FOXP3 Expression Is Upregulated in CD4+T Cells in Progressive HIV-1 Infection and Is a Marker of Disease Severity

Background

Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection.

Methodology

FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution.

Principal Findings

HIV infected individuals had significantly higher frequencies of CD4⁺FOXP3⁺ T cells (median of 8.11%; range 1.33%–26.27%) than healthy controls (median 3.72%; range 1.3–7.5%; P = 0.002), despite having lower absolute counts of CD4⁺FOXP3⁺ T cells. There was a significant positive correlation between the frequency of CD4⁺FOXP3⁺ T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = −0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/µl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%–26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4⁺ T cells following antigenic or other stimulation.

Conclusions/Significance

FOXP3 expression in the CD4⁺ T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression.     

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.     

Tumor-Derived Microvesicles Induce,Expand and Up-Regulate Biological Activities of Human Regulatory T Cells (Treg)

Background

Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4⁺CD25^highFOXP3⁺ Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/expansion/and activation of human Treg.

Methodology/Principal Findings

TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4⁺CD25^neg T cells into CD4⁺CD25^highFOXP3⁺ Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-β1, CTLA-4, granzyme B and perforin expression (p<0.05) and mediated stronger suppression of responder cell (RC) proliferation (p<0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-β1 and/or IL-10 significantly inhibited TMV ability to expand Treg.

Conclusions/Significance

This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV with Treg represent a newly-defined mechanism that might be involved in regulating peripheral tolerance by tumors and in supporting immune evasion of human cancers.     

A Decreased Frequency of Regulatory T Cells in Patients with Common Variable Immunodeficiency

Introduction

Common variable immunodeficiency disorder (CVID) is a heterogeneous syndrome, characterized by deficient antibody production and recurrent bacterial infections in addition abnormalities in T cells. CD4⁺CD25^high regulatory T cells (Treg) are essential modulators of immune responses, including down-modulation of immune response to pathogens, allergens, cancer cells and self-antigens.

Objective

In this study we set out to investigate the frequency of Treg cells in CVID patients and correlate with their immune activation status.

Materials and Methods

Sixteen patients (6 males and 10 females) with CVID who had been treated with regular intravenous immunoglobulin and 14 controls were enrolled. Quantitative analyses of peripheral blood mononuclear cells (PBMC) were performed by multiparametric flow cytometry using the following cell markers: CD38, HLA-DR, CCR5 (immune activation); CD4, CD25, FOXP3, CD127, and OX40 (Treg cells); Ki-67 and IFN-γ (intracellular cytokine).

Results

A significantly lower proportion of CD4⁺CD25^highFOXP3 T cells was observed in CVID patients compared with healthy controls (P<0.05). In addition to a higher proportion of CD8⁺ T cells from CVID patients expressing the activation markers, CD38⁺ and HLA-DR⁺ (P<0.05), we observed no significant correlation between Tregs and immune activation.

Conclusion

Our results demonstrate that a reduction in Treg cells could have impaired immune function in CVID patients.     

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.     

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.     

Intracerebral human regulatory T cells: analysis of CD4+ CD25+ FOXP3+ T cells in brain lesions and cerebrospinal fluid of multiple sclerosis patients

Impaired suppressive capacity of CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Treg) from peripheral blood of patients with multiple sclerosis (MS) has been reported by multiple laboratories. It is, however, currently unresolved whether Treg dysfunction in MS patients is limited to reduced control of peripheral T cell activation since most studies analyzed peripheral blood samples only. Here, we assessed early active MS lesions in brain biopsies obtained from 16 patients with MS by FOXP3 immunohistochemistry. In addition, we used six-color flow cytometry to determine numbers of Treg by analysis of FOXP3/CD127 expression in peripheral blood and cerebrospinal fluid (CSF) of 17 treatment-naïve MS patients as well as quantities of apoptosis sensitive CD45RO^hiCD95^hi cells in circulating and CSF Treg subsets. Absolute numbers of FOXP3⁺ and CD4⁺ cells were rather low in MS brain lesions and Treg were not detectable in 30% of MS biopsies despite the presence of CD4⁺ cell infiltrates. In contrast, Treg were detectable in all CSF samples and Treg with a CD45RO^hiCD95^hi phenotype previously shown to be highly apoptosis sensitive were found to be enriched in the CSF compared to peripheral blood of MS patients. We suggest a hypothetical model of intracerebral elimination of Treg by CD95L-mediated apoptosis within the MS lesion.     

