Regulatory T Cells Suppress Natural Killer Cells during Plasmid DNA Vaccination in Mice,Blunting the CD8+ T Cell Immune Response by the Cytokine TGFβ

Background

CD4⁺CD25⁺ regulatory T cells (Tregs) suppress adaptive T cell-mediated immune responses to self- and foreign-antigens. Tregs may also suppress early innate immune responses to vaccine antigens and might decrease vaccine efficacy. NK and NKT cells are the first responders after plasmid DNA vaccination and are found at the site of inoculation. Earlier reports demonstrated that NKT cells could improve plasmid DNA efficacy, a phenomenon not found for NK cells. In fact, it has been shown that under certain disease conditions, NK cells are suppressed by Tregs via their release of IL-10 and/or TGFβ. Therefore, we tested the hypothesis that NK cell function is suppressed by Tregs in the setting of plasmid DNA vaccination.

Methodology/Principal Findings

In this study we show that Tregs directly inhibit NK cell function during plasmid DNA vaccination by suppressing the potentially 10-fold, NK cell-mediated, augmentation of plasmid DNA antigen-specific CD8⁺ T cells. We found that this phenomenon is dependent on the secretion of cytokine TGFβ by Tregs, and independent of IL-10.

Conclusions

Our data indicate a crucial function for Tregs in blocking plasmid DNA vaccine-elicited immune responses, revealing potentially novel strategies for improving the efficiency of plasmid DNA vaccines including chemical- or antibody-induced localized blockage of Treg-mediated suppression of NK cells at the site of plasmid DNA vaccine inoculation.     

Effects of natalizumab treatment on Foxp3+ T regulatory cells

Background

Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients.

Methodology

A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.

Principal Findings

Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25^highCD127^lowFoxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.

Conclusions

We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.     

CD39+ Regulatory T cells suppress generation and differentiation of Th17 cells in human malignant pleural effusion via a LAP-dependent mechanism

Background

Both regulatory T cells (Tregs) and T helper IL-17-producing cells (Th17 cells) have been found to be involved in human malignancies, however, the possible implication of Tregs in regulating generation and differentiation of Th17 cells in malignant pleural effusion remains to be elucidated.

Methods

The numbers of both CD39⁺Tregs and Th17 cells in malignant pleural effusion and peripheral blood from patients with lung cancer were determined by flow cytometry. The regulation and mechanism of Tregs on generation and differentiation of Th17 cells were explored.

Results

Both CD39⁺Tregs and Th17 cells were increased in malignant pleural effusion when compared with blood, and the numbers of CD39⁺Tregs were correlated negatively with those of Th17 cells. It was also noted that high levels of IL-1β, IL-6, and TGF-β1 could be observed in malignant pleural effusion when compared the corresponding serum, and that pleural CD39⁺Tregs could express latency-associated peptide on their surface. When naïve CD4⁺ T cells were cocultured with CD39⁺Tregs, Th17 cell numbers decreased as CD39⁺Treg numbers increased, addition of the anti-latency-associated peptide mAb to the coculture reverted the inhibitory effect exerted by CD39⁺Tregs.

Conclusions

Therefore, the above results indicate that CD39⁺Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.     

Trafficking of high avidity HER-2/neu-specific T cells into HER-2/neu-expressing tumors after depletion of effector/memory-like regulatory T cells

Background

Cancer vaccines are designed to activate and enhance cancer-antigen-targeted T cells that are suppressed through multiple mechanisms of immune tolerance in cancer-bearing hosts. T regulatory cell (Treg) suppression of tumor-specific T cells is one barrier to effective immunization. A second mechanism is the deletion of high avidity tumor-specific T cells, which leaves a less effective low avidity tumor specific T cell repertoire available for activation by vaccines. Treg depleting agents including low dose cyclophosphamide (Cy) and antibodies that deplete CD25-expressing Tregs have been used with limited success to enhance the potency of tumor-specific vaccines. In addition, few studies have evaluated mechanisms that activate low avidity cancer antigen-specific T cells. Therefore, we developed high and low avidity HER-2/neu-specific TCR transgenic mouse colonies specific for the same HER-2/neu epitope to define the tolerance mechanisms that specifically affect high versus low avidity tumor-specific T cells.

