Kinetics of IL-6 Production Defines T Effector Cell Responsiveness to Regulatory T Cells in Multiple Sclerosis

In multiple sclerosis (MS) autoaggressive T effector cells (Teff) are not efficiently controlled by regulatory T cells (Treg) but the underlying mechanisms are incompletely understood. Proinflammatory cytokines are key factors facilitating Teff activity in chronic inflammation. Here we investigated the influence of IL-6 on Treg sensitivity of Teff from therapy-naïve MS patients with or without active disease. Compared to healthy volunteers and independent of disease course CD4⁺ and especially CD8⁺ MS-Teff were insensitive against functional active Treg from healthy controls. This unresponsiveness was caused by accelerated production of IL-6, elevated IL-6 receptor expression and phosphorylation of protein kinase B (PKB)/c-Akt in MS-Teff. In a positive feedback loop, IL-6 itself induced its accelerated synthesis and enhanced phosphorylation of PKB/c-Akt that finally mediated Treg resistance. Furthermore, accelerated IL-6 release especially by CD8⁺ Teff prevented control of surrounding Teff, described here as “bystander resistance”. Blockade of IL-6 receptor signaling or direct inhibition of PKB/c-Akt phosphorylation restored Treg responsiveness of Teff and prevented bystander resistance. In Teff of healthy controls (HC) exogenous IL-6 also changed the kinetics of IL-6 production and induced Treg unresponsiveness. This modulation was only transient in Teff from healthy volunteers, whereas accelerated IL-6 production in MS-Teff maintained also in absence of IL-6. Hence, we showed that the kinetics of IL-6 production instead of elevated IL-6 levels defines the Teff responsiveness in early Treg-T cell communication in MS independent of their disease course and propose IL-6 and associated PKB/c-Akt activation as effective therapeutic targets for modulation of Teff activity in MS.     

Homeostasis and Function of Regulatory T Cells in HIV/SIV Infection

Regulatory T cells (Tregs) play a pivotal role in the maintenance of tolerance as well as in the control of immune activation, particularly during chronic infections. In the setting of HIV infection, the majority of studies have reported an increase in Treg frequency but a decrease in absolute number in all immune compartments of HIV-infected individuals. Several nonexclusive mechanisms have been postulated to explain this preferential Treg accumulation, including peripheral survival, increased proliferation, increased peripheral conversion, and tissue redistribution. The role played by Tregs during HIV infection is still poorly understood, as two opposing hypotheses have been proposed. A detrimental role of Tregs during HIV infection was suggested based on the evidence that Tregs suppress virus-specific immune responses. Conversely, Tregs could be beneficial by limiting immune activation, thus controlling the availability of HIV targets as well as preventing immune-based pathologies. Despite the technical difficulties, getting a better understanding of the mechanisms regulating Treg dynamics remains important, as it will help determine whether we can successfully manipulate Treg function or number to the advantage of the infected host. The aim of this review is thus to discuss the recent findings on Treg homeostasis and function in the setting of HIV infection.     

Epitope-Specific Regulatory CD4 T Cells Reduce Virus-Induced Illness while Preserving CD8 T-Cell Effector Function at the Site of Infection