Functional Improvement of Regulatory T Cells from Rheumatoid Arthritis Subjects Induced by Capsular Polysaccharide Glucuronoxylomannogalactan

Objective

Regulatory T cells (Treg) play a critical role in the prevention of autoimmunity, and the suppressive activity of these cells is impaired in rheumatoid arthritis (RA). The aim of the present study was to investigate function and properties of Treg of RA patients in response to purified polysaccharide glucuronoxylomannogalactan (GXMGal).

Methods

Flow cytometry and western blot analysis were used to investigate the frequency, function and properties of Treg cells.

Results

GXMGal was able to: i) induce strong increase of FOXP3 on CD4⁺ T cells without affecting the number of CD4⁺CD25⁺FOXP3⁺ Treg cells with parallel increase in the percentage of non-conventional CD4⁺CD25⁻FOXP3⁺ Treg cells; ii) increase intracellular levels of TGF-β1 in CD4⁺CD25⁻FOXP3⁺ Treg cells and of IL-10 in both CD4⁺CD25⁺FOXP3⁺ and CD4⁺CD25⁻FOXP3⁺ Treg cells; iii) enhance the suppressive activity of CD4⁺CD25⁺FOXP3⁺ and CD4⁺CD25⁻FOXP3⁺ Treg cells in terms of inhibition of effector T cell activity and increased secretion of IL-10; iv) decrease Th1 response as demonstrated by inhibition of T-bet activation and down-regulation of IFN-γ and IL-12p70 production; v) decrease Th17 differentiation by down-regulating pSTAT3 activation and IL-17A, IL-23, IL-21, IL-22 and IL-6 production.

Conclusion

These data show that GXMGal improves Treg functions and increases the number and function of CD4⁺CD25⁻FOXP3⁺ Treg cells of RA patients. It is suggested that GXMGal may be potentially useful for restoring impaired Treg functions in autoimmune disorders and for developing Treg cell-based strategies for the treatment of these diseases.     

T regulatory cells in cord blood--FOXP3 demethylation as reliable quantitative marker

Background

Regulatory T-cells (Tregs), characterized as CD4+CD25^hi T-cells expressing FOXP3, play a crucial role in controlling healthy immune development during early immune maturation. Recently, FOXP3 demethylation was suggested to be a novel marker for natural Tregs in adults. In cord blood, the role and function of Tregs and its demethylation is poorly understood. We assessed FOXP3 demethylation in cord blood in relation to previously used Treg markers such as CD4⁺CD25^hi, FOXP3 mRNA, protein expression, and suppressive Treg function.

Methodology

Cord blood mononuclear cells (CBMC) were isolated from 70 healthy neonates, stimulated for 3 days with the microbial stimulus lipid A (LpA), and allergen Dermatophagoides pteronyssinus (Derp1). Tregs (CD4+CD25^hi, intracellular, mRNA FOXP3 expression, isolated cells), DNA methylation of the FOXP3-locus and suppressive Treg function were assessed.

Principal Findings

Demethylation of FOXP3 in whole blood was specific for isolated CD4+CD25^hi Tregs. Demethylation of FOXP3 was positively correlated with unstimulated and LpA-stimulated FOXP3 mRNA-expression (p≤0.05), and CD4⁺CD25^hi T-cells (p≤0.03). Importantly, increased FOXP3 demethylation correlated with more efficient suppressive capacity of Tregs (r = 0.72, p = 0.005). Furthermore, FOXP3 demethylation was positively correlated with Th2 cytokines (IL-5, IL-13) following LpA-stimulation (p = 0.006/0.04), with Th2 and IL-17 following Derp1+LpA-stimulations (p≤0.009), but not Th1 cytokines (IFN-γ).

Conclusions

FOXP3 demethylation reliable quantifies Tregs in cord blood. FOXP3 demethylation corresponds well with the suppressive potential of Tregs. The resulting strict correlation with functionally suppressive Tregs and the relative ease of measurement render it into a valuable novel marker for large field studies assessing Tregs as qualitative marker indicative of functional activity.     