Methodology/Principal Findings

High and low avidity CD8⁺ T cell receptor (TCR) transgenic mice specific for the breast cancer antigen HER-2/neu (neu) were developed to provide a purified source of naïve, tumor-specific T cells that can be used to study tolerance mechanisms. Adoptive transfer studies into tolerant FVB/N-derived HER-2/neu transgenic (neu-N) mice demonstrated that high avidity, but not low avidity, neu-specific T cells are inhibited by Tregs as the dominant tolerizing mechanism. High avidity T cells persisted, produced IFNγ, trafficked into tumors, and lysed tumors after adoptive transfer into mice treated with a neu-specific vaccine and low dose Cy to deplete Tregs. Analysis of Treg subsets revealed a Cy-sensitive CD4⁺Foxp3⁺CD25^low tumor-seeking migratory phenotype, characteristic of effector/memory Tregs, and capable of high avidity T cell suppression.

Conclusion/Significance

Depletion of CD25^low Tregs allows activation of tumor-clearing high avidity T cells. Thus, the development of agents that specifically deplete Treg subsets should translate into more effective immunotherapies while avoiding autoimmunity.     

Efficient Isolation of Mouse Liver NKT Cells by Perfusion

Background

NKT cell is a population of unconventional T cells that mediate both innate and adaptive T cell responses. Since NKT cells are most abundant in the liver, much of NKT biology has been learnt from studies of NKT cells isolated from liver. This is a cumbersome procedure with variations in cell yield.

Results

Based on recent evidence that NKT cells reside in liver vascular compartment, we developed a simple method to isolate NKT cells by perfusion with PBS-containing 10 mM of EDTA. The number and cell surface phenotype of liver NKT cells recovered by perfusion and by the traditional method were comparable. The yield of other lymphocytes was also comparable.

Conclusion/Significance

Our data demonstrated that liver lymphocytes can be efficiently isolated by simple perfusion. These data provide a convenient method to isolate liver lymphocyte while preserving liver tissue for other analysis.     

Impaired survival of regulatory T cells in pulmonary sarcoidosis

Background

Impaired regulatory T cell (Treg) function is thought to contribute to ongoing inflammatory responses in sarcoidosis, but underlying mechanisms remain unclear. Moreover, it is not known if increased apoptotic susceptibility of Tregs may contribute to an impaired immunosuppressive function in sarcoidosis. Therefore, the aim of this study is to analyze proportions, phenotype, survival, and apoptotic susceptibility of Tregs in sarcoidosis.

Methods

Patients with pulmonary sarcoidosis (n = 58) were included at time of diagnosis. Tregs were analyzed in broncho-alveolar lavage fluid and peripheral blood of patients and healthy controls (HC).

Results

In sarcoidosis patients no evidence was found for a relative deficit of Tregs, neither locally nor systemically. Rather, increased proportions of circulating Tregs were observed, most prominently in patients developing chronic disease. Sarcoidosis circulating Tregs displayed adequate expression of FoxP3, CD25 and CTLA4. Remarkably, in sarcoidosis enhanced CD95 expression on circulating activated CD45RO⁺ Tregs was observed compared with HC, and proportions of these cells were significantly increased. Specifically sarcoidosis Tregs - but not Th cells - showed impaired survival compared with HC. Finally, CD95L-mediated apoptosis was enhanced in sarcoidosis Tregs.

Conclusion

In untreated patients with active pulmonary sarcoidosis, Tregs show impaired survival and enhanced apoptotic susceptibility towards CD95L. Increased apoptosis likely contributes to the insufficient immunosuppressive function of sarcoidosis Tregs. Further research into this field will help determine whether improvement of Treg survival holds a promising new therapeutic approach for chronic sarcoidosis patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0265-8) contains supplementary material, which is available to authorized users.     

Regulatory T cells enhance susceptibility to experimental trypanosoma congolense infection independent of mouse genetic background

Background

BALB/c mice are highly susceptible while C57BL/6 are relatively resistant to experimental Trypanosoma congolense infection. Although regulatory T cells (Tregs) have been shown to regulate the pathogenesis of experimental T. congolense infection, their exact role remains controversial. We wished to determine whether Tregs contribute to distinct phenotypic outcomes in BALB/c and C57BL/6 mice and if so how they operate with respect to control of parasitemia and production of disease-exacerbating proinflammatory cytokines.