The role of epitope-specific regulatory CD4 T cells in modulating CD8 T-cell-mediated immunopathology during acute viral infection has not been well defined. In the murine model of respiratory syncytial virus (RSV) infection, CD8 T cells play an important role in both viral clearance and immunopathology. We have previously characterized two RSV epitope-specific CD4 T-cell responses with distinct phenotypic properties. One of them, the IA^bM₂₀₉-specific subset, constitutively expresses FoxP3 and modulates CD8 T-cell function in vitro. We show here that the IA^bM₂₀₉-specific CD4 T-cell response regulates CD8 T-cell function in vivo and is associated with diminished RSV-induced illness without affecting viral clearance at the site of infection. Achieving the optimal balance of regulatory and effector T-cell function is an important consideration for designing future vaccines.A subset of CD4 T cells with regulatory function (Treg) has been shown to play an important role in modulating adaptive immune responses. Natural Tregs are characterized by the expression of FoxP3 and participate in reducing the activation of CD8 T-cell responses in peripheral lymphoid organs (11, 20, 35). This modulation can diminish the ability of adaptive immune responses to control systemic infections (4). However, the presence of natural regulatory CD4 T cells can have a beneficial effect on immune-mediated pathology, particularly at the site of infection. Tregs have been shown to limit pulmonary inflammation and lung injury induced by pneumocystis infection (29) and to modulate herpes simplex virus-induced inflammatory lesions of the eye (46). Natural Tregs also reduce the symptoms of West Nile virus infections in both humans and mice; Treg-deficient mice were more likely to develop lethal infection (25). Viral infection can also induce antigen-specific CD4 T cells that express FoxP3 (27), and their role in protective immunity and immunopathology needs more detailed investigation.T lymphocytes are key components of adaptive immunity against respiratory syncytial virus (RSV) infection. Children with T-cell deficiencies have delayed virus clearance and are more susceptible to fatal RSV infection (10, 18). The absence of T cells infiltrating into lung is associated with fatal RSV infections in children without recognized underlying disease (49). In the murine model, CD8 T cells play a major role in RSV clearance, presumably through direct cytotoxicity to infected cells and the generation of immunocompetent molecules (2, 15, 43); depletion of CD8 T cells in mice results in delayed viral clearance (14). The CD8 T-cell response also induces immunopathology in primary infection of mice (15, 32, 48). Transferring high doses of CD8 T cells facilitates virus clearance but also causes hemorrhagic pneumonia and enhanced disease (6, 14). These studies demonstrate that while CD8 T cells are required for viral clearance, they are responsible for immunopathology. We have described the pattern of CD8 T-cell responses that occur in mice that are the F₁ hybrid (H-2^d/b) between BALB/c (H-2^d) and C57BL/6 (H-2^b) (39). These mice respond both to the well-characterized K^dM2₈₂ epitope (24) and to a more recently described D^bM₁₈₇ epitope (38). Both CD8 T-cell responses are dominant in the parent strains but assume a hierarchy (K^dM2₈₂ > D^bM₁₈₇) in the F₁ hybrid (39). This model infection allows the analysis of factors that determine T-cell response hierarchy and provides multiple endpoints for the assessment of factors that modify or regulate CD8 T-cell responses.We recently described epitope-specific CD4 T-cell responses distinguished by novel major histocompatibility complex (MHC) class II tetramers in RSV infection. The IA^bM₂₀₉-specific CD4 T cells have a high frequency of FoxP3 expression and suppress RSV-specific CD8 T-cell cytokine production in vitro (27). To investigate the regulatory role of IA^bM₂₀₉-specific CD4 T cells in vivo, we sought to determine how immunizing mice with the CD4 T-cell epitope peptide M₂₀₉ would affect the RSV-specific CD8 T-cell response. We show here that the IA^bM₂₀₉-specific CD4 T cells have a regulatory effect on the dominant CD8 T-cell response to RSV infection, reducing both the magnitude and effector cytokine production in peripheral lymphoid organs while allowing effector functions at the site of infection to clear virus with normal kinetics. Viral clearance was thus achieved with less illness.     

Mycobacterium tuberculosis Complex Enhances Susceptibility of CD4 T Cells to HIV through a TLR2-Mediated Pathway

Among HIV-infected individuals, co-infection with Mycobacterium tuberculosis is associated with faster progression to AIDS. We investigated the hypothesis that M. bovis BCG and M. tuberculosis (Mtb complex) could enhance susceptibility of CD4+ cells to HIV infection. Peripheral blood mononuclear cells (PBMCs) collected from healthy donors were stimulated with M. bovis BCG, M. tuberculosis CDC1551 and M. smegmatis MC(2)155, and stimulated CD4+ cells were infected with R5-and X4-tropic single replication-competent pseudovirus. CD4+ cells stimulated with Mtb complex showed enhanced infection with R5- and X4-tropic HIV, compared to unstimulated cells or cells stimulated with M. smegmatis (p<0.01). Treatment with TLR2 siRNA reversed the increased susceptibility of CD4+ cells with R5- and X4-tropic virus induced by Mtb complex. These findings suggest that TB infection and/or BCG vaccination may be a risk factor for HIV acquisition.     

