Cutting edge: regulatory T cells do not mediate suppression via programmed cell death pathways

Regulatory T cells (Tregs) play a critical role in the immune system to regulate peripheral tolerance and prevent autoimmunity. However, the relative importance of different mechanisms of Treg function remains obscure. In this article, we reveal a limited role for programmed cell death pathways in mediating Treg suppression of conventional T cells. We show that Tregs are able to suppress the proliferation of conventional T cells that are resistant to apoptosis (Bim(-/-), Bim(-/-)Puma(-/-), Bcl-2 transgenic) or receptor-interacting serine-threonine kinase-dependent necrosis (also referred to as regulated necrosis or necroptosis) (Ripk3(-/-)) in several in vitro and in vivo assays. These data suggest that programmed cell death pathways, such as apoptosis and receptor-interacting serine-threonine kinase-dependent necrosis, are not required for Treg-mediated suppression.     

Cutting edge: ligation of the glucocorticoid-induced TNF receptor enhances autoreactive CD4+ T cell activation and experimental autoimmune encephalomyelitis

The glucocorticoid-induced TNFR (GITR) is expressed at high levels on resting CD4(+)CD25(+) T regulatory (T(R)) cells and regulates their suppressive phenotype. Accordingly, we show that anti-GITR mAb treatment of SJL mice with proteolipid protein 139-151-induced experimental autoimmune encephalomyelitis significantly exacerbated clinical disease severity and CNS inflammation, and induced elevated levels of Ag-specific T cell proliferation and cytokine production. Interestingly, prior depletion of T(R) cells failed to result in exacerbated experimental autoimmune encephalomyelitis suggesting alternative targets for the anti-GITR mAb treatment. Importantly, naive CD4(+)CD25(-) T cells up-regulated GITR expression in an activation-dependent manner and anti-GITR mAb treatment enhanced the level of CD4(+) T cell activation, proliferation, and cytokine production in the absence of T(R) cells both in vivo and in vitro. Taken together, these findings suggest a dual functional role for GITR as GITR cross-linking both inactivates T(R) cells and increases CD4(+)CD25(-) T cell effector function, thus enhancing T cell immunity.     

Cutting edge: suppression of T cell chemotaxis by sphingosine 1-phosphate

Murine CD4 and CD8 T cells express predominantly types 1 and 4 sphingosine 1-phosphate (S1P) G protein-coupled receptors (designated S1P1 and S1P4 or previously endothelial differentiation gene-encoded 1 and 6) for S1P, which has a normal plasma concentration of 0.1-1 microM. S1P now is shown to enhance chemotaxis of CD4 T cells to CCL-21 and CCL-5 by up to 2.5-fold at 10 nM to 0.1 microM, whereas 0.3-3 microM S1P inhibits this chemotaxis by up to 70%. Chemotaxis of S1P(1), but not S1P(4), transfectants to CXCL1 and CXCL4 was similarly affected by S1P. Activation of CD4 T cells, which decreases S1P receptor expression, suppressed effects of S1P on chemotaxis. Pretreatment of labeled CD4 T cells with S1P before reintroduction into mice inhibited by a maximum of 75% their migration into chemokine-challenged s.c. air pouches. The S1P-S1P(1) receptor axis thus controls recruitment of naive T cells by maintaining their response threshold to diverse lymphotactic factors.     

Cutting edge: Human regulatory T cells require IL-35 to mediate suppression and infectious tolerance

Human regulatory T cells (T(reg)) are essential for the maintenance of immune tolerance. However, the mechanisms they use to mediate suppression remain controversial. Although IL-35 has been shown to play an important role in T(reg)-mediated suppression in mice, recent studies have questioned its relevance in human T(reg). In this study, we show that human T(reg) express and require IL-35 for maximal suppressive capacity. Substantial upregulation of EBI3 and IL12A, but not IL10 and TGFB, was observed in activated human T(reg) compared with conventional T cells (T(conv)). Contact-independent T(reg)-mediated suppression was IL-35 dependent and did not require IL-10 or TGF-β. Lastly, human T(reg)-mediated suppression led to the conversion of the suppressed T(conv) into iTr35 cells, an IL-35-induced T(reg) population, in an IL-35-dependent manner. Thus, IL-35 contributes to human T(reg)-mediated suppression, and its conversion of suppressed target T(conv) into IL-35-induced T(reg) may contribute to infectious tolerance.     

Cutting edge: in vivo trogocytosis as a mechanism of double negative regulatory T cell-mediated antigen-specific suppression

Recent data have demonstrated that treatment with alphabeta-TCR(+)CD3(+)CD4(-)CD8(-)NK1.1(-) double negative (DN) regulatory T cells (Tregs) inhibits autoimmune diabetes and enhances allotransplant and xenotransplant survival in an Ag-specific fashion. However, the mechanisms whereby DN Tregs suppress Ag-specific immune responses remain largely unknown. In this study, we demonstrate that murine DN Tregs acquire alloantigen in vivo via trogocytosis and express it on their cell surface. Trogocytosis requires specific interaction of MHC-peptide on APCs and Ag-specific TCR on DN Tregs, as blocking this interaction prevents DN Treg-mediated trogocytosis. Acquisition of alloantigen by DN Tregs was required for their ability to kill syngeneic CD8(+) T cells. Importantly, DN Tregs that had acquired alloantigen were cytotoxic toward Ag-specific, but not Ag-nonspecific, syngeneic CD8(+) T cells. These data provide new insight into how Tregs mediate Ag-specific T cell suppression and may enhance our ability to use DN Tregs as a therapy for transplant rejection and autoimmune diseases.     

Cutting edge: profound defect in T cell responses in TNF receptor-associated factor 2 dominant negative mice

TNFR-associated factor 2 (TRAF2) is an adapter protein that links several members of the TNFR family to downstream signaling pathways. Mice expressing a dominant negative form of TRAF2 in their lymphoid cells (TRAF2.DN mice) have a profound defect in T cell responses to allogeneic APC. In contrast, APC from wild-type or TRAF2.DN mice show an equivalent level of stimulation in a MLR. Ab production and class switch are unimpaired in TRAF2.DN mice. Thus, defects in the TRAF.DN mice appear to be limited to T cells. TRAF2.DN mice demonstrate an impaired T cell response to influenza virus, including decreased secondary expansion of IFN-gamma-secreting T cells as well as a decrease in CTL activity. CD4 T cell production of IL-2 was also dramatically impaired in TRAF2.DN mice. These studies suggest an essential role of TRAF2-linked receptors in secondary CD4 and CD8 T cell responses and have important implications for transplantation.     

Cutting edge: direct suppression of B cells by CD4+ CD25+ regulatory T cells

Regulatory T cells (Tregs) can potentially migrate to the B cell areas of secondary lymphoid tissues and suppress T cell-dependent B cell Ig response. T cell-dependent Ig response requires B cell stimulation by Th cells. It has been unknown whether Tregs can directly suppress B cells or whether they must suppress Th cells to suppress B cell response. We report here that Foxp3+ Tregs are found in T-B area borders and within germinal centers of human lymphoid tissues and can directly suppress B cell Ig response. Although Tregs can effectively suppress T cells, they can also directly suppress B cell response without the need to first suppress Th cells. The direct suppression of B cell Ig production by Tregs is accompanied by inhibition of Ig class switch recombination.     

T cell costimulation by the TNF ligand BAFF.

The TNF ligand family member BAFF (B cell activating factor belonging to the TNF family, also called Blys, TALL-1, zTNF-4, or THANK) is an important survival factor for B cells [corrected]. In this study, we show that BAFF is able to regulate T cell activation. rBAFF induced responses (thymidine incorporation and cytokine secretion) of T cells, suboptimally stimulated through their TCR. BAFF activity was observed on naive, as well as on effector/memory T cells (both CD4+ and CD8+ subsets), indicating that BAFF has a wide function on T cell responses. Analysis of the signal transduced by BAFF into T cells shows that BAFF has no obvious effect on T cell survival upon activation, but is able to deliver a complete costimulation signal into T cells. Indeed, BAFF is sufficient to induce IL-2 secretion and T cell division, when added to an anti-TCR stimulation. This highlights some differences in the BAFF signaling pathway in T and B cells. In conclusion, our results indicate that BAFF may play a role in the development of T cell responses, in addition to its role in B cell homeostasis.     