Methodology/Findings

BALB/c and C57BL/6 mice were infected intraperitoneally (i.p) with 10³ T. congolense clone TC13 and both the kinetics of Tregs expansion and intracellular cytokine profiles in the spleens and livers were monitored directly ex vivo by flow cytometry. In some experiments, mice were injected with anti-CD25 mAb prior or post T. congolense infection or adoptively (by intravenous route) given highly enriched naïve CD25⁺ T lymphocytes prior to T. congolense infection and the inflammatory cytokine/chemokine levels and survival were monitored. In contrast to a transient and non significant increase in the percentages and absolute numbers of CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) in C57BL/6 mouse spleens and livers, a significant increase in the percentage and absolute numbers of Tregs was observed in spleens of infected BALB/c mice. Ablation or increasing the number of CD25⁺ cells in the relatively resistant C57BL/6 mice by anti-CD25 mAb treatment or by adoptive transfer of CD25⁺ T cells, respectively, ameliorates or exacerbates parasitemia and production of proinflammatory cytokines.

Conclusion

Collectively, our results show that regulatory T cells contribute to susceptibility in experimental murine trypanosomiasis in both the highly susceptible BALB/c and relatively resistant C57BL/6 mice.     

T cells from Programmed Death-1 deficient mice respond poorly to Mycobacterium tuberculosis infection

Background

Programmed Death-1 (PD-1; CD279) receptor molecule is widely believed to be a negative regulator predominantly expressed by exhausted/activated mouse T cells. Upon interaction with its ligands, PD-L1 and PD-L2, PD-1 inhibits activation of T cells and cytokine production, which has been documented in various viral and fungal infections as well as in vitro studies. Therefore, inhibition of T cell responses by PD-1 resulted in disease resistance in a variety of mouse infection models studied heretofore.

Methodology/Principal Findings

Here, we report that PD-1 deficient (PD-1^−/−) mice infected with Mycobacterium tuberculosis (M. tb) H37Rv by the aerosol route have increased susceptibility as compared with their wild type littermates. Surprisingly, M. tb antigen-specific T cell proliferation was dramatically reduced in PD-1 deficient animals compared with wild-type littermates, and this was due to increased numbers of regulatory T cells (Tregs) and recruitment of mesenchymal stem cells. Furthermore, PD-1^−/− mice exhibited decreases in the autophagy-induced LC3-B marker protein in macrophages.

Conclusions/Significance

Our findings suggest that PD-1 does not play an inhibitory role during M. tb infection and instead promotes mycobacterial clearance in mice.     

iNKT Cells Control Mouse Spontaneous Carcinoma Independently of Tumor-Specific Cytotoxic T Cells

Background

CD1d-restricted invariant NKT (iNKT) cells are a subset of T lymphocytes endowed with innate effector functions that aid in the establishment of adaptive T and B cell immune responses. iNKT cells have been shown to play a spontaneous protective role against experimental tumors. Yet, the interplay between iNKT and tumor-specific T cells in cancer immune surveillance/editing has never been addressed. The transgenic adenocarcinoma of the mouse prostate (TRAMP) is a realistic model of spontaneous oncogenesis, in which the tumor-specific cytotoxic T cell (CTL) response undergoes full tolerance upon disease progression.

Principal Findings

We report here that lack of iNKT cells in TRAMP mice resulted in the appearance of more precocious and aggressive tumors that significantly reduced animal survival. TRAMP mice bearing or lacking iNKT cells responded similarly to a tumor-specific vaccination and developed tolerance to a tumor-associated antigen at comparable rate.

Conclusions

Hence, our data argue for a critical role of iNKT cells in the immune surveillance of carcinoma that is independent of tumor-specific CTL.     

Tunable Chemokine Production by Antigen Presenting Dendritic Cells in Response to Changes in Regulatory T Cell Frequency in Mouse Reactive Lymph Nodes

Background

Although evidence exists that regulatory T cells (Tregs) can suppress the effector phase of immune responses, it is clear that their major role is in suppressing T cell priming in secondary lymphoid organs. Recent experiments using two photon laser microscopy indicate that dendritic cells (DCs) are central to Treg cell function and that the in vivo mechanisms of T cell regulation are more complex than those described in vitro.