Fingolimod Increases CD39-Expressing Regulatory T Cells in Multiple Sclerosis Patients

Background

Multiple sclerosis (MS) likely results from an imbalance between regulatory and inflammatory immune processes. CD39 is an ectoenzyme that cleaves ATP to AMP and has been suggested as a novel regulatory T cells (Treg) marker. As ATP has numerous proinflammatory effects, its degradation by CD39 has anti-inflammatory influence. The purpose of this study was to explore regulatory and inflammatory mechanisms activated in fingolimod treated MS patients.

Methods and Findings

Peripheral blood mononuclear cells (PBMCs) were isolated from relapsing-remitting MS patients before starting fingolimod and three months after therapy start. mRNA expression was assessed in ex vivo PBMCs. The proportions of CD8, B cells, CD4 and CD39-expressing cells were analysed by flow cytometry. Treg proportion was quantified by flow cytometry and methylation-specific qPCR. Fingolimod treatment increased mRNA levels of CD39, AHR and CYP1B1 but decreased mRNA expression of IL-17, IL-22 and FOXP3 mRNA in PBMCs. B cells, CD4⁺ cells and Treg proportions were significantly reduced by this treatment, but remaining CD4⁺ T cells were enriched in FOXP3⁺ cells and in CD39-expressing Tregs.

Conclusions

In addition to the decrease in circulating CD4⁺ T cells and CD19⁺ B cells, our findings highlight additional immunoregulatory mechanisms induced by fingolimod.     

Functional Modulation of Regulatory T Cells by IL-2

The suppressive function of regulatory T cells (Tregs) is critical to the maintenance of immune homeostasis in vivo and yet, the specific identification of Tregs by phenotypic markers is not perfect. Tregs were originally identified in the CD4⁺CD25⁺ fraction of T cells, but FoxP3 expression was later included as an additional marker of Tregs as FoxP3 expression was identified as being critical to the development and function of these cells. Intracellular expression of FoxP3 makes it difficult in using to isolate live and not permeabilized cells for functional assays. As such CD4⁺CD25⁺ fraction is still frequently used for functional assays of Tregs. Although, the CD4⁺CD25⁺ fraction substantially overlaps with the FoxP3⁺ fraction, the minor mismatch between CD4⁺CD25⁺ and FoxP3⁺ fractions may confound the functional characteristics of Tregs. In this study, we isolated CD4⁺FoxP3⁺ as well as CD4⁺CD25⁺ fractions from Foxp3 knock-in mice, and compared their proliferative and suppressive activity in the presence or absence of various concentrations of IL-2. Our results showed comparable patterns of proliferative and suppressive responses for both fractions, except that contrary to the CD4⁺CD25⁺ fraction the FoxP3⁺ fraction did not proliferate in an autocrine fashion even in response to a strong stimulation. In presence of exogenous IL-2, both CD4⁺CD25⁺ and CD4⁺FoxP3⁺ fractions were more sensitive than the CD4⁺CD25^- responder cells in proliferative responsiveness. In addition, a low dose IL-2 enhanced whereas a high dose abrogated the suppressive activities of the CD4⁺CD25⁺ and CD4⁺FoxP3⁺ fractions. These results may provide an additional understanding of the characteristics of the various fractions of isolated Tregs based on phenotype and function and the role of varying levels of exogenous IL-2 on the suppressive activity of these cells.     

The Clinical Correlation of Regulatory T Cells and Cyclic Adenosine Monophosphate in Enterovirus 71 Infection

Background

Brainstem encephalitis (BE) and pulmonary edema (PE) are notable complications of enterovirus 71 (EV71) infection.

Objective

This study investigated the immunoregulatory characterizations of EV71 neurological complications by disease severity and milrinone treatment.

