Nabila Seddiki

Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells

Abnormalities in CD4+CD25+Foxp3+ regulatory T (T reg) cells have been implicated in susceptibility to allergic, autoimmune, and immunoinflammatory conditions. However, phenotypic and functional assessment of human T reg cells has been hampered by difficulty in distinguishing between CD25-expressing activated and regulatory T cells. Here, we show that expression of CD127, the α chain of the interleukin-7 receptor, allows an unambiguous flow cytometry–based distinction to be made between CD127lo T reg cells and CD127hi conventional T cells within the CD25+CD45RO+RA− effector/memory and CD45RA+RO− naive compartments in peripheral blood and lymph node. In healthy volunteers, peripheral blood CD25+CD127lo cells comprised 6.35 ± 0.26% of CD4+ T cells, of which 2.05 ± 0.14% expressed the naive subset marker CD45RA. Expression of FoxP3 protein and the CD127lo phenotype were highly correlated within the CD4+CD25+ population. Moreover, both effector/memory and naive CD25+CD127lo cells manifested suppressive activity in vitro, whereas CD25+CD127hi cells did not. Cell surface expression of CD127 therefore allows accurate estimation of T reg cell numbers and isolation of pure populations for in vitro studies and should contribute to our understanding of regulatory abnormalities in immunopathic diseases.

Proliferation of weakly suppressive regulatory CD4+ T cells is associated with over-active CD4+ T-cell responses in HIV-positive patients with mycobacterial immune restoration disease.

The role of Treg in patients with late‐stage HIV disease, who commence combination antiretroviral therapy (cART) and develop pathogen‐specific immunopathology manifesting as immune restoration disease (IRD) remains unclear. We hypothesised that Treg could be defective in either numbers and/or function and therefore unable to ensure the physiological equilibrium of the immune system in patients with IRD. Phenotypic and functional CD4+ T‐cell subsets of eight late‐stage HIV patients with nadir CD4 count <50 cells/μL, who developed mycobacterial IRD upon commencing cART were compared with six therapy naive HIV+ patients (nadir CD4 count <50 cells/μL), who did not develop an IRD after cART. Mycobacterium‐avium‐specific CD4+ T cells from IRD patients produced high levels of IFN‐γ and IL‐2 compared with controls (p<0.001). Surprisingly, we found a significant expansion of CD127loFoxp3+CD25+ Treg in IRD patients and a higher ratio of Treg to effector/memory subsets (p<0.001). In vitro suppression assays demonstrