Ross S. Francis

Exogenous IFN-γ ex vivo shapes the alloreactive T-cell repertoire by inhibition of Th17 responses and generation of functional Foxp3+ regulatory T cells†

Interferon (IFN)‐γ was originally characterized as a pro‐inflammatory cytokine with T helper type 1‐inducing activity, but subsequent work has demonstrated that mice deficient in IFN‐γ or IFN‐γ receptor show exacerbated inflammatory responses and accelerated allograft rejection, suggesting that IFN‐γ also has important immunoregulatory functions. Here, we demonstrate that ex vivo IFN‐γ conditioning of CD4 T cells driven by allogeneic immature dendritic cells (DC) results in the emergence of a Foxp3+ regulatory T‐cell (Treg)‐ dominant population that can prevent allograft rejection. The development of this population involves conversion of non‐Treg precursors, preferential induction of activation‐induced cell death within the non‐Treg population and suppression of Th2 and Th17 responses. The suppressive activity of IFN‐γ is dependent on the transcription factor signal transducer and activator of transcription 1 and is mediated by induced nitric oxide. These data indicate not only how IFN‐γ could be used to shape beneficial immune responses ex vivo for possible cell therapy but also provide some mechanistic insights that may be relevant to exacerbated inflammatory responses noted in several autoimmune and transplant models with IFN‐γ deficiency.

Induction of transplantation tolerance converts potential effector T cells into graft‐protective regulatory T cells

Naturally occurring FOXP3+CD4+ Treg have a crucial role in self‐tolerance. The ability to generate similar populations against alloantigens offers the possibility of preventing transplant rejection without indefinite global immunosuppression. Exposure of mice to donor alloantigens combined with anti‐CD4 antibody induces operational tolerance to cardiac allografts, and generates Treg that prevent skin and islet allograft rejection in adoptive transfer models. If protocols that generate Treg in vivo are to be developed in the clinical setting it will be important to know the origin of the Treg population and the mechanisms responsible for their generation. In this study, we demonstrate that graft‐protective Treg arise in vivo both from naturally occurring FOXP3+CD4+ Treg and from non‐regulatory FOXP3−CD4+ cells. Importantly, tolerance induction also inhibits CD4+ effector cell priming and T cells from tolerant mice have impaired effector function in vitro. Thus, adaptive tolerance induction shapes the immune response to alloantigen by converting potential effector cells into graft‐protective Treg and by expanding alloreactive naturally occurring Treg. In relation to clinical tolerance induction, the data indicate that while the generation of alloreactive Treg may be critical for long‐term allograft survival without chronic immunosuppression, successful protocols will also require strategies that target potential effector cells.

Functional Regulatory T Cells Produced by Inhibiting Cyclic Nucleotide Phosphodiesterase Type 3 Prevent Allograft Rejection

A clinically approved agent can generate ex vivo graft-reactive, functional mouse and human regulatory T cells. Regulatory T cells Go cAMPing As summer approaches, campers yearn to leave their homes and sleep out under the stars. However, camping isn’t as blissful at it seems from the comfort of a couch in an air-conditioned home: Bugs, heat, and lack of facilities all take their toll on the sensitive adventurer. Transplanted organs face similar challenges in a new host. Not only must they acclimatize to a new environment, but they have to avoid attack from the recipient’s immune system. One way to dodge damage is with the help of immunosuppressive regulatory T cells (Tregs). Feng et al. now find that stimulating these Tregs in the presence of PDE (phosphodiesterase 3) inhibition enriches functional allospecific Tregs, which then can prevent transplant rejection. Adenosine 3′,5′-monophosphate (cAMP) not only directly inhibits effector T cells, it also promotes production of Tregs. Feng et al. hypothesize that inhibiting PDEs, which break down cAMP, would increase cAMP concentrations in Tregs in culture and promote expansion of these cells in the presence of allogeneic dendritic cells. In the mouse PDE3 inhibition with cilostamide in conjunction with allostimulation enriched for functional alloreactive Tregs; these cells inhibited effector T cell function in vitro and prevented graft rejection. PDE3 inhibition also generates human Tregs and these inhibit T cell proliferation and prevent the rejection of human vessel transplants in a humanized mouse transplant model. The ability to selectively enhance allospecific Treg proliferation using cilostamide, a clinically approved agent, should decrease the need for general immunosuppression in transplant recipients. Allospecific Tregs expanded with cilostamide may provide a transplanted organ with an environment that is less like the great outdoors and instead has most of the comforts of home. Regulatory T cells (Tregs) manipulated ex vivo have potential as cellular therapeutics in autoimmunity and transplantation. Although it is possible to expand naturally occurring Tregs, an attractive alternative possibility, particularly suited to solid organ and bone marrow transplantation, is the stimulation of total T cell populations with defined allogeneic antigen-presenting cells (APCs) under conditions that lead to the generation or expansion of donor-reactive, adaptive Tregs. Here we demonstrate that stimulation of mouse CD4+ T cells by immature allogeneic dendritic cells combined with pharmacological inhibition of phosphodiesterase 3 (PDE) resulted in a functional enrichment of Foxp3+ T cells. Without further manipulation or selection, the resultant population delayed skin allograft rejection mediated by polyclonal CD4+ effectors or donor-reactive CD8+ T cell receptor transgenic T cells and inhibited both effector cell proliferation and T cell priming for interferon-γ production. Notably, PDE inhibition also enhanced the enrichment of human Foxp3+ CD4+ T cells driven by allogeneic APCs. These cells inhibited T cell proliferation in a standard in vitro mixed lymphocyte assay and, moreover, attenuated the development of vasculopathy mediated by autologous peripheral blood mononuclear cells in a functionally relevant humanized mouse transplant model. These data establish a method for the ex vivo generation of graft-reactive, functional mouse and human Tregs that uses a clinically approved agent, making pharmacological PDE inhibition a potential strategy for Treg-based therapies.

