E. Nova-Lamperti

Regulatory T-cell therapy in the induction of transplant tolerance: the issue of subpopulations.

Clinical tolerance induction to permit minimization or cessation of immunosuppressive drugs is one of the key research goals in solid organ transplantation. The use of ex vivo expanded or manipulated immunologic cells, including CD4CD25FOXP3 regulatory T cells (Tregs), to achieve this aim is already a reality, with several trials currently recruiting patients. Tregs are a highly suppressive, nonredundant, population of regulatory cells that prevent the development of autoimmune diseases in mammals. Data from transplanted humans and animal models support the notion that Tregs can mediate both induction and adoptive transfer of transplantation tolerance. However, human Tregs are highly heterogeneous and include subpopulations with the potential to produce the proinflammatory cytokine interleukin-17, which has been linked to transplant rejection. Tregs are also small in number in the peripheral circulation, thus they require ex vivo expansion before infusion into man. Selection of the most appropriate Treg population for cell therapy is, therefore, a critical step in ensuring successful clinical outcomes. In this review, we discuss Treg subpopulations, their subdivision based on nonmutually exclusive criteria of origin, expression of immunologic markers and function, availability in the peripheral blood of patients awaiting transplantation, and their suitability for programs of cell-based therapy.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Angiogenesis: Potencial Clinical Application

Mesenchymal stem cells (MSCs) are adult multipotent stem cells that are able to differentiate into multiple specialized cell types including osteocytes, adipocytes, and chondrocytes. MSCs exert different functions in the body and have recently been predicted to have a major clinical/therapeutic potential. However, the mechanisms of self-renewal and tissue regeneration are not completely understood. It has been shown that the biological effect depends mainly on its paracrine action. Furthermore, it has been reported that the secretion of soluble factors and the release of extracellular vesicles, such as exosomes, could mediate the cellular communication to induce cell-differentiation/self-renewal. This review provides an overview of MSC-derived exosomes in promoting angiogenicity and of the clinical relevance in a therapeutic approach.

Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment?

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell–related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group—namely, the tolerant recipients—were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS‐independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross‐validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

IL-10-produced by human transitional B-cells down-regulates CD86 expression on B-cells leading to inhibition of CD4+T-cell responses

A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function, the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore, under this stimulatory condition, CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10, which in turn leads to decreased T-cell proliferation and TNF-α production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation, which correlates with lower CD86 expression compared to patients with chronic rejection. Hence, the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production.

An Atlas of Human Regulatory T Helper-like Cells Reveals Features of Th2-like Tregs that Support a Tumorigenic Environment

Summary Regulatory T cells (Tregs) play a pivotal role in maintaining immunological tolerance, but they can also play a detrimental role by preventing antitumor responses. Here, we characterized T helper (Th)-like Treg subsets to further delineate their biological function and tissue distribution, focusing on their possible contribution to disease states. RNA sequencing and functional assays revealed that Th2-like Tregs displayed higher viability and autocrine interleukin-2 (IL-2)-mediated activation than other subsets. Th2-like Tregs were preferentially found in tissues rather than circulation and exhibited the highest migratory capacity toward chemokines enriched at tumor sites. These cellular responses led us to hypothesize that this subset could play a role in maintaining a tumorigenic environment. Concurrently, Th2-like Tregs were enriched specifically in malignant tissues from patients with melanoma and colorectal cancer compared to healthy tissue. Overall, our results suggest that Th2-like Tregs may contribute to a tumorigenic environment due to their increased cell survival, higher migratory capacity, and selective T-effector suppressive ability.

Increased CD40 ligation and reduced BCR signalling leads to higher IL-10 production in B-cells from tolerant kidney transplant patients

Background An increased percentage of peripheral transitional B cells producing IL-10 has been observed in patients tolerant to kidney allografts. In healthy volunteers, the balance between the CD40 and B-cell receptor (BCR) signalling modulated IL-10 production by B cells, with stimulation via the BCR decreasing CD40-mediated IL-10 production. In this study, we evaluate whether in tolerant kidney transplant patients, the increased IL-10 production by B cells was due to an altered CD40 and/or BCR signalling. Methods B cells obtained from a new cohort of tolerant renal transplant recipients and those from age- and sex-matched healthy volunteers were activated via CD40 and BCR, either alone or in combination. Results In tolerant patients, we observed higher percentages of B cells producing IL-10 after CD40 ligation and higher expression of CD40L on activated T cells compared with healthy controls. Furthermore, B cells from tolerant recipients had reduced extracellular signal-regulated kinase signalling after BCR-mediated activation compared with healthy controls. In keeping with this, combining BCR signalling with CD40 ligation did not reduce IL-10 secretion as was observed in healthy control transitional B cells. Conclusions Altogether, our data suggest that the altered response of B cells in tolerant recipients may contribute to long-term stable graft acceptance.

