Regulatory T Cell Suppression of Gag-Specific CD8+ T Cell Polyfunctional Response After Therapeutic Vaccination of HIV-1-Infected Patients on ART

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4⁺ T cells (Treg), thereby masking enhancement of HIV-1-specific CD8⁺ T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4⁺CD25^hiFOXP3⁺ Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8⁺ T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine–pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8⁺ T cell vaccine response by enzyme linked immunosorbent assay for interferon γ production. Although there was no significant change in CD8⁺ T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8⁺ T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection.     

Commensal-Induced Regulatory T Cells Mediate Protection against Pathogen-Stimulated NF-κB Activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-κB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-κB activation was quantified using biophotonic imaging. CD4⁺CD25⁺Foxp3⁺ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4⁺ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-κB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis–fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4⁺CD25⁺Foxp3⁺ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4⁺CD25⁺ T cells transferred the NF-κB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-κB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.     

Regulatory T Cell Suppressive Potency Dictates the Balance between Bacterial Proliferation and Clearance during Persistent Salmonella Infection

The pathogenesis of persistent infection is dictated by the balance between opposing immune activation and suppression signals. Herein, virulent Salmonella was used to explore the role and potential importance of Foxp3-expressing regulatory T cells in dictating the natural progression of persistent bacterial infection. Two distinct phases of persistent Salmonella infection are identified. In the first 3–4 weeks after infection, progressively increasing bacterial burden was associated with delayed effector T cell activation. Reciprocally, at later time points after infection, reductions in bacterial burden were associated with robust effector T cell activation. Using Foxp3 ^GFP reporter mice for ex vivo isolation of regulatory T cells, we demonstrate that the dichotomy in infection tempo between early and late time points is directly paralleled by drastic changes in Foxp3⁺ Treg suppressive potency. In complementary experiments using Foxp3 ^DTR mice, the significance of these shifts in Treg suppressive potency on infection outcome was verified by enumerating the relative impacts of regulatory T cell ablation on bacterial burden and effector T cell activation at early and late time points during persistent Salmonella infection. Moreover, Treg expression of CTLA-4 directly paralleled changes in suppressive potency, and the relative effects of Treg ablation could be largely recapitulated by CTLA-4 in vivo blockade. Together, these results demonstrate that dynamic regulation of Treg suppressive potency dictates the course of persistent bacterial infection.     

Regulatory Function of a Novel Population of Mouse Autoantigen-Specific Foxp3? Regulatory T Cells Depends on IFN-γ, NO,and Contact with Target Cells

Background

Both naturally arising Foxp3⁺ and antigen-induced Foxp3⁻ regulatory T cells (Treg) play a critical role in regulating immune responses, as well as in preventing autoimmune diseases and graft rejection. It is known that antigen-specific Treg are more potent than polyclonal Treg in suppressing pathogenic immune responses that cause autoimmunity and inflammation. However, difficulty in identifying and isolating a sufficient number of antigen-specific Treg has limited their use in research to elucidate the mechanisms underlying their regulatory function and their potential role in therapy.

Methodology/Principal Findings

Using a novel class II MHC tetramer, we have isolated a population of CD4⁺ Foxp3⁻ T cells specific for the autoantigen glutamic acid decarboxylase p286–300 peptide (NR286 T cells) from diabetes-resistant non-obese resistant (NOR) mice. These Foxp3⁻ NR286 T cells functioned as Treg that were able to suppress target T cell proliferation in vitro and inhibit type 1 diabetes in animals. Unexpected results from mechanistic studies in vitro showed that their regulatory function was dependent on not only IFN-gamma and nitric oxide, but also on cell contact with target cells. In addition, separating NR286 Treg from target T cells in transwell assays abolished both production of NO and suppression of target T cells, regardless of whether IFN-γ was produced in cell cultures. Therefore, production of NO, not IFN-gamma, was cell contact dependent, suggesting that NO may function downstream of IFN-gamma in mediating regulatory function of NR286 Treg.

Conclusions/Significance

These studies identified a unique population of autoantigen-specific Foxp3⁻ Treg that can exert their regulatory function dependent on not only IFN-γ and NO but also cell contact with target cells.     

A Cancer Vaccine Induces Expansion of NY-ESO-1-Specific Regulatory T Cells in Patients with Advanced Melanoma

Cancer vaccines are designed to expand tumor antigen-specific T cells with effector function. However, they may also inadvertently expand regulatory T cells (Treg), which could seriously hamper clinical efficacy. To address this possibility, we developed a novel assay to detect antigen-specific Treg based on down-regulation of surface CD3 following TCR engagement, and used this approach to screen for Treg specific to the NY-ESO-1 tumor antigen in melanoma patients treated with the NY-ESO-1/ISCOMATRIX^TM cancer vaccine. All patients tested had Treg (CD25^bright FoxP3⁺ CD127^neg) specific for at least one NY-ESO-1 epitope in the blood. Strikingly, comparison with pre-treatment samples revealed that many of these responses were induced or boosted by vaccination. The most frequently detected response was toward the HLA-DP4-restricted NY-ESO-1_157–170 epitope, which is also recognized by effector T cells. Notably, functional Treg specific for an HLA-DR-restricted epitope within the NY-ESO-1_115–132 peptide were also identified at high frequency in tumor tissue, suggesting that NY-ESO-1-specific Treg may suppress local anti-tumor immune responses. Together, our data provide compelling evidence for the ability of a cancer vaccine to expand tumor antigen-specific Treg in the setting of advanced cancer, a finding which should be given serious consideration in the design of future cancer vaccine clinical trials.     