Plasmodium falciparum–Mediated Induction of Human CD25hiFoxp3hi CD4 T Cells Is Independent of Direct TCR Stimulation and Requires IL-2, IL-10 and TGFβ

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) regulate disease-associated immunity and excessive inflammatory responses, and numbers of CD4⁺CD25⁺Foxp3⁺ Tregs are increased during malaria infection. The mechanisms governing their generation, however, remain to be elucidated. In this study we investigated the role of commonly accepted factors for Foxp3 induction, TCR stimulation and cytokines such as IL-2, TGFβ and IL-10, in the generation of human CD4⁺CD25⁺Foxp3⁺ T cells by the malaria parasite Plasmodium falciparum. Using a co-culture system of malaria-infected red blood cells (iRBCs) and peripheral blood mononuclear cells from healthy individuals, we found that two populations of Foxp3^hi and Foxp3^int CD4⁺CD25^hi T cells with a typical Treg phenotype (CTLA-4⁺, CD127^low, CD39⁺, ICOS⁺, TNFRII⁺) were induced. Pro-inflammatory cytokine production was confined to the Foxp3^int subset (IFNγ, IL-4 and IL-17) and inversely correlated with high relative levels of Foxp3^hi cells, consistent with Foxp3^hi CD4 T cell–mediated inhibition of parasite-induced effector cytokine T cell responses. Both Foxp3^hi and Foxp3^int cells were derived primarily from proliferating CD4⁺CD25⁻ T cells with a further significant contribution from CD25⁺Foxp3⁺ natural Treg cells to the generation of the Foxp3^hi subset. Generation of Foxp3^hi, but not Foxp3^int, cells specifically required TGFβ1 and IL-10. Add-back experiments showed that monocytes expressing increased levels of co-stimulatory molecules were sufficient for iRBC-mediated induction of Foxp3 in CD4 T cells. Foxp3 induction was driven by IL-2 from CD4 T cells stimulated in an MHC class II–dependent manner. However, transwell separation experiments showed that direct contact of monocytes with the cells that acquire Foxp3 expression was not required. This novel TCR-independent and therefore antigen-non specific mechanism for by-stander CD4⁺CD25^hiFoxp3⁺ cell induction is likely to reflect a process also occurring in vivo as a consequence of immune activation during malaria infection, and potentially a range of other infectious diseases.     

Cord blood derived CD4+ CD25(high) T cells become functional regulatory T cells upon antigen encounter

Background: Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these “excessive” responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself.Methods: Cord blood (>37 week of gestation) and peripheral blood (healthy controls) were obtained and different Treg cell subsets were isolated. The suppressive potential of Treg populations after antigen exposure was evaluated via functional inhibition assays ([³H]thymidine incorporation assay and CFSE staining) with or without allergen stimulation. The frequency and markers of CD4⁺CD25^highFoxP3⁺ T cells were characterized by mRNA analysis and flow cytometry.Results: Cord blood derived CD4⁺CD25^high cells did not show substantial suppressor capacity upon TCR activation, in contrast to CD4⁺CD25^high cells freshly purified from adult blood. This could not be explained by a lower frequency of FoxP3⁺CD4⁺CD25^highcells or FOXP3 mRNA expression. However, after antigen-specific stimulation in vitro, these cells showed strong proliferation and expansion and gained potent suppressive properties. The efficiency of their suppressive capacity can be enhanced in the presence of endotoxins. If T-cells were sorted according to their CD127 expression, a tiny subset of Treg cells (CD4⁺CD25⁺CD127^low) is highly suppressive even without prior antigen exposure.Conclusion: Cord blood harbors a very small subset of CD4⁺CD25^high Treg cells that requires antigen-stimulation to show expansion and become functional suppressive Tregs.     

Influence of Membrane CD25 Stability on T Lymphocyte Activity: Implications for Immunoregulation

Background

CD25, a component of the IL-2 receptor, is important in T cell proliferation, activation induced cell death, as well as the actions of both regulatory (Treg) and effector (Teff) T cells. Recent genome wide association studies have implicated the CD25 locus as an important region for genetic susceptibility to a number of autoimmune disorders, with serum levels of soluble CD25 receptor (sCD25) serving as a potential phenotypic marker for this association. However, the functional impact of CD25 cleavage, as well as the influence of sCD25 on immunoregulatory activities, remain largely unknown and form the basis of this effort.