Principal Findings

Here we have sought to determine whether and how modulation of Treg numbers modifies the lymph node (LN) microenvironment. We found that pro-inflammatory chemokines—CCL2 (MCP-1) and CCL3 (MIP-la)—are secreted in the LN early (24 h) after T cell activation, that this secretion is dependent on antigen-specific DC–T cell interactions, and that it was inversely related to the frequency of Tregs specific for the same antigen. Furthermore, we demonstrate that Tregs modify the chemoattractant properties of antigen-presenting DCs, which, as the frequency of Tregs increases, fail to produce CCL2 and CCL3 and to attract antigen-specific T cells.

Conclusions

These results substantiate a major role of Tregs in LN patterning during antigen-specific immune responses.     

CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis

Background

Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.

Methodology/Principal Findings

In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.

Conclusions/Significance

The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.     

Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis

Introduction

Regulatory T (Treg) cells play a crucial role in preventing autoimmune diseases and are an ideal target for the development of therapies designed to suppress inflammation in an antigen-specific manner. Type 1 regulatory T (Tr1) cells are defined by their capacity to produce high levels of interleukin 10 (IL-10), which contributes to their ability to suppress pathological immune responses in several settings. The aim of this study was to evaluate the therapeutic potential of collagen type II–specific Tr1 (Col-Treg) cells in two models of rheumatoid arthritis (RA) in mice.

Methods

Col-Treg clones were isolated and expanded from collagen-specific TCR transgenic mice. Their cytokine secretion profile and phenotype characterization were studied. The therapeutic potential of Col-Treg cells was evaluated after adoptive transfer in collagen-antibody– and collagen-induced arthritis models. The in vivo suppressive mechanism of Col-Treg clones on effector T-cell proliferation was also investigated.

Results

Col-Treg clones are characterized by their specific cytokine profile (IL-10^highIL-4^negIFN-γ^int) and mediate contact-independent immune suppression. They also share with natural Tregs high expression of GITR, CD39 and granzyme B. A single infusion of Col-Treg cells reduced the incidence and clinical symptoms of arthritis in both preventive and curative settings, with a significant impact on collagen type II antibodies. Importantly, injection of antigen-specific Tr1 cells decreased the proliferation of antigen-specific effector T cells in vivo significantly.

Conclusions

Our results demonstrate the therapeutic potential of Col-Treg cells in two models of RA, providing evidence that Col-Treg could be an efficient cell-based therapy for RA patients whose disease is refractory to current treatments.     

Y27, a novel derivative of 4-hydroxyquinoline-3-formamide,prevents the development of murine systemic lupus erythematosus-like diseases in MRL/lpr autoimmune mice and BDF1 hybrid mice

Introduction

Naturally occurring CD4⁺CD25⁺ regulatory T (Treg) cells are central to the maintenance of peripheral tolerance. Impaired activity and/or a lower frequency of these cells lead to systemic lupus erythematosus (SLE). Manipulating the number or activity of Treg cells is to be a promising strategy in treating it and other autoimmune diseases. We have examined the effects of Y27, a novel derivative of 4-hydroxyquinoline-3-formamide, on SLE-like symptoms in MRL/lpr autoimmune mice and BDF1 hybrid mice. Whether the beneficial effect of Y27 involves modulation of CD4⁺CD25⁺ Treg cells has also been investigated.

Methods

Female MRL/lpr mice that spontaneously develop lupus were treated orally by gavage with Y27 for 10 weeks, starting at 10 weeks of age. BDF1 mice developed a chronic graft-versus-host disease (GVHD) by two weekly intravenous injections of parental female DBA/2 splenic lymphocytes, characterized by immunocomplex-mediated glomerulonephritis resembling SLE. Y27 was administered to chronic GVHD mice for 12 weeks. Nephritic symptoms were monitored and the percentage of CD4⁺CD25⁺FoxP3⁺ Treg peripheral blood leukocyte was detected with mouse regulatory T cell staining kit by flowcytometry. Purified CD4⁺CD25⁺ Tregs were assessed for immune suppressive activity using the mixed lymphocyte reaction.

