Finding a Way Out: S1P Signaling and Immune Cell Migration.

The signaling lipid sphingosine 1-phosphate (S1P) plays critical roles in an immune response. Drugs targeting S1P signaling have been remarkably successful in treatment of multiple sclerosis, and they have shown promise in clinical trials for colitis and psoriasis. One mechanism of these drugs is to block lymphocyte exit from lymph nodes, where lymphocytes are initially activated, into circulation, from which lymphocytes can reach sites of inflammation. Indeed, S1P can be considered a circulation marker, signaling to immune cells to help them find blood and lymphatic vessels, and to endothelial cells to stabilize the vasculature. That said, S1P plays pleiotropic roles in the immune response, and it will be important to build an integrated view of how S1P shapes inflammation. S1P can function so effectively because its distribution is exquisitely tightly controlled. Here we review how S1P gradients regulate immune cell exit from tissues, with particular attention to key outstanding questions in the field.

Gradients of the signaling lipid S1P in lymph nodes position natural killer cells and regulate their interferon-γ response.

The lymph node periphery is an important site for many immunological functions, from pathogen containment to the differentiation of helper T cells, yet the cues that position cells in this region are largely undefined. Here, through the use of a reporter for the signaling lipid S1P (sphingosine 1-phosphate), we found that cells sensed higher concentrations of S1P in the medullary cords than in the T cell zone and that the S1P transporter SPNS2 on lymphatic endothelial cells generated this gradient. Natural killer (NK) cells are located at the periphery of the lymph node, predominantly in the medulla, and we found that expression of SPNS2, expression of the S1P receptor S1PR5 on NK cells, and expression of the chemokine receptor CXCR4 were all required for NK cell localization during homeostasis and rapid production of interferon-γ by NK cells after challenge. Our findings elucidate the spatial cues for NK cell organization and reveal a previously unknown role for S1P in positioning cells within the medulla.

Have Cytokines, Will Travel.

In many contexts, innate lymphoid cells (ILCs) are primarily tissue resident. By contrast, in a recent issue of Science, Huang et al. (2018) show that inflammatory type 2 ILCs migrate from the intestine to the lungs and that this movement is guided by sphingosine-1-phosphate receptors.

Monocyte-derived S1P in the lymph node regulates immune responses.

The lipid chemoattractant sphingosine 1-phosphate (S1P) guides cells out of tissues, where the concentration of S1P is relatively low, into circulatory fluids, where the concentration of S1P is high1. For example, S1P directs the exit of T cells from lymph nodes, where T cells are initially activated, into lymph, from which T cells reach the blood and ultimately inflamed tissues1. T cells follow S1P gradients primarily using S1P receptor 1 (ref. 1). Recent studies have described how S1P gradients are established at steady state, but little is known about the distribution of S1P in disease or about how changing levels of S1P may affect immune responses. Here we show that the concentration of S1P increases in lymph nodes during an immune response. We found that haematopoietic cells, including inflammatory monocytes, were an important source of this S1P, which was an unexpected finding as endothelial cells provide S1P to lymph1. Inflammatory monocytes required the early activation marker CD69 to supply this S1P, in part because the expression of CD69 was associated with reduced levels of S1pr5 (which encodes S1P receptor 5). CD69 acted as a 'stand-your-ground' signal, keeping immune cells at a site of inflammation by regulating both the receptors and the gradients of S1P. Finally, increased levels of S1P prolonged the residence time of T cells in the lymph nodes and exacerbated the severity of experimental autoimmune encephalomyelitis in mice. This finding suggests that residence time in the lymph nodes might regulate the differentiation of T cells, and points to new uses of drugs that target S1P signalling.

Exit Strategies: S1P Signaling and T Cell Migration.

Whereas the role of sphingosine 1-phosphate receptor 1 (S1PR1) in T cell egress and the regulation of S1P gradients between lymphoid organs and circulatory fluids in homeostasis are increasingly well understood, much remains to be learned about S1P signaling and distribution during an immune response. Recent data suggest that the role of S1PR1 in directing cells from tissues into circulatory fluids is reprised again and again, particularly in guiding activated T cells from non-lymphoid tissues into lymphatics. Conversely, S1P receptor 2 (S1PR2), which antagonizes migration towards chemokines, confines cells within tissues. Here we review the current understanding of the roles of S1P signaling in activated T cell migration. In this context, we outline open questions, particularly regarding the shape of S1P gradients in different tissues in homeostasis and inflammation, and discuss recent strategies to measure S1P.