Cutting edge: antigen-independent CD8 T cell proliferation

Recent analyses of CD8 T cell responses to Listeria monocytogenes infection demonstrate that the duration of in vivo T cell proliferation is not determined by the amount or duration of Ag presentation. However, the extent to which T lymphocytes are capable of proliferating in the absence of Ag is unknown. Herein we demonstrate that CD8 T lymphocytes undergo up to eight rounds of proliferation in the absence of Ag following transient, 2.5-h in vitro antigenic stimulation. Ag-independent expansion of CD8 T cells is driven by IL-2 and is further augmented by IL-7 or IL-15. These experiments clearly demonstrate that CD8 T cells undergo prolonged proliferation following transient Ag exposure and support the notion that in vivo CD8 T cell expansion following infection can be uncoupled from Ag presentation.     

Cutting edge: estrogen drives expansion of the CD4+CD25+ regulatory T cell compartment

CD4(+)CD25(+) regulatory T cells are crucial to the maintenance of tolerance in normal individuals. However, the factors regulating this cell population and its function are largely unknown. Estrogen has been shown to protect against the development of autoimmune disease, yet the mechanism is not known. We demonstrate that estrogen (17-beta-estradiol, E2) is capable of augmenting FoxP3 expression in vitro and in vivo. Treatment of naive mice with E2 increased both CD25(+) cell number and FoxP3 expression level. Further, the ability of E2 to protect against autoimmune disease (experimental autoimmune encephalomyelitis) correlated with its ability to up-regulate FoxP3, as both were reduced in estrogen receptor alpha-deficient animals. Finally, E2 treatment and pregnancy induced FoxP3 protein expression to a similar degree, suggesting that high estrogen levels during pregnancy may help to maintain fetal tolerance. In summary, our data suggest E2 promotes tolerance by expanding the regulatory T cell compartment.     

Cutting edge: selective impairment of CD8+ T cell function in mice lacking the TNF superfamily member LIGHT

Interactions of LIGHT and its receptors, herpesvirus entry mediator on T cells and lymphotoxin beta receptor on stromal cells, are implicated in the regulation of lymphoid organogenesis, costimulation of T cells, and activation of dendritic cells. In this work we report that LIGHT-deficient mice had normal lymphoid organs with T cells and APCs that normally responded to Ag stimulation and normally stimulated T cells. Although the number of Vbeta8(+) T cells in naive LIGHT(+/+) and LIGHT(-/-) mice was identical, Vbeta8(+)CD8(+) T cell proliferation in response to staphylococcal enterotoxin B was significantly lower in LIGHT(-/-) mice. Consistently, induction and cytokine secretion of CD8(+) CTL to MHC class I-restricted peptide was also reduced in LIGHT(-/-) mice. However, the proliferative response of Vbeta8(+)CD4(+) T cells to staphylococcal enterotoxin B was comparable in LIGHT(-/-) and LIGHT(+/+) mice. Our results suggest that LIGHT is required for activation of normal CD8(+) T cells but not CD4(+) T cells.     

Cutting edge: Evidence for a dynamically driven T cell signaling mechanism

T cells use the αβ TCR to bind peptides presented by MHC proteins (pMHC) on APCs. Formation of a TCR-pMHC complex initiates T cell signaling via a poorly understood process, potentially involving changes in oligomeric state, altered interactions with CD3 subunits, and mechanical stress. These mechanisms could be facilitated by binding-induced changes in the TCR, but the nature and extent of any such alterations are unclear. Using hydrogen/deuterium exchange, we demonstrate that ligation globally rigidifies the TCR, which via entropic and packing effects will promote associations with neighboring proteins and enhance the stability of existing complexes. TCR regions implicated in lateral associations and signaling are particularly affected. Computational modeling demonstrated a high degree of dynamic coupling between the TCR constant and variable domains that is dampened upon ligation. These results raise the possibility that TCR triggering could involve a dynamically driven, allosteric mechanism.     

Cutting edge: IL-12 induces CD4+CD25- T cell activation in the presence of T regulatory cells

IL-12p40 cytokines have been implicated in the development of organ-specific autoimmune diseases as well as pathogen-specific adaptive immunity. In addition to inducing IFN-gamma, IL-12 stimulates effector CD4(+) T cells to express adhesion molecules and homing receptors that facilitate their migration to sites of inflammation. In this study, we expand upon those observations by demonstrating an alternative pathway by which IL-12 could promote Th1 inflammatory responses in mice, namely, by restoring proliferation and cytokine expression by effector T cells in the presence of CD4(+)CD25(+) regulatory T cells (Treg). This effect of IL-12 was not replicated by IL-23 or IFN-gamma and was dependent on signaling through the IL-12R expressed on CD25(-) responder cells, but not on Treg. Our studies suggest that IL-12 could act in concert with other proinflammatory factors to stimulate CD4(+)CD25(-) T cell activation in the presence of Treg.     