Results

The life-span of MRL/lpr mice treated with Y27 for 10 weeks was significantly prolonged, proteinuria and renal lesion severity were ameliorated, and blood urea nitrogen, triglyceride and serum anti-double-stranded DNA antibodies were decreased. Similar results were found in chronic GVHD mice. Administration of Y27 had little impact on percentage of the peripheral blood lymphocyte CD4⁺CD25⁺Foxp3⁺ Treg cells in both groups of mice. In contrast, the suppressive capacity of CD4⁺CD25⁺ Treg cells in splenocytes was markedly augmented in Y27-treated mice ex vivo.

Conclusions

Experimental evidence of the protect effects of Y27 against autoimmune nephritis has been shown. The mechanism may involve enhancement of the suppressive capacity of CD4⁺CD25⁺ Treg cells.     

HCV+ Hepatocytes Induce Human Regulatory CD4+ T Cells through the Production of TGF-β

Background

Hepatitis C Virus (HCV) is remarkably efficient at establishing persistent infection and is associated with the development of chronic liver disease. Impaired T cell responses facilitate and maintain persistent HCV infection. Importantly, CD4⁺ regulatory T cells (Tregs) act by dampening antiviral T cell responses in HCV infection. The mechanism for induction and/or expansion of Tregs in HCV is unknown.

Methodology/Principal Findings

HCV-expressing hepatocytes were used to determine if hepatocytes are able to induce Tregs. The infected liver environment was modeled by establishing the co-culture of the human hepatoma cell line, Huh7.5, containing the full-length genome of HCV genotype 1a (Huh7.5-FL) with activated CD4⁺ T cells. The production of IFN-γ was diminished following co-culture with Huh7.5-FL as compared to controls. Notably, CD4⁺ T cells in contact with Huh7.5-FL expressed an increased level of the Treg markers, CD25, Foxp3, CTLA-4 and LAP, and were able to suppress the proliferation of effector T cells. Importantly, HCV⁺ hepatocytes upregulated the production of TGF-β and blockade of TGF-β abrogated Treg phenotype and function.

Conclusions/Significance

These results demonstrate that HCV infected hepatocytes are capable of directly inducing Tregs development and may contribute to impaired host T cell responses.     

In Vivo Dynamical Interactions between CD4 Tregs,CD8 Tregs and CD4+CD25? Cells in Mice

Background

Regulatory T cells (Tregs) were shown to be central in maintaining immunological homeostasis and preventing the development of autoimmune diseases. Several subsets of Tregs have been identified to date; however, the dynamics of the interactions between these subsets, and their implications on their regulatory functions are yet to be elucidated.

Methodology/Principal Findings

We employed a combination of mathematical modeling and frequent in vivo measurements of several T cell subsets. Healthy BALB/c mice received a single injection of either hCDR1 - a tolerogenic peptide previously shown to induce Tregs, a control peptide or vehicle alone, and were monitored for 16 days. During this period, splenocytes from the treated mice were analyzed for the levels of CD4, CD25, CD8, CD28 and Foxp3. The collected data were then fitted to mathematical models, in order to test competing hypotheses regarding the interactions between the followed T cell subsets. In all 3 treatment groups, a significant, lasting, non-random perturbation of the immune system could be observed. Our analysis predicted the emergence of functional CD4 Tregs based on inverse oscillations of the latter and CD4⁺CD25⁻ cells. Furthermore, CD4 Tregs seemed to require a sufficiently high level of CD8 Tregs in order to become functional, while conversion was unlikely to be their major source. Our results indicated in addition that Foxp3 is not a sufficient marker for regulatory activity.

Conclusions/Significance

In this work, we unraveled the dynamics of the interplay between CD4, CD8 Tregs and effector T cells, using, for the first time, a mathematical-mechanistic perspective in the analysis of Treg kinetics. Furthermore, the results obtained from this interdisciplinary approach supported the notion that CD4 Tregs need to interact with CD8 Tregs in order to become functional. Finally, we generated predictions regarding the time-dependent function of Tregs, which can be further tested empirically in future work.     

Ncf1 (p47phox) is essential for direct regulatory T cell mediated suppression of CD4+ effector T cells

Background

Multiple mechanisms have been advanced to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated suppression of CD4+ effector T cells (Teffs) but none appear to completely explain suppression. Previous data indicates that Tregs may affect the microenvironment redox state. Given the inherent redox sensitivity of T cells, we tested the hypothesis that oxidants may mediate the direct suppression of Teffs by Tregs.