Study Design

Patients <18 years with virologically confirmed EV71 infections were enrolled and divided into 2 groups: the hand, foot, and mouth disease (HFMD) or BE group, and the autonomic nervous system (ANS) dysregulation or PE group. Cytokine and cyclic adenosine monophosphate (cAMP) levels, and the regulatory T cell (Tregs) profiles of the patients were determined.

Results

Patients with ANS dysregulation or PE exhibited significantly low frequency of CD4⁺CD25⁺Foxp3⁺ and CD4⁺Foxp3⁺ T cells compared with patients with HFMD or BE. The expression frequency of CD4⁻CD8⁻ was also significantly decreased in patients with ANS dysregulation or PE. Among patients with ANS dysregulation or PE, the expression frequency of CD4⁺Foxp3⁺ increased markedly after milrinone treatment, and was associated with reduction of plasma levels IL-6, IL-8 and IL-10. Plasma concentrations of cAMP were significantly decreased in patients with ANS dysregulation or PE compared with patients with HFMD or BE; however, cAMP levels increased after milrinone treatment.

Conclusions

These findings suggested decreased different regulatory T populations and cAMP expression correlate with increased EV71 disease severity. Improved outcome after milrinone treatment may associate with increased regulatory T populations, cAMP expression and modulation of cytokines levels.     

CD134 Costimulation Couples the CD137 Pathway to Induce Production of Supereffector CD8 T Cells That Become IL-7 Dependent

The TNFR superfamily members 4-1BB (CD137) and OX40 (CD134) are costimulatory molecules that potently boost CD8 and CD4 T cell responses. Concomitant therapeutic administration of agonist anti-CD137 and -CD134 mAbs mediates rejection of established tumors and fosters powerful CD8 T cell responses. To reveal the mechanism, the role of CD137 expression by specific CD8 T cells was determined to be essential for optimal clonal expansion and accumulation of effector cells. Nonetheless, dual costimulation induced production of supereffector CD8 T cells when either the specific T cells or the host alone bore CD137. Perhaps surprisingly, the total absence of CD137 prevented anti-CD134 augmentation of supereffector differentiation demonstrating an unappreciated link between these related pathways. Ultimately, it was reasoned that these powerful dual costimulatory responses involved common gamma family members, and we show substantial increases of CD25 and IL-7Ralpha-chain expression by the specific CD8 T cells. To investigate this further, it was shown that IL-7 mediated T cell accumulation, but importantly, a gradual and preferential effect of survival was directed toward supereffector CD8 T cells. In fact, a clear enhancement of effector differentiation was demonstrated to be proportional to the increasing amount of IL-7Ralpha expression by the specific CD8 T cells. Therefore, dual costimulation through CD137 and CD134 drives production and survival of supereffector CD8 T cells through a distinct IL-7-dependent pathway.     

Concurrent Generation of Effector and Central Memory CD8 T Cells during Vaccinia Virus Infection

It is generally thought that during the contraction phase of an acute anti-viral T cell reponse, the effector T cells that escape activation-induced cell death eventually differentiate into central memory T cells over the next several weeks. Here we report that antigen-specific CD8T cells with the phenotype and function of central memory cells develop concomitantly with effector T cells during vaccinia virus (vv) infection. As soon as 5 days after an intraperitoneal infection with vv, we could identify a subset of CD44^hi and CD62L⁺ vv-specific CD8 T cells in the peritoneal exudate lymphocytes. This population constituted approximately 10% of all antigen-specific T cells and like central memory T cells, they also expressed high levels of CCR7 and IL-7R but expressed little granzyme B. Importantly, upon adoptive transfer into naïve congenic hosts, CD62L⁺, but not CD62L⁻ CD8 T cells were able to expand and mediate a rapid recall response to a new vv challenge initiated 6 weeks after transfer, confirming that the CD62L⁺ vv-specific CD8 T cells are bonafide memory cells. Our results are thus consistent with the branched differentiation model, where effector and memory cells develop simultaneously. These results are likely to have implications in the context of vaccine design, particularly those based on vaccinia virus recombinants.     