P21 activated kinase‐1 (PAK1) in macrophages is required for promotion of Th17 cell response during helminth infection

CD4⁺T cells differentiate into distinct functional effector and inhibitory subsets are facilitated by distinct cytokine cues present at the time of antigen recognition. Maintaining a balance between T helper 17 (Th17) and regulatory T (Treg) cells are critical for the control of the immunopathogenesis of liver diseases. Here, by using the mouse model of helminth Schistosoma japonicum (S japonicum) infection, we show that the hepatic mRNA levels of P21‐activated kinase 1 (PAK1), a key regulator of the actin cytoskeleton, adhesion and cell motility, are significantly increased and associated with the development of liver pathology during S japonicum infection. In addition, PAK1‐deficient mice are prone to suppression of Th17 cell responses but increased Treg cells. Furthermore, PAK1 enhances macrophage activation through promoting IRF1 nuclear translocation in an NF‐κB‐dependent pathway, resulting in promoting Th17 cell differentiation through inducing IL‐6 production. These findings highlight the importance of PAK1 in macrophages fate determination and suggest that PAK1/IRF1 axis‐dependent immunomodulation can ameliorate certain T cell–based immune pathologies.     

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.     

Regulatory T cells in acute and chronic human Chikungunya infection

Chikungunya virus (CHIKV) infection generates strong immune responses that are associated with the disease pathophysiology. Regulatory T cells (Treg-cluster of differentiation (CD)-4⁺CD25^highforkhead box P3 (FOXP3⁺)) are essential for the induction and maintenance of peripheral tolerance. Thus, they play key roles in determining the patient prognosis by preventing excessive immune responses via different suppression immune mechanisms. However, the regulatory mechanisms involved in human CHIKV infection are still poorly understood. Here, we characterize for the first time the Treg cell molecule-associated-mechanism during acute and chronic human Chikungunya disease. Here, we assessed the Treg cell population and molecule-associated mechanism in the peripheral blood samples of acute and chronic patients with Chikungunya. Our results indicate that CHIKV infection is associated with reduced frequency of Tregs, along with the impaired expression and production of Treg functional markers, including CD39, CD73, perforin, granzyme, programmed death 1 (PD-1), cytotoxic T lymphocyte antigen (CTLA)-4, and transforming growth factor (TGF)-β. This observation suggests that Treg cells possess the poor regulatory capacity in both acute and chronic phases of the disease. Taken together, these data provide significant evidence that the imbalanced response of Treg cells plays an essential role in establishing the pathogenesis of Chikungunya.     

Role of Lymphocyte Activation Gene-3 (Lag-3) in Conventional and Regulatory T Cell Function in Allogeneic Transplantation

Lag-3 has emerged as an important molecule in T cell biology. We investigated the role of Lag-3 in conventional T cell (Tcon) and regulatory T cell (Treg) function in murine GVHD with the hypothesis that Lag-3 engagement diminishes alloreactive T cell responses after bone marrow transplantation. We demonstrate that Lag-3 deficient Tcon (Lag-3^−/− Tcon) induce significantly more severe GVHD than wild type (WT) Tcon and that the absence of Lag-3 on CD4 but not CD8 T cells is responsible for exacerbating GVHD. Lag-3^−/− Tcon exhibited increased activation and proliferation as indicated by CFSE and bioluminescence imaging analyses and higher levels of activation markers such as CD69, CD107a, granzyme B, and Ki-67 as well as production of IL-10 and IFN-g early after transplantation. Lag-3^−/− Tcon were less responsive to suppression by WT Treg as compared to WT Tcon. The absence of Lag-3, however, did not impair Treg function as both Lag-3^−/− and WT Treg equally suppress the proliferation of Tcon in vitro and in vivo and protect against GVHD. Further, we demonstrate that allogeneic Treg acquire recipient MHC class II molecules through a process termed trogocytosis. As MHC class II is a ligand for Lag-3, we propose a novel suppression mechanism employed by Treg involving the acquisition of host MHC-II followed by the engagement of Lag-3 on T cells. These studies demonstrate for the first time the biologic function of Lag-3 expression on conventional and regulatory T cells in GVHD and identify Lag-3 as an important regulatory molecule involved in alloreactive T cell proliferation and activation after bone marrow transplantation.     