Methodology/Principal Findings

Tregs and Teffs were isolated from the spleens of wild type (WT) C57BL/6 mice or Ncf1(p47phox)-deficient C57BL/6 mice which lack NADPH oxidase function. Teffs were labeled with CFSE and co-cultured with unlabeled Tregs at varying Treg:Teff ratios in the presence of anti-CD3/CD28 coated beads for 3 days in suppression assays. Treg-mediated suppression was quantified by flow cytometric analysis of CFSE dilution in Teffs. The presence of the antioxidants n-acetylcysteine (NAC) or 2-mercaptoethanol or inhibitors of NADPH oxidase (diphenyleneiodonium and VAS-2870) resulted in reduced WT Treg-mediated suppression. The observed suppression was in part dependent upon TGFβ as it was partially blocked with neutralizing antibodies. The suppression of Teff proliferation induced by exogenous TGFβ treatment could be overcome with NAC. Ncf1-deficient Teff were slightly but significantly less sensitive than WT Teff to suppression by exogenous TGFβ. Ncf1-deficient Tregs suppressed Ncf1-deficient Teff very poorly compared to wild type controls. There was partial but incomplete reconstitution of suppression in assays with WT Tregs and Ncf1-deficient Teff.

Conclusions/Significance

We present evidence that NADPH oxidase derived ROS plays a role in the direct Treg mediated suppression of CD4+ effector T cells in a process that is blocked by thiol-containing antioxidants, NADPH oxidase inhibitors or a lack of Ncf1 expression in Tregs and Teffs. Oxidants may represent a potential new target for therapeutic modulation of Treg function.     

Resting regulatory CD4 T cells: a site of HIV persistence in patients on long-term effective antiretroviral therapy

Background

In HIV-infected patients on long-term HAART, virus persistence in resting long-lived CD4 T cells is a major barrier to curing the infection. Cell quiescence, by favouring HIV latency, reduces the risk of recognition and cell destruction by cytotoxic lymphocytes. Several cell-activation-based approaches have been proposed to disrupt cell quiescence and then virus latency, but these approaches have not eradicated the virus. CD4⁺CD25⁺ regulatory T cells (Tregs) are a CD4⁺ T-cell subset with particular activation properties. We investigated the role of these cells in virus persistence in patients on long-term HAART.

Methodology/Principal Findings

We found evidence of infection of resting Tregs (HLADR⁻CD69⁻CD25^hiFoxP3⁺CD4⁺ T cells) purified from patients on prolonged HAART. HIV DNA harbouring cells appear more abundant in the Treg subset than in non-Tregs. The half-life of the Treg reservoir was estimated at 20 months. Since Tregs from patients on prolonged HAART showed hyporesponsiveness to cell activation and inhibition of HIV-specific cytotoxic T lymphocyte-related functions upon activation, therapeutics targeting cell quiescence to induce virus expression may not be appropriate for purging the Treg reservoir.

Conclusions

Our results identify Tregs as a particular compartment within the latent reservoir that may require a specific approach for its purging.     

Reduced Cellular Susceptibility to In Vitro HIV Infection Is Associated with CD4+ T Cell Quiescence

Background

HIV preferentially establishes productive infection in activated CD4+ T cells. Since proportions of activated CD4+ T cells vary between individuals, this study aimed to determine if individuals with a greater proportion of activated CD4+ T cells would be more susceptible to in vitro HIV infection.

Methodology/Principal Findings

Unstimulated peripheral blood mononuclear cells (PBMC) from various donors were inoculated with HIV_ML1956 in vitro. HIV replication was evaluated by HIV p24 ELISA of culture supernatants and intracellular staining for HIV p24, which was detected by flow cytometry. Baseline T cell phenotypes and infected cell phenotypes were also evaluated by flow cytometry. Ex vivo phenotyping at the time of blood draw showed that elevated T cell activation and reduced Tregs were associated with increased cellular susceptibility to in vitro infection. Furthermore, the infected CD4+ T cell population was enriched for activated cells.

Conclusion/Significance

These data suggest that CD4+ T cell quiescence provides an environment less conducive to the establishment of HIV infection by limiting the pool of activated target cells.