CD4+ Regulatory T Cells Generated in Vitro with IFN-γ and Allogeneic APC Inhibit Transplant Arteriosclerosis

We have developed a method to generate alloreactive regulatory T cells in vitro in the presence of interferon (IFN)-gamma and donor antigen presenting cells (APCs). We hypothesized that these IFN-gamma-conditioned T cells (Tcon) would reduce transplantation-associated arteriosclerosis. Tcon were generated from mouse (CBA.Ca, H-2(k)) CD4(+) T cells cultured in the presence of IFN-gamma for 14 days. These cultures were pulsed with bone marrow-derived B6 (H-2(b)) APC. 1 x 10(5) CD25(-)CD4(+) effector T cells from naive H-2(k) mice were then cotransferred with 4 x 10(5) Tcon into CBA-rag(-/-) mice. One day later, these mice received a fully allogenic B6 CD31(-/-) abdominal aorta transplant. Transfer of CD25(-)CD4(+) effectors resulted in 29.7 +/- 14.5% luminal occlusion of allogeneic aortic grafts after 30 days. Cotransfer of Tcon reduced this occlusion to 11.7 +/- 13.1%; P < 0.05. In addition, the CD31(-) donor endothelium was fully repopulated by CD31(+) recipient endothelial cells in the absence of Tcon, but not in the presence of Tcon. In some experiments, we cotransplanted B6 skin with aortic grafts to ensure enhanced reactivation of the regulatory cells, which led to an additional reduction in vasculopathy (1.9 +/- 3.0% luminal occlusion). In the presence of Tcon, CD4(+) T cell infiltration into grafts was markedly reduced by a regulatory mechanism that included reduced priming and proliferation of CD25(-)CD4(+) effectors. These data illustrate the potential of ex vivo generated regulatory T cells for the inhibition of transplant-associated vasculopathy.

Indoxyl sulphate and kidney disease: causes, consequences and interventions

In the last decade, chronic kidney disease (CKD), defined as reduced renal function (glomerular filtration rate (GFR) < 60 mL/min per 1.73 m2) and/or evidence of kidney damage (typically manifested as albuminuria) for at least 3 months, has become one of the fastest‐growing public health concerns worldwide. CKD is characterized by reduced clearance and increased serum accumulation of metabolic waste products (uremic retention solutes). At least 152 uremic retention solutes have been reported. This review focuses on indoxyl sulphate (IS), a protein‐bound, tryptophan‐derived metabolite that is generated by intestinal micro‐organisms (microbiota). Animal studies have demonstrated an association between IS accumulation and increased fibrosis, and oxidative stress. This has been mirrored by in vitro studies, many of which report cytotoxic effects in kidney proximal tubular cells following IS exposure. Clinical studies have associated IS accumulation with deleterious effects, such as kidney functional decline and adverse cardiovascular events, although causality has not been conclusively established. The aims of this review are to: (i) establish factors associated with increased serum accumulation of IS; (ii) report effects of IS accumulation in clinical studies; (iii) critique the reported effects of IS in the kidney, when administered both in vivo and in vitro; and (iv) summarize both established and hypothetical therapeutic options for reducing serum IS or antagonizing its reported downstream effects in the kidney.

BK Polyomavirus: Clinical Aspects, Immune Regulation, and Emerging Therapies

SUMMARY BK polyomavirus (BKV) causes frequent infections during childhood and establishes persistent infections within renal tubular cells and the uroepithelium, with minimal clinical implications. However, reactivation of BKV in immunocompromised individuals following renal or hematopoietic stem cell transplantation may cause serious complications, including BKV-associated nephropathy (BKVAN), ureteric stenosis, or hemorrhagic cystitis. Implementation of more potent immunosuppression and increased posttransplant surveillance has resulted in a higher incidence of BKVAN. Antiviral immunity plays a crucial role in controlling BKV replication, and our increasing knowledge about host-virus interactions has led to the development of improved diagnostic tools and clinical management strategies. Currently, there are no effective antiviral agents for BKV infection, and the mainstay of managing reactivation is reduction of immunosuppression. Development of immune-based therapies to combat BKV may provide new and exciting opportunities for the successful treatment of BKV-associated complications.

Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients

Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as “off-the-shelf” therapeutics as well as autologous T-cell therapies for transplant patients.

Cryptococcal infections in solid organ transplant recipients over a 15-year period at a state transplant center

The aim of this research paper was to determine the incidence, risk factors, and clinical outcome of solid organ transplant (SOT) recipients diagnosed and treated for cryptococcosis at our institution.

Transient massive proteinuria after gelatin-derived plasma expander (Gelofusine®) administration.

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Optimising assessment of kidney function when managing localised renal masses

Increased early and incidental detection, improved surgical techniques and technological advancement mean that the management of renal mass lesions is constantly evolving. The treatment of choice for renal mass lesions has historically been radical nephrectomy. Partial nephrectomy is now recommended for localised renal masses, owing to favourable renal functional outcomes. Ablative renal surgery confers a significant risk of chronic kidney disease. There are few studies assessing long term outcomes of nephrectomy on renal outcomes, and virtually no studies assessing long term outcomes for less invasive therapies such as ablation. Unless a renal mass is clearly benign on imaging, management decisions will be made with an assumption of malignancy. The content of this review applies to both benign and malignant renal mass lesions. We advocate for improved strategies for kidney function assessment and risk stratification, early targeted referral, and regular screening for chronic kidney disease for all patients after surgery.