Early Growth Response Genes 2 and 3 Regulate the Expression of Bcl6 and Differentiation of T Follicular Helper Cells

T follicular helper (Tfh) cells support differentiation of B cells to plasma cells and high affinity antibody production in germinal centers (GCs), and Tfh differentiation requires the function of B cell lymphoma 6 (BCL6). We have now discovered that early growth response gene 2 (EGR2) and EGR3 directly regulate the expression of Bcl6 in Tfh cells, which is required for their function in regulation of GC formation. In the absence of EGR2 and -3, the expression of BCL6 in Tfh cells is defective, leading to impaired differentiation of Tfh cells, resulting in a failure to form GCs following virus infection and defects in production of antiviral antibodies. Enforced expression of BCL6 in EGR2/3-deficient CD4 T cells partially restored Tfh differentiation and GC formation in response to virus infection. Our findings demonstrate a novel function of EGR2/3 that is important for Tfh cell development and Tfh cell-mediated B cell immune responses.     

Foxp3+ Regulatory T Cells among Tuberculosis Patients: Impact on Prognosis and Restoration of Antigen Specific IFN-γ Producing T Cells

CD4⁺CD25⁺Foxp3⁺ Regulatory T cells (Treg) and programmed death-1 (PD-1) molecules have emerged as pivotal players in immune suppression of chronic diseases. However, their impact on the disease severity, therapeutic response and restoration of immune response in human tuberculosis remains unclear. Here, we describe the possible role of Treg cells, their M. tuberculosis driven expansion and contribution of PD-1 pathway to the suppressive function of Treg cells among pulmonary tuberculosis (PTB) patients. Multicolor flow cytometry, cell culture, cells sorting and ELISA were employed to execute the study. Our results showed significant increase in frequency of antigen-reactive Treg cells, which gradually declined during successful therapy and paralleled with decline of M. tuberculosis–specific IL-10 along with elevation of IFN-γ production, and raising the IFN-γ/IL-4 ratio. Interestingly, persistence of Treg cells tightly correlated with MDR tuberculosis. Also, we show that blocking PD-1/PD-L1 pathway abrogates Treg-mediated suppression, suggesting that the PD-1/PD-L1 pathway is required for Treg-mediated suppression of the antigen-specific T cells. Treg cells possibly play a role in dampening the effector immune response and abrogating PD-1 pathway on Treg cells significantly rescued protective T cell response, suggesting its importance in immune restoration among tuberculosis patients.     

Polyclonal B Cell Differentiation and Loss of Gastrointestinal Tract Germinal Centers in the Earliest Stages of HIV-1 Infection

Background

The antibody response to HIV-1 does not appear in the plasma until approximately 2–5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1–specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4⁺ T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.

Methods and Findings

In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT) B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI) included not only HIV-1–specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1–induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer''s patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis.

Conclusions

Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1–induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors'' Summary     

An Early Reduction in Treg Cells Correlates with Enhanced Local Inflammation in Cutaneous Leishmaniasis in CCR6-Deficient Mice

Resistance to Leishmania major infection is dependent on the development of a cell-mediated Th1 immune response in resistant C57BL/6 mice whereas Th2-prone BALB/c mice develop non-healing lesions after infection. The chemokine receptor CCR6 is shared by anti-inflammatory regulatory T cells and pro-inflammatory Th17 cells. In a recent study we showed that C57BL/6 mice deficient in CCR6 exhibited enhanced footpad swelling and impaired T helper cell migration indicated by reduced recruitment of total T helper cells into the skin after infection and a reduced delayed type hypersensitivity reaction. Based on these findings we tested whether the lack of CCR6 alters Treg or Th17 cell responses during the course of Leishmania major infection. When we analyzed T cell subsets in the lymph nodes of CCR6-deficient mice, Th17 cell numbers were not different. However, reduced numbers of Treg cells paralleled with a stronger IFNγ response. Furthermore, the early increase in IFNγ-producing cells correlated with increased local tissue inflammation at later time points. Our data indicate an important role of CCR6 for Treg cells and a redundant role for Th17 cells in a Th1 cell-driven anti-parasitic immune response against Leishmania major parasites in resistant C57BL/6 mice.     

In Situ Immune Response in Human Chromoblastomycosis – A Possible Role for Regulatory and Th17 T Cells

Background

Chromoblastomycosis is a chronic fungal infection that affects skin and subcutaneous tissue. Lesions can be classified in tumorous, verrucous, cicatricial and plaque type. The cellular immune response in the severe form of the disease seems to correlate with a Th2 pattern of cytokines. The humoral immune response also seems to play a role. We intended to explore the populations of regulatory T cells and the Th17 pattern.

Methodology

Twenty-three biopsies of verrucous form were obtained from patients with clinical, culture and histopathological diagnostic of chromoblastomycosis, without treatment. It was performed an immunohistochemistry method to detect Foxp3, CD25, TGF-β, IL-6, IL-17 and IL-23.

Principal findings

IL-17 was the only cytokine with high expression in CBM when compared to normal skin. The expression of Treg cells, TGF- β, IL-6 and IL-23 were similar to normal skin.

Conclusions/Significance

The constitution of a local immune response with high expression of IL-17 and low expression of other cytokines could be at least in part, an attempt to help the immune system against fungal infection. On the other hand, high levels of local immune response mediated by Th17 profile could overcome the role of Treg cells. The inefficient immunomodulation as a consequence of the unbalance by Treg/Th17 cells seems to corroborate with the less effective immune response against fungi.