Cutting edge: requirement for TRAF6 in the induction of T cell anergy

TRAF6, TNFR-associated factor 6, is a key adaptor downstream from the TNF receptor and TLR superfamily members. T cell-specific deletion of TRAF6 (TRAF6-DeltaT) was recently shown to result in the development of multiorgan inflammatory disease and the resistance of responder T cells to suppression by CD4+CD25+ regulatory T cells. In this study we examined the role of TRAF6 in an additional mechanism of peripheral tolerance, anergy. We have determined that the loss of TRAF6 restores the ability of CD28-/- T cells to proliferate and produce IL-2. Consistent with this, TRAF6-DeltaT T cells were resistant to anergizing signals both in vitro and in vivo. Resistance to anergy was correlated with decreased expression of Cbl-b. These findings reveal that in addition to its role in rendering T cells susceptible to control by CD4+CD25+ regulatory T cells, TRAF6 is essential for the induction of T cell anergy, implicating TRAF6 as a critical mediator of peripheral tolerance.     

Cutting edge: Th17 and regulatory T cell dynamics and the regulation by IL-2 in the tumor microenvironment

Th17 cells play an active role in inflammation and autoimmune diseases. However, the nature and regulation of Th17 in the context of tumor immunity remain unknown. In this study, we show that parallel to regulatory T (Treg) cells, IL-17(+) CD4(+) and CD8(+) T cells are kinetically induced in multiple tumor microenvironments in mice and humans. Treg cells play a crucial role in tumor immune pathogenesis and temper immune therapeutic efficacy. IL-2 is crucial for the production and function of Treg cells. We now show that IL-2 reduces IL-17(+) T cell differentiation in the tumor microenvironment accompanied with an enhanced Treg cell compartment in vitro and in vivo. Altogether, our work demonstrates a dynamic differentiation of IL-17(+) T cells in the tumor microenvironment, reveals a novel role for IL-2 in controlling the balance between IL-17(+) and Treg cells, and provides new insight of IL-17(+) T cells in tumor immune pathology and therapy.     

Cutting edge: mechanisms of IL-2-dependent maintenance of functional regulatory T cells

IL-2 controls the survival of regulatory T cells (Tregs), but it is unclear whether IL-2 also directly affects Treg suppressive capacity in vivo. We have found that eliminating Bim-dependent apoptosis in IL-2- and CD25-deficient mice restored Treg numbers but failed to cure their lethal autoimmune disease, demonstrating that IL-2-dependent survival and suppressive activity can be uncoupled in Tregs. Treatment with IL-2-anti-IL-2-Ab complexes enhanced the numbers and suppressive capacity of IL-2-deprived Tregs with striking increases in CD25, CTLA-4, and CD39/CD73 expression. Although cytokine treatment induced these suppressive mechanisms in both IL-2(-/-) and IL-2(-/-)Bim(-/-) mice, it only reversed autoimmune disease in the latter. Our results suggest that successful IL-2 therapy of established autoimmune diseases will require a threshold quantity of Tregs present at the start of treatment and show that the suppressive capacity of Tregs critically depends on IL-2 even when Treg survival is independent of this cytokine.     

Cutting edge: fasting-induced hypoleptinemia expands functional regulatory T cells in systemic lupus erythematosus

Fasting is beneficial in the prevention and amelioration of the clinical manifestations of autoimmune diseases including systemic lupus erythematosus. The mechanisms responsible for these effects are not well understood. During fasting, there is a dramatic reduction of the levels of circulating leptin, an adipokine with proinflammatory effects. Leptin also inhibits CD4(+)CD25(+)Foxp3(+) regulatory T cells, which are known to contribute significantly to the mechanisms of peripheral immune tolerance. In this study, we show that fasting-induced hypoleptinemia in (NZB × NZW)F(1) lupus-prone mice induced an expansion of functional regulatory T cells that was reversed by leptin replacement. The specificity of the findings was indicated by the lack of these effects in leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice. These observations help to explain the beneficial effects of fasting in autoimmunity and could be exploited for leptin-based immune intervention in systemic lupus erythematosus.