Methodology/Principal Findings

The generation of sCD25 by Treg (CD4⁺CD25⁺) and Teff (CD4⁺CD25⁻) cells was examined during in vitro suppression assays, efforts that demonstrated constitutive and stable surface CD25 expression on Treg throughout the period of in vitro assessment. In contrast, Teff cells increased CD25 expression during the process of in vitro suppression, with supernatant sCD25 levels correlating to the amount of cellular proliferation. Interestingly, under serum-free conditions, Tregs partially lost their characteristic anergic and suppressive properties. sCD25 supplementation at physiological concentrations to serum free in vitro suppression assays reduced Teff proliferation without specifically influencing suppression. Indeed, sCD25 production within these cultures correlated with cell death.

Conclusions/Significance

These results support the notion that sCD25 functions as both a surrogate marker of T cell activation as well as an indicator of subsequent cellular death. In addition, the role of CD25 in immunomodulation is likely dependent on the local inflammatory milieu, with molecules capable of modulating surface CD25 expression playing a key role in defining immune responsiveness.     

Human Regulatory T Cell Suppressive Function Is Independent of Apoptosis Induction in Activated Effector T Cells

Background

CD4⁺CD25⁺FOXP3⁺ Regulatory T cells (Treg) play a central role in the immune balance to prevent autoimmune disease. One outstanding question is how Tregs suppress effector immune responses in human. Experiments in mice demonstrated that Treg restrict effector T cell (Teff) responses by deprivation of the growth factor IL-2 through Treg consumption, resulting in apoptosis of Teff.

Principal Findings

In this study we investigated the relevance of Teff apoptosis induction to human Treg function. To this end, we studied naturally occurring Treg (nTreg) from peripheral blood of healthy donors, and, to investigate Treg function in inflammation in vivo, Treg from synovial fluid of Juvenile Idiopathic Arthritis (JIA) patients (SF-Treg). Both nTreg and SF-Treg suppress Teff proliferation and cytokine production efficiently as predicted. However, in contrast with murine Treg, neither nTreg nor SF-Treg induce apoptosis in Teff. Furthermore, exogenously supplied IL-2 and IL-7 reverse suppression, but do not influence apoptosis of Teff.

Significance

Our functional data here support that Treg are excellent clinical targets to counteract autoimmune diseases. For optimal functional outcome in human clinical trials, future work should focus on the ability of Treg to suppress proliferation and cytokine production of Teff, rather than induction of Teff apoptosis.     

Accumulation of CCR4+ CTLA-4hi FOXP3+CD25hi Regulatory T Cells in Colon Adenocarcinomas Correlate to Reduced Activation of Conventional T Cells

Background

Colorectal cancer usually gives rise to a specific anti-tumor immune response, but for unknown reasons the resulting immunity is not able to clear the tumor. Recruitment of activated effector lymphocytes to the tumor is important for efficient anti-tumor responses, while the presence of regulatory T cells (Treg) down-modulate tumor-specific immunity. We therefore aimed to determine homing mechanisms and activation stage of Treg and effector T cell infiltrating colon tumors compared to cells from the unaffected mucosa in patients suffering from colon adenocarcinoma.

Methodology/Principal Findings

Lymphocytes were isolated from unaffected and tumor mucosa from patients with colon adenocarcinoma, and flow cytometry, immunohistochemistry, and quantitative PCR was used to investigate the homing mechanisms and activation stage of infiltrating Treg and conventional lymphocytes. We detected significantly higher frequencies of CD25^highFOXP3⁺CD127^low putative Treg in tumors than unaffected mucosa, which had a complete demethylation in the FOXP3 promotor. Tumor-associated Treg had a high expression of CTLA-4, and some appeared to be antigen experienced effector/memory cells based on their expression of αEβ7 (CD103). There were also significantly fewer activated T cells and more CTLA-4⁺ conventional T cells susceptible to immune regulation in the tumor-associated mucosa. In contrast, CD8⁺granzyme B⁺ putative cytotoxic cells were efficiently recruited to the tumors. The frequencies of cells expressing α4β7 and the Th1 associated chemokine receptor CXCR3 were significantly decreased among CD4⁺ T cells in the tumor, while frequencies of CD4⁺CCR4⁺ lymphocytes were significantly increased.