Paricalcitol Reduces Peritoneal Fibrosis in Mice through the Activation of Regulatory T Cells and Reduction in IL-17 Production

Fibrosis is a significant health problem associated with a chronic inflammatory reaction. The precise mechanisms involved in the fibrotic process are still poorly understood. However, given that inflammation is a major causative factor, immunomodulation is a possible therapeutic approach to reduce fibrosis. The vitamin D receptor (VDR) that is present in all hematopoietic cells has been associated with immunomodulation. We investigated whether the intraperitoneal administration of paricalcitol, a specific activator of the VDR, modulates peritoneal dialysis fluid (PDF)-induced peritoneal fibrosis. We characterized the inflammatory process in the peritoneal cavity of mice treated or not treated with paricalcitol and analyzed the ensuing fibrosis. The treatment reduced peritoneal IL-17 levels, which strongly correlated with a significantly lower peritoneal fibrotic response. In vitro studies demonstrate that both CD4⁺ and CD8⁺ regulatory T cells appear to impact the regulation of IL-17. Paricalcitol treatment resulted in a significantly increased frequency of CD8⁺ T cells showing a regulatory phenotype. The frequency of CD4⁺ Tregs tends to be increased, but it did not achieve statistical significance. However, paricalcitol treatment increased the number of CD4⁺ and CD8⁺ Treg cells in vivo. In conclusion, the activation of immunological regulatory mechanisms by VDR signaling could prevent or reduce fibrosis, as shown in peritoneal fibrosis induced by PDF exposure in mice.     

4-1BB Signaling in Conventional T Cells Drives IL-2 Production That Overcomes CD4+CD25+FoxP3+ T Regulatory Cell Suppression

Costimulation with the recombinant SA-4-1BBL agonist of 4-1BB receptor on conventional CD4⁺ T cells (Tconvs) overcomes the suppression mediated by naturally occurring CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs). The mechanistic basis of this observation has remained largely unknown. Herein we show that Tconvs, but not Tregs, are the direct target of SA-4-1BBL-mediated evasion of Treg suppression. IL-2 produced by Tconvs in response to 4-1BB signaling is both necessary and sufficient for overcoming Treg suppression. Supernatant from Tconvs stimulated with SA-4-1BBL contains high levels of IL-2 and overcomes Treg suppression in ex vivo Tconv:Treg cocultures. Removal of IL-2 from such supernatant restores Treg suppression and repletion of Tconv:Treg cocultures with exogenous recombinant IL-2 overcomes suppression. This study establishes 4-1BB signaling as a key circuit that regulates physical and functional equilibrium between Tregs and Tconvs with important implications for immunotherapy for indications where a fine balance between Tregs and Teffs plays a decisive role.     

Abrogation of CD40-CD154 Signaling Impedes the Homeostasis of Thymic Resident Regulatory T Cells by Altering the Levels of IL-2, but Does Not Affect Regulatory T Cell Development

A greater understanding of the function of the human immune system at the single-cell level in healthy individuals is critical for discerning aberrant cellular behavior that occurs in settings such as autoimmunity, immunosenescence, and cancer. To achieve this goal, a systems-level approach capable of capturing the response of the interdependent immune cell types to external stimuli is required. In this study, an extensive characterization of signaling responses in multiple immune cell subpopulations within PBMCs from a cohort of 60 healthy donors was performed using single-cell network profiling (SCNP). SCNP is a multiparametric flow cytometry-based approach that enables the simultaneous measurement of basal and evoked signaling in multiple cell subsets within heterogeneous populations. In addition to establishing the interindividual degree of variation within a broad panel of immune signaling responses, the possible association of any observed variation with demographic variables including age and race was investigated. Using half of the donors as a training set, multiple age- and race-associated variations in signaling responses in discrete cell subsets were identified, and several were subsequently confirmed in the remaining samples (test set). Such associations may provide insight into age-related immune alterations associated with high infection rates and diminished protection following vaccination and into the basis for ethnic differences in autoimmune disease incidence and treatment response. SCNP allowed for the generation of a functional map of healthy immune cell signaling responses that can provide clinically relevant information regarding both the mechanisms underlying immune pathological conditions and the selection and effect of therapeutics.     

