Identifying the 'Achilles heel' of type 1 diabetes.

When Thetis dipped her son Achilles into the River Styx to make him immortal, she held him by the heel, which was not submerged, and thus created a weak spot that proved deadly for Achilles. Millennia later, Achilles heel is part of today's lexicon meaning an area of weakness or a vulnerable spot that causes failure. Also implied is that an Achilles heel is often missed, forgotten or under-appreciated until it is under attack, and then failure is fatal. Paris killed Achilles with an arrow 'guided by the Gods'. Understanding the pathogenesis of type 1 diabetes (T1D) in order to direct therapy for prevention and treatment is a major goal of research into T1D. At the International Congress of the Immunology of Diabetes Society, 2018, five leading experts were asked to present the case for a particular cell/element that could represent 'the Achilles heel of T1D'. These included neutrophils, B cells, CD8+ T cells, regulatory CD4+ T cells, and enteroviruses, all of which have been proposed to play an important role in the pathogenesis of type 1 diabetes. Did a single entity emerge as 'the' Achilles heel of T1D? The arguments are summarized here, to make this case.

Methods to manufacture regulatory T cells for cell therapy.

Regulatory T cell (Treg ) therapy has shown promise in early clinical trials for treating graft-versus-host disease, transplant rejection and autoimmune disorders. A challenge has been to isolate sufficiently pure Tregs and expand them to a clinical dose. However, there has been considerable progress in the development and optimization of these methods, resulting in a variety of manufacturing protocols being tested in clinical trials. In this review, we summarize methods that have been used to manufacture Tregs for clinical trials, including the choice of cell source and protocols for cell isolation and expansion. We also discuss alternative culture or genome editing methods for modulating Treg specificity, function or stability that could be applied to future clinical manufacturing protocols to increase the efficacy of Treg therapy.

Tailoring the homing capacity of human Tregs for directed migration to sites of Th1-inflammation or intestinal regions.

Cell-based therapy with CD4+ FOXP3+ regulatory T cells (Tregs) is a promising strategy to limit organ rejection and graft-vs-host disease. Ongoing clinical applications have yet to consider how human Tregs could be modified to direct their migration to specific inflammation sites and/or tissues for more targeted immunosuppression. We show here that stable, homing-receptor-tailored human Tregs can be generated from thymic Tregs isolated from pediatric thymus or adult blood. To direct migration to Th1-inflammatory sites, addition of interferon-γ and IL-12 during Treg expansion produced suppressive, epigenetically stable CXCR3+ TBET+ FOXP3+ T helper (Th)1-Tregs. CXCR3 remained expressed after injection in vivo and Th1-Tregs migrated efficiently towards CXCL10 in vitro. To induce tissue-specific migration, addition of retinoic acid (RA) during Treg expansion induced expression of the gut-homing receptors α4ß7-integrin and CCR9. FOXP3+ RA-Tregs had elevated expression of the functional markers latency-associated peptide and glycoprotein A repetitions predominant, increased suppressive capacity in vitro and migrated efficiently to healthy and inflamed intestine after injection into mice. Homing-receptor-tailored Tregs were epigenetically stable even after long-term exposure to inflammatory conditions, suppressive in vivo and characterized by Th1- or gut-homing-specific transcriptomes. Tailoring human thymic Treg homing during in vitro expansion offers a new and clinically applicable approach to improving the potency and specificity of Treg therapy.

How antigen specificity directs regulatory T-cell function: self, foreign and engineered specificity.

Regulatory T cells (Tregs) are a suppressive subset of T cells that have important roles in maintaining self-tolerance and preventing immunopathology. The T-cell receptor (TCR) and its antigen specificity play a dominant role in the differentiation of cells to a Treg fate, either in the thymus or in the periphery. This review focuses on the effects of the TCR and its antigen specificity on Treg biology. The role of Tregs with specificity for self-antigen has primarily been studied in the context of autoimmune disease, although recent studies have focused on their role in steady-state conditions. The role of Tregs that are specific for pathogens, dietary antigens and allergens is much less studied, although recent data suggest a significant and previously underappreciated role for Tregs during memory responses to a wide range of foreign antigens. The development of TCR- or chimeric antigen receptor (CAR)-transduced T cells means we are now able to engineer Tregs with disease-relevant antigen specificities, paving the way for ensuring specificity with Treg-based therapies. Understanding the role that antigens play in driving the generation and function of Tregs is critical for defining the pathophysiology of many immune-mediated diseases, and developing new therapeutic interventions.

Obesity-Associated Adipose Tissue Inflammation and Transplantation.