Reduced Tcr Signaling Contributes to Impaired Th17 Responses in Tolerant Kidney Transplant Recipients

Background The development of spontaneous kidney transplant tolerance has been associated with numerous B cell–related immune alterations. We have previously shown that tolerant recipients exhibit reduced B-cell receptor signalling and higher IL-10 production than healthy volunteers. However, it is unclear whether cluster of differentiation (CD)4+ T cells from tolerant recipients also display an anti-inflammatory profile that could contribute to graft maintenance. Methods CD4+ T cells were isolated from kidney transplant recipients who were identified as being tolerant recipients, patients with chronic rejection or healthy volunteers. CD4+ T cells from the 3 groups were compared in terms of their gene expression profile, phenotype, and functionally upon activation. Results Gene expression analysis of transcription factors and signalling proteins, in addition to surface proteins expression and cytokine production, revealed that tolerant recipients possessed fewer Th17 cells and exhibited reduced Th17 responses, relative to patients with chronic rejection or healthy volunteers. Furthermore, impaired T-cell receptor signalling and altered cytokine cooperation by monocytes contributed to the development of Th17 cells in tolerant recipients. Conclusions These data suggest that defective proinflammatory Th17 responses may contribute to the prolonged graft survival and stable graft function, which is observed in tolerant recipients in the absence of immunosuppressive agents.

Human retinoic acid–regulated CD161+ regulatory T cells support wound repair in intestinal mucosa

Repair of tissue damaged during inflammatory processes is key to the return of local homeostasis and restoration of epithelial integrity. Here we describe CD161+ regulatory T (Treg) cells as a distinct, highly suppressive population of Treg cells that mediate wound healing. These Treg cells were enriched in intestinal lamina propria, particularly in Crohn’s disease. CD161+ Treg cells had an all-trans retinoic acid (ATRA)-regulated gene signature, and CD161 expression on Treg cells was induced by ATRA, which directly regulated the CD161 gene. CD161 was co-stimulatory, and ligation with the T cell antigen receptor induced cytokines that accelerated the wound healing of intestinal epithelial cells. We identified a transcription-factor network, including BACH2, RORγt, FOSL2, AP-1 and RUNX1, that controlled expression of the wound-healing program, and found a CD161+ Treg cell signature in Crohn’s disease mucosa associated with reduced inflammation. These findings identify CD161+ Treg cells as a population involved in controlling the balance between inflammation and epithelial barrier healing in the gut.Treg cells are essential for enforcing peripheral tolerance but can also influence tissue regeneration. Afzali and colleagues use high-dimensional analysis to describe a distinct population of CD161+ human Treg cells involved in wound healing of the intestinal mucosa.

Expanded regulatory T cells induce alternatively activated monocytes with a reduced capacity to expand T helper-17 cells

Regulatory T cells (Tregs) are essential in maintaining peripheral immunological tolerance by modulating several subsets of the immune system including monocytes. Under inflammatory conditions, monocytes migrate into the tissues, where they differentiate into dendritic cells or tissue-resident macrophages. As a result of their context-dependent plasticity, monocytes have been implicated in the development/progression of graft-vs-host disease (GvHD), autoimmune diseases and allograft rejection. In the last decade, Tregs have been exploited for their use in cell therapy with the aim to induce tolerance after solid organ transplantation and for the treatment of autoimmune diseases and GvHD. To date, safety and feasibility of Treg infusion has been demonstrated; however, many questions of how these cells induce tolerance have been raised and need to be answered. As monocytes constitute the major cellular component in inflamed tissues, we have developed an in vitro model to test how Tregs modulate their phenotype and function. We demonstrated that expanded Tregs can drive monocytes toward an alternatively activated state more efficiently than freshly isolated Tregs. The effect of expanded Tregs on monocytes led to a reduced production of pro-inflammatory cytokines (IL-6 and tumor necrosis factor-α) and NF-κB activation. Furthermore, monocytes co-cultured with expanded Tregs downregulated the expression of co-stimulatory and MHC-class II molecules with a concomitant upregulation of M2 macrophage specific markers, CD206, heme oxygenase-1, and increased interleukin-10 production. Importantly, monocytes co-cultured with expanded Tregs showed a reduced capacity to expand IL-17-producing T cells compared with monocyte cultured with freshly isolated Tregs and conventional T cells. The capacity to decrease the expansion of pro-inflammatory Th-17 was not cytokine mediated but the consequence of their lower expression of the co-stimulatory molecule CD86. Our data suggest that expanded Tregs have the capacity to induce phenotypical and functional changes in monocytes that might be crucial for tolerance induction in transplantation and the prevention/treatment of GvHD and autoimmune diseases.

Vascular Regeneration by Endothelial Progenitor Cells in Health and Diseases

Human endothelial progenitor cells (hEPCs) are adult stem cells, located in the bone marrow and peripheral blood. These cells can be differentiated into mature endothelial cells, which are involved in processes of angiogenesis and vessel regeneration. Different phenotypes and subtypes of endothelial progenitor cells (EPCs), such as early and late EPCs, have been described according to their functionality. Thus, it has been shown that early EPCs release cytokines that promote tissue regeneration and neovasculogenesis, whereas late EPC and endothelial colony forming cells (ECFCs) contribute to the formation of blood vessels and stimulate tube formation. It has been demonstrated that the number of circulating hEPC is decreased in individuals with hypercholesterolemia, hypertension, and/or diabetes. In addition, the number and the migratory activity of these cells are inversely correlated with risk factors such as hypertension, hypercholes‐ terolemia, diabetes, and metabolic syndrome. On the other hand, the number of circulating hEPC is increased in hypoxia or acute myocardial infarction (AMI). hEPCs have been used for cell‐based therapies due to their capacity to contribute in the re‐ endothelialization of injured blood vessels and neovascularization in ischemic tissues. This chapter provides an overview of the key role of hEPC in promoting angiogenesis and their potential use for cell therapy.