In Vitro HIV Infection Impairs the Capacity of Myeloid Dendritic Cells to Induce Regulatory T Cells

Myeloid dendritic cells (mDCs) are the antigen-presenting cells best capable of promoting peripheral induction of regulatory T cells (Tregs), and are among the first targets of HIV. It is thus important to understand whether HIV alters their capacity to promote Treg conversion. Monocyte-derived DCs (moDCs) from uninfected donors induced a Treg phenotype (CD25(+)FOXP3(+)) in autologous conventional T cells. These converted FOXP3(+) cells suppressed the proliferation of responder T cells similarly to circulating Tregs. In contrast, the capacity of moDCs to induce CD25 or FOXP3 was severely impaired by their in vitro infection with CCR5-utilizing virus. MoDC exposure to inactivated HIV was sufficient to impair FOXP3 induction. This DC defect was not dependent on IL-10, TGF-β or other soluble factors, but was due to preferential killing of Tregs by HIV-exposed/infected moDCs, through a caspase-dependent pathway. Importantly, similar results were obtained with circulating primary myeloid DCs. Upon infection in vitro, these mDCs also killed Treg through mechanisms at least partially caspase-dependent, leading to a significantly lower proportion of induced Tregs. Taken together, our data suggest that Treg induction may be defective when DCs are exposed to high levels of virus, such as during the acute phase of infection or in AIDS patients.     

Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background

The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4⁺ CD25^highFoxp3⁺ regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-γ chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.

Methodology/Principal Findings

CD4⁺CD25⁺ and CD4⁺CD25^neg T cells were isolated from PBMC of normal controls (n = 21) using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4⁺CD25^high and CD4⁺CD25^neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4⁺CD25^neg or CD8⁺CD25^neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4⁺CD25⁺ T cells in the presence of 1–100 nM RAPA (p<0.001). RAPA-expanded Treg were largely CD4⁺CD25^highFoxp3⁺ cells and were resistant to apoptosis, while CD4⁺CD25^neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4⁺CD25^neg cells. Activated Treg±RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway.

Conclusions/Significance

RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.     

IL-33 Induces IL-9 Production in Human CD4+ T Cells and Basophils

IL-33, an IL-1 family member and ligand for the IL-1 receptor-related protein ST2, has been associated with induction of Th2 cytokines such as IL-4, IL-5, and IL-13. Here, we report that IL-33 can initiate IL-9 protein secretion in vitro in human CD4+ T cells and basophils isolated from peripheral blood. TGF-β has been described as a critical factor for IL-9 induction in Th2 cells; however, we found that TGF-β also induces co-production of IL-9 in purified, naïve (>99%) CD4⁺CD45RA⁺CD45RO⁻CD25⁻ T cells differentiated towards a Th1 profile. Subsequently, it was demonstrated that TGF-β is important, although not an absolute requirement, for IL-9 production in CD4+ T cells. IL-9 production by purified (>95%) human basophils, cultured for 24 h with IL-3 or IL-33, was found, with a strong synergy between the two, likely to be explained by the IL-3 upregulated ST2 expression. Collectively, these data indicate that barrier functioning cells are important for the regulation of IL-9 production by immune cells in inflamed tissue.     

Infection with Feline Immunodeficiency Virus Directly Activates CD4+ CD25+ T Regulatory Cells

Lentivirus infection activates CD4⁺ CD25⁺ T regulatory (Treg) cells. Activation of Treg cells may be due to direct virus infection or chronic antigenic stimulation. Herein we demonstrate that in vitro feline immunodeficiency virus (FIV) infection, but not UV-inactivated virus, activates Treg cells as measured by immunosuppressive function and upregulation of GARP, FoxP3, and membrane-bound transforming growth factor β (TGF-β). These data demonstrate for the first time that AIDS lentiviruses infect and activate Treg cells, potentially contributing to immune dysfunction.