Treatment of Uveitis by In Situ Administration of Ex Vivo-Activated Polyclonal Regulatory T Cells.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cell therapy is a promising approach for the treatment of autoimmune diseases. To be effective, Treg cells should be in an activated state in the target tissue. This can be achieved by systemic administration of Ag-specific Treg cells, which are difficult to produce in conditions that can be translated to the clinic. In this paper, we propose an alternative approach consisting of in situ injection of preactivated polyclonal Treg cells that would exert bystander suppression in the target tissue. We show that polyclonal Treg cells suppressed uveitis in mice as efficiently as Ag-specific Treg cells but only when preactivated and administered in the vitreous. Uveitis control was correlated with an increase of IL-10 and a decrease of reactive oxygen species produced by immune cell infiltrates in the eye. Thus, our results reveal a new mechanism of Treg cell-mediated suppression and a new Treg cell therapy approach.

Heterogeneous CD3 expression levels in differing T cell subsets correlate with the in vivo anti-CD3-mediated T cell modulation.

The tolerogenic anti-CD3ε monoclonal Abs (anti-CD3) are promising compounds for the treatment of type 1 diabetes. Anti-CD3 administration induces transient T cell depletion both in preclinical and in clinical studies. Notably, the said depletion mainly affects CD4(+) but not CD8(+) T cells. Moreover, type 1 diabetes reversal in preclinical models is accompanied by the selective expansion of CD4(+)Foxp3(+) T regulatory (Treg) cells, which are fundamental for the long-term maintenance of anti-CD3-mediated tolerance. The mechanisms that lead to this immune-shaping by affecting mainly CD4(+) T effector cells while sparing CD4(+)Foxp3(+) Treg cells have still to be fully elucidated. This study shows that CD3 expression levels differ from one T cell subset to another. CD4(+)Foxp3(-) T cells contain higher amounts of CD3 molecules than do CD4(+)Foxp3(+) and CD8(+) T cells in both mice and humans. The said differences correlate with the anti-CD3-mediated immune resetting that occurs in vivo after anti-CD3 administration in diabetic NOD mice. Additionally, transcriptome analysis demonstrates that CD4(+)Foxp3(+) Treg cells are significantly less responsive than are CD4(+)Foxp3(-) T cells to anti-CD3 treatment at a molecular level. Thus, heterogeneity in CD3 expression seems to confer to the various T cell subsets differing susceptibility to the in vivo tolerogenic anti-CD3-mediated modulation. These data shed new light on the molecular mechanism that underlies anti-CD3-mediated immune resetting and thus may open new opportunities to improve this promising treatment.

Effector T cells boost regulatory T cell expansion by IL-2, TNF, OX40, and plasmacytoid dendritic cells depending on the immune context.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a major role in peripheral tolerance. Multiple environmental factors and cell types affect their biology. Among them, activated effector CD4(+) T cells can boost Treg cell expansion through TNF or IL-2. In this study, we further characterized this effector T (Teff) cell-dependent Treg cell boost in vivo in mice. This phenomenon was observed when both Treg and Teff cells were activated by their cognate Ag, with the latter being the same or different. Also, when Treg cells highly proliferated on their own, there was no additional Treg cell boost by Teff cells. In a condition of low inflammation, the Teff cell-mediated Treg cell boost involved TNF, OX40L, and plasmacytoid dendritic cells, whereas in a condition of high inflammation, it involved TNF and IL-2. Thus, this feedback mechanism in which Treg cells are highly activated by their Teff cell counterparts depends on the immune context for its effectiveness and mechanism. This Teff cell-dependent Treg cell boost may be crucial to limit inflammatory and autoimmune responses.

SPNS2 enables T cell egress from lymph nodes during an immune response.

T cell expression of sphingosine 1-phosphate (S1P) receptor 1 (S1PR1) enables T cell exit from lymph nodes (LNs) into lymph, while endothelial S1PR1 expression regulates vascular permeability. Drugs targeting S1PR1 treat autoimmune disease by trapping pathogenic T cells within LNs, but they have adverse cardiovascular side effects. In homeostasis, the transporter SPNS2 supplies lymph S1P and enables T cell exit, while the transporter MFSD2B supplies most blood S1P and supports vascular function. It is unknown whether SPNS2 remains necessary to supply lymph S1P during an immune response, or whether in inflammation other compensatory transporters are upregulated. Here, using a model of dermal inflammation, we demonstrate that SPNS2 supplies the S1P that guides T cells out of LNs with an ongoing immune response. Furthermore, deletion of Spns2 is protective in a mouse model of multiple sclerosis. These results support the therapeutic potential of SPNS2 inhibitors to achieve spatially specific modulation of S1P signaling.