Obesity is often associated with the development of adipose tissue (AT) inflammation, resulting in metabolic dysfunction and an increased risk for developing type 2 diabetes. It is also associated with multiple chronic diseases, including cardiovascular, liver, and kidney disease, and thus can contribute to organ failure. Several studies have investigated whether there is a correlation between obesity and outcomes in transplantation, but there is currently very limited information on the specific role of AT inflammation in the rejection process or on the overall function of the transplanted organ. Here, we provide a brief review of the current understanding of the cellular mechanisms that control obesity-associated AT inflammation and summarize knowledge about how obesity affects clinical outcomes following solid organ or hematopoietic stem cell transplantation. We also highlight opportunities for more research to better understand how obesity affects outcomes of transplantation.

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells.

Regulatory T cell (Treg)-based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3(+) Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood.

Translational mini-review series on Th17 cells: function and regulation of human T helper 17 cells in health and disease.

T helper (Th) cell have a central role in modulating immune responses. While Th1 and Th2 cells have long been known to regulate cellular and humoral immunity, Th17 cells have been identified only recently as a Th lineage that regulates inflammation via production of distinct cytokines such as interleukin (IL)-17. There is growing evidence that Th17 cells are pathological in many human diseases, leading to intense interest in defining their origins, functions and developing strategies to block their pathological effects. The cytokines that regulate Th17 differentiation have been the focus of much debate, due primarily to inconsistent findings from studies in humans. Evidence from human disease suggests that their in vivo development is driven by specialized antigen-presenting cells. Knowledge of how Th17 cells interact with other immune cells is limited, but recent data suggest that Th17 cells may not be subject to strict cellular regulation by T regulatory cells. Notably, Th17 cells and T regulatory cells appear to share common developmental pathways and both cell types retain significant plasticity. Herein, we will discuss the molecular and cellular regulation of Th17 cells with an emphasis on studies in humans.

Phenotypic and functional differences between human CD4+CD25+ and type 1 regulatory T cells.

T regulatory (Tr) cells have an essential role in the induction and maintenance of tolerance to both and foreign self-antigens. Many types of T cells with regulatory activity have been described in mice and humans, and those within the CD4+ subset have been extensively characterized. CD4+ Type-1 regulatory T (Tr1) cells produce high levels of IL-10 and mediate IL-10-dependent suppression, whereas the effects of naturally occurring CD4+CD25+ Tr cells appear to be cell-contact-dependent. Tr1 cells arise in the periphery upon encountering antigen in a tolerogenic environment. In contrast, it appears that CD4+CD25+ Tr cells can either arise directly in the thymus or be induced by antigen in the periphery. We have been interested in defining the phenotype and function of different subsets of CD4+ Tr cells present in human peripheral blood, with the ultimate aim of designing therapeutic strategies to harness their immunoregulatory effects. This review will discuss the similarities and differences between human Tr1 and naturally occurring CD4+CD25+ Tr cells, as well as evidence that indicates that they have nonoverlapping, but synergistic roles in immune homeostasis.

Recombinant human interleukin 10 suppresses gliadin dependent T cell activation in ex vivo cultured coeliac intestinal mucosa.

BACKGROUND: Enteropathy in coeliac disease (CD) is sustained by a gliadin specific Th1 response. Interleukin (IL)-10 can downregulate Th1 immune responses. AIM: We investigated the ability of recombinant human (rh) IL-10 to suppress gliadin induced Th1 response. PATIENTS AND METHODS: IL-10 RNA transcripts were analysed by competitive reverse transcription-polymerase chain reaction in duodenal biopsies from untreated and treated CD patients, non-coeliac enteropathies (NCE), and controls. CD biopsies were cultured with a peptic-tryptic digest of gliadin with or without rhIL-10. The proportion of CD80+ and CD25+ cells in the lamina propria, epithelial expression of Fas, intraepithelial infiltration of CD3+ cells, as well as cytokine synthesis (interferon gamma (IFN-gamma) and IL-2) were measured. Short term T cell lines (TCLs) obtained from treated CD biopsies cultured with gliadin with or without rhIL-10 were analysed by ELISPOT for gliadin specific production of IFN-gamma. RESULTS: In untreated CD and NCE, IL-10 RNA transcripts were significantly upregulated. In ex vivo organ cultures, rhIL-10 downregulated gliadin induced cytokine synthesis, inhibited intraepithelial migration of CD3+ cells, and reduced the proportion of lamina propria CD25+ and CD80+ cells whereas it did not interfere with epithelial Fas expression. In short term TCLs, rhIL-10 abrogated the IFN-gamma response to gliadin. CONCLUSIONS: rhIL-10 suppresses gliadin specific T cell activation. It may interfere with the antigen presenting capacity of lamina propria mononuclear cells as it reduces the expression of CD80. Interestingly, rhIL-10 also induces a long term hyporesponsiveness of gliadin specific mucosal T cells. These results offer new perspectives for therapeutic strategies in coeliac patients based on immune modulation by IL-10.