Conclusions/Significance

This study shows that CCR4⁺CTLA4^hi Treg accumulate in colon tumors, while the frequencies of activated conventional Th1 type T cells are decreased. The altered lymphocyte composition in colon tumors will probably diminish the ability of the immune system to effectively attack tumor cells, and reducing the Treg activity is an important challenge for future immunotherapy protocols.     

A Crucial Role for Kupffer Cell-Derived Galectin-9 in Regulation of T Cell Immunity in Hepatitis C Infection

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (∼70–80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-γ, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4⁺CD25⁺FoxP3⁺CD127^low regulatory T cells, contraction of CD4⁺ effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.     

Increased Sensitivity of CD4+ T-Effector Cells to CD4+CD25+ Treg Suppression Compensates for Reduced Treg Number in Asymptomatic HIV-1 Infection

Background

In HIV infection, uncontrolled immune activation and disease progression is attributed to declining CD4+CD25+FoxP3+ regulatory T-cell (Treg) numbers. However, qualitative aspects of Treg function in HIV infection, specifically the balance between Treg cell suppressive potency versus suppressibility of effector cells, remain poorly understood. This report addresses this issue.

Methodology/Principal Findings

A classic suppression assay to measure CD4+CD45RO+CD25hi Treg cells to suppress the proliferation of CD4+CD45RO+CD25− effectors cells (E) following CD3/CD28 polyclonal stimulation was employed to compare the suppressive ability of healthy volunteers (N = 27) and chronic, asymptomatic, treatment naïve, HIV-infected subjects (N = 14). HIV-infected subjects displayed significantly elevated Treg-mediated suppression compared to healthy volunteers (p = 0.0047). Cross-over studies comparing Treg cell potency from HIV-infected versus control subjects to suppress the proliferation of a given population of allogeneic effector cells demonstrated increased sensitivity of CD4+CD25− effector cells from HIV-infected subjects to be suppressed, associated with reduced production of the Treg counter-regulatory cytokine, IL-17, rather than an increase in the suppressive potential of their CD4+CD25+ Treg cells. However, compared to controls, HIV+ subjects had significantly fewer absolute numbers of circulating CD4+CD25+FoxP3+ Treg cells. In vitro studies highlighted that one mechanism for this loss could be the preferential infection of Treg cells by HIV.

Conclusions/Significance

Together, novel data is provided to support the contention that elevated Treg-mediated suppression may be a natural host response to HIV 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.     

Transduction of Human T Cells with a Novel T-Cell Receptor Confers Anti-HCV Reactivity

Hepatitis C Virus (HCV) is a major public health concern, with no effective vaccines currently available and 3% of the world''s population being infected. Despite the existence of both B- and T-cell immunity in HCV-infected patients, chronic viral infection and HCV-related malignancies progress. Here we report the identification of a novel HCV TCR from an HLA-A2-restricted, HCV NS3:1073–1081-reactive CTL clone isolated from a patient with chronic HCV infection. We characterized this HCV TCR by expressing it in human T cells and analyzed the function of the resulting HCV TCR-transduced cells. Our results indicate that both the HCV TCR-transduced CD4⁺ and CD8⁺ T cells recognized the HCV NS3:1073–1081 peptide-loaded targets and HCV⁺ hepatocellular carcinoma cells (HCC) in a polyfunctional manner with cytokine (IFN-γ, IL-2, and TNF-α) production as well as cytotoxicity. Tumor cell recognition by HCV TCR transduced CD8⁻ Jurkat cells and CD4⁺ PBL-derived T cells indicated this TCR was CD8-independent, a property consistent with other high affinity TCRs. HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.     

Abnormally High Levels of Virus-Infected IFN-γ+CCR4+CD4+CD25+ T Cells in a Retrovirus-Associated Neuroinflammatory Disorder

Background

Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified.

Principal Findings

Here, we demonstrate that CD4⁺CD25⁺CCR4⁺ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-γ production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4⁺CD25⁺CCR4⁺ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-γ-producing CD4⁺CD25⁺CCR4⁺Foxp3⁻ T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity.

Conclusions

We have defined a unique T cell subset—IFN-γ⁺CCR4⁺CD4⁺CD25⁺ T cells—that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.