Tumor Necrosis Factor Receptor Superfamily in T Cell Priming and Effector Function.

The tumor necrosis factor receptor superfamily (TNFRSF) and their ligands mediate lymphoid tissue development and homeostasis in addition to key aspects of innate and adaptive immune responses. T cells of the adaptive immune system express a number of TNFRSF members that are used to receive signals at different instructive stages and produce several tumor necrosis factor superfamily (TNFSF) members as effector molecules. There is also one example of a TNFRSF member serving as a ligand for negative regulatory checkpoint receptors. In most cases, the ligands in afferent and efferent phases are membrane proteins and thus the interaction with TNFRSF members must take place in immunological synapses and other modes of cell-cell interaction. A particular feature of the TNFRSF-mediated signaling is the prominent use of linear ubiquitin chains as scaffolds for signaling complexes that activate nuclear factor κ-B and Fos/Jun transcriptional regulators. This review will focus on the signaling mechanisms triggered by TNFRSF members in their role as costimulators of early and late phases of T cell instruction and the delivery mechanism of TNFSF members through the immunological synapses of helper and cytotoxic effector cells.

Loss of immune tolerance to IL-2 in type 1 diabetes.

Type 1 diabetes (T1D) is characterized by a chronic, progressive autoimmune attack against pancreas-specific antigens, effecting the destruction of insulin-producing ß-cells. Here we show interleukin-2 (IL-2) is a non-pancreatic autoimmune target in T1D. Anti-IL-2 autoantibodies, as well as T cells specific for a single orthologous epitope of IL-2, are present in the peripheral blood of non-obese diabetic (NOD) mice and patients with T1D. In NOD mice, the generation of anti-IL-2 autoantibodies is genetically determined and their titre increases with age and disease onset. In T1D patients, circulating IgG memory B cells specific for IL-2 or insulin are present at similar frequencies. Anti-IL-2 autoantibodies cloned from T1D patients demonstrate clonality, a high degree of somatic hypermutation and nanomolar affinities, indicating a germinal centre origin and underscoring the synergy between cognate autoreactive T and B cells leading to defective immune tolerance.

Role of cytokines in thymus- versus peripherally derived-regulatory T cell differentiation and function.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are essential players in the control of immune responses. Recently, accordingly to their origin, two main subsets of Tregs have been described: thymus-derived Tregs (tTregs) and peripherally derived Tregs (pTregs). Numerous signaling pathways including the IL-2/STAT5 or the TGF-ß/Smad3 pathways play a crucial role in segregating the two lineages. Here, we review some of the information existing on the distinct requirements of IL-2, TGF-ß, and TNF-α three major cytokines involved in tTreg and pTreg generation, homeostasis and function. Today it is clear that signaling via the IL-2Rß chain (CD122) common to IL-2 and IL-15 is required for proper differentiation of tTregs and for tTreg and pTreg survival in the periphery. This notion has led to the development of promising therapeutic strategies based on low-dose IL-2 administration to boost the patients' own Treg compartment and dampen autoimmunity and inflammation. Also, solid evidence points to TGF-ß as the master regulator of pTreg differentiation and homeostasis. However, therapeutic administration of TGF-ß is difficult to implement due to toxicity and safety issues. Knowledge on the role of TNF-α on the biology of Tregs is fragmentary and inconsistent between mice and humans. Moreover, emerging results from the clinical use of TNF-α inhibitors indicate that part of their anti-inflammatory effect may be dependent on their action on Tregs. Given the profusion of clinical trials testing cytokine administration or blocking to modulate inflammatory diseases, a better knowledge of the effects of cytokines on tTregs and pTregs biology is necessary to improve the efficiency of these immunotherapies.

Fetal pancreas transplants are dependent on prolactin for their development and prevent type 1 diabetes in syngeneic but not allogeneic mice.