Type 1 T regulatory cells.

Suppression by T regulatory (Tr) cells is essential for induction of tolerance. Many types of Tr cells have been described in a number of systems, and their biology has been the subject of intensive investigation. Although many aspects of the mechanisms by which these cells exert their effects remain to be elucidated, it is well established that Tr cells suppress immune responses via cell-to-cell interactions and/or the production of interleukin (IL)-10 and transforming growth factor (TGF)-beta. Type-1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL-10 and TGF-beta. Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4+ T cells in the presence of IL-10 in vitro. Tr1 cells have a low proliferative capacity, which can be overcome by IL-15. Tr1 cells suppress naive and memory T helper type 1 or 2 responses via production of IL-10 and TGF-beta. Further characterisation of Tr1 cells at the molecular level will define their mechanisms of action and clarify their relationship with other subsets of Tr cells. The use of Tr1 cells to identify novel targets for the development of new therapeutic agents, and as a cellular therapy to modulate peripheral tolerance, can be foreseen.

Human cd25(+)cd4(+) t regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function.

Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4(+) Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25(+)CD4(+) Tr cells in humans has not been reported. Here we show that human CD25(+)CD4(+) Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor-mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte-associated antigen (CTLA)-4. Human CD25(+)CD4(+) Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-beta, low levels of interferon (IFN)-gamma, and no IL-4 or IL-2. Importantly, CD25(+)CD4(+) Tr cells strongly inhibited the proliferative responses of both naive and memory CD4(+) T cells to alloantigens, but neither IL-10, TGF-beta, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25(+)CD4(+) Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25(+)CD4(+) Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell-mediated diseases.

IFN-alpha and IL-10 induce the differentiation of human type 1 T regulatory cells.

CD4(+) T regulatory type 1 (Tr1) cells suppress Ag-specific immune responses in vitro and in vivo. Although IL-10 is critical for the differentiation of Tr1 cells, the effects of other cytokines on differentiation of naive T cells into Tr1 cells have not been investigated. Here we demonstrate that endogenous or exogenous IL-10 in combination with IFN-alpha, but not TGF-beta, induces naive CD4(+) T cells derived from cord blood to differentiate into Tr1 cells: IL-10(+)IFN-gamma(+)IL-2(-/low)IL-4(-). Naive CD4(+) T cells derived from peripheral blood require both exogenous IL-10 and IFN-alpha for Tr1 cell differentiation. The proliferative responses of the Tr1-containing lymphocyte populations, following activation with anti-CD3 and anti-CD28 mAbs, were reduced. Similarly, cultures containing Tr1 cells displayed reduced responses to alloantigens via a mechanism that was partially mediated by IL-10 and TGF-beta. More importantly, Tr1-containing populations strongly suppressed responses of naive T cells to alloantigens. Collectively, these results show that IFN-alpha strongly enhances IL-10-induced differentiation of functional Tr1 cells, which represents a first major step in establishing specific culture conditions to generate T regulatory cells for biological and biochemical analysis, and for cellular therapy to induce peripheral tolerance in humans.

The role of different subsets of T regulatory cells in controlling autoimmunity.

T regulatory cells-in addition to clonal deletion and anergy-are essential for the downregulation of T cell responses to both foreign and self antigens, and for the prevention of autoimmunity. Recent progress has been made in characterising the different subsets of T regulatory cells, the factors that drive their differentiation, and their mode of action. The resolution of these mechanisms will make it possible to use T regulatory cells therapeutically in human autoimmune diseases.

T-regulatory 1 cells: a novel subset of CD4 T cells with immunoregulatory properties.

Clonal deletion and clonal anergy are well established mechanisms of peripheral tolerance. A role has also been described for clonal suppression by regulatory cells in the induction of peripheral tolerance to a variety of antigens. However, it has been difficult to isolate regulatory cells and to define their mechanism of action. We have recently reported the in vitro generation and characterization of a novel subset of CD4(+) T cells that have regulatory properties and are able to suppress antigen-specific immune responses in vitro and in vivo. These T-regulatory 1 (Tr1) cells are defined by their unique profile of cytokine production and make high levels of IL-10 and TGF-beta, but no IL-4 or IL-2. The IL-10 and TGF-beta produced by these cells mediate the inhibition of primary naive T cells in vitro. There is also evidence that Tr1 cells exist in vivo, and we have documented the presence of high IL-10-producing CD4(+) T cells in patients with severe combined immunodeficiency who have received allogeneic stem-cell transplants. These findings support the notion that Tr1 cells are involved in the regulation of peripheral tolerance and that they could potentially be used as a cellular therapy to modulate immune responses in vivo.

