diminished by the printing process. Conclusion Firstly, our data suggests nTregs and iTregs can be safely bioprinted with minimal effect on viability or functional marker expression. Secondly, we demonstrate that hydrogel modification with IL-2 has a positive impact on the survival of bio-printed nTregs. Finally, this study serves as proof of principle for the capacity of immune cells to survive within hydrogel structures. Figure. No caption available.