Transplantation of adult pancreatic islets has been proposed to cure type 1 diabetes (T1D). However, it is rarely considered in the clinic because of its transient effect on disease, the paucity of donors, and the requirement for strong immunosuppressive treatment to prevent allogeneic graft rejection. Transplantation of fetal pancreases (FPs) may constitute an attractive alternative because of potential abundant donor sources, possible long-term effects due to the presence of stem cells maintaining tissue integrity, and their supposed low immunogenicity. In this work, we studied the capacity of early FPs from mouse embryos to develop into functional pancreatic islets producing insulin after transplantation in syngeneic and allogeneic recipients. We found that as few as two FPs were sufficient to control T1D in syngeneic mice. Surprisingly, their development into insulin-producing cells was significantly delayed in male compared with female recipients, which may be explained by lower levels of prolactin in males. Finally, allogeneic FPs were rapidly rejected, even in the context of minor histocompatibility disparities, with massive graft infiltration with T and myeloid cells. This work suggests that FP transplantation as a therapeutic option of T1D needs to be further assessed and would require immunosuppressive treatment.

Limitations of IL-2 and rapamycin in immunotherapy of type 1 diabetes.

Administration of low-dose interleukin-2 (IL-2) alone or combined with rapamycin (RAPA) prevents hyperglycemia in NOD mice. Also, low-dose IL-2 cures recent-onset type 1 diabetes (T1D) in NOD mice, partially by boosting pancreatic regulatory T cells (Treg cells). These approaches are currently being evaluated in humans. Our objective was to study the effect of higher IL-2 doses (250,000-500,000 IU daily) as well as low-dose IL-2 (25,000 IU daily) and RAPA (1 mg/kg daily) (RAPA/IL-2) combination. We show that, despite further boosting of Treg cells, high doses of IL-2 rapidly precipitated T1D in prediabetic female and male mice and increased myeloid cells in the pancreas. Also, we observed that RAPA counteracted IL-2 effects on Treg cells, failed to control IL-2-boosted NK cells, and broke IL-2-induced tolerance in a reversible way. Notably, the RAPA/IL-2 combination failure to cure T1D was associated with an unexpected deleterious effect on glucose homeostasis at multiple levels, including ß-cell division, glucose tolerance, and liver glucose metabolism. Our data help to understand the therapeutic limitations of IL-2 alone or RAPA/IL-2 combination and could lead to the design of improved therapies for T1D.

Pathogenic T cells have a paradoxical protective effect in murine autoimmune diabetes by boosting Tregs.

CD4+CD25+Foxp3+ Tregs play a major role in prevention of autoimmune diseases. The suppressive effect of Tregs on effector T cells (Teffs), the cells that can mediate autoimmunity, has been extensively studied. However, the in vivo impact of Teff activation on Tregs during autoimmunity has not been explored. In this study, we have shown that CD4+ Teff activation strongly boosts the expansion and suppressive activity of Tregs. This helper function of CD4+ T cells, which we believe to be novel, was observed in the pancreas and draining lymph nodes in mouse recipients of islet-specific Teffs and Tregs. Its physiological impact was assessed in autoimmune diabetes. When islet-specific Teffs were transferred alone, they induced diabetes. Paradoxically, when the same Teffs were cotransferred with islet-specific Tregs, they induced disease protection by boosting Treg expansion and suppressive function. RNA microarray analyses suggested that TNF family members were involved in the Teff-mediated Treg boost. In vivo experiments showed that this Treg boost was partially dependent on TNF but not on IL-2. This feedback regulatory loop between Teffs and Tregs may be critical to preventing or limiting the development of autoimmune diseases.

IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells.

Regulatory T cells (T reg cells) play a major role in controlling the pathogenic autoimmune process in type 1 diabetes (T1D). Interleukin 2 (IL-2), a cytokine which promotes T reg cell survival and function, may thus have therapeutic efficacy in T1D. We show that 5 d of low-dose IL-2 administration starting at the time of T1D onset can reverse established disease in NOD (nonobese diabetic) mice, with long-lasting effects. Low-dose IL-2 increases the number of T reg cells in the pancreas and induces expression of T reg cell-associated proteins including Foxp3, CD25, CTLA-4, ICOS (inducible T cell costimulator), and GITR (glucocorticoid-induced TNF receptor) in these cells. Treatment also suppresses interferon gamma production by pancreas-infiltrating T cells. Transcriptome analyses show that low-dose IL-2 exerts much greater influence on gene expression of T reg cells than effector T cells (T eff cells), suggesting that nonspecific activation of pathogenic T eff cells is less likely. We provide the first preclinical data showing that low-dose IL-2 can reverse established T1D, suggesting that this treatment merits evaluation in patients with T1D.