Heterodimerization of the alpha and beta chains of the interleukin-3 (IL-3) receptor is necessary and sufficient for IL-3-induced mitogenesis.

The high-affinity receptor for interleukin-3 (IL-3) is a complex of the IL-3-binding subunit (alpha(IL-3)) and a larger beta chain-beta(c), or, in the mouse, beta(c) or its close relative beta(IL-3). There is evidence that the critical event that initiates signaling is not the approximation of the cytoplasmic domains of alpha(IL-3) and beta(IL-3), but is, rather, the formation of a beta-beta homodimer. Many of these studies involved the analyses of receptor chimeras where the cytoplasmic domains were derived from alpha(IL-3), beta(c) or beta(IL-3), and the extracellular domains were derived from other cytokine receptors, such as the erythropoietin receptor (EpoR). However, evidence that the EpoR may also associate with other receptors clouds the interpretation of these experiments. Therefore, we reevaluated the structure of the functional IL-3R using chimeric receptors with extracellular domains derived not from members of the cytokine-receptor family, but from CD8 or CD16. We show, by expression of these chimeras in Ba/F3 or CTLL-2 cells, that mitogenic signals were only generated by heterodimerization of the cytoplasmic domains of alpha(IL-3) and beta(IL-3). Homodimers of either alpha(IL-3) or beta(IL-3), alone or in combination, were nonfunctional. Furthermore, the ability of heterodimers to stimulate mitogenesis correlated with their ability to induce tyrosine phosphorylation of JAK-2. These data suggest that the physiological activation of the IL-3R involves the generation of simple heterodimers of alpha(IL-3) and beta(IL-3).

Interleukin-4 synergizes with Raf-1 to promote long-term proliferation and activation of c-jun N-terminal kinase.

This report shows that interleukin-4 (IL-4), which plays a key role in regulating immune responses, fails to support cellular growth. We investigated whether this failure of IL-4 to promote growth was because of its unique inability to activate the Ras/Raf/Erk pathway. Consistent with other reports, expression in Ba/F3, a factor-dependent hematopoietic cell line, of either activated Q61KN-Ras or a hormone-inducible activated Raf-1, resulted in suppression of apoptosis but not in long-term growth. However, in the presence of IL-4, Ba/F3 cells that expressed either Q61KN-Ras or activated Raf-1 grew continuously at a rate comparable with that stimulated by IL-3. Investigation of the biochemical events associated with the stimulation of long-term growth showed that, as expected, the presence of activated Raf-1 resulted in an increased activity of extracellular signal regulated kinase (ERK) mitogen-activated protein kinase (MAPK) but not of c-jun N-terminal kinase/stress-activated protein kinase (JNK). However, surprisingly, if IL-4 was present, cells expressing active Raf-1 exhibited increases in JNK activity. These observations point to a novel mechanism for JNK activation involving synergy between Raf-1 and pathways activated by IL-4 and suggest that in hematopoietic cells proliferation is correlated not only with "mitogen activated" ERK activity, but also with JNK activity.

IL-4 inhibits the production of TNF-alpha and IL-12 by STAT6-dependent and -independent mechanisms.

IL-4 promotes allergic responses and inhibits the production of proinflammatory cytokines by monocytes and macrophages. The promotion of allergic responses by IL-4 has been shown to be absolutely dependent on the transcription factor STAT6. We report here that the inhibitory effects of IL-4 on the production of TNF-alpha or IL-12 by macrophages had both STAT6-dependent and -independent components, depending on the stimuli. IL-4 failed to inhibit the release of TNF-alpha or IL-12 from STAT6 null macrophages stimulated with LPS alone. However, IL-4 still induced significant inhibition of the production of TNF-alpha and IL-12 from STAT6 null macrophages that were stimulated with the more physiologically relevant combination of LPS and IFN-gamma. These data show that STAT6 is required for the IL-4-mediated inhibition of the production of TNF-alpha and IL-12 stimulated by LPS alone, but that IL-4 also activates distinct, STAT6 independent mechanism(s) that inhibit the IFN-gamma-mediated enhancement of IL-12 and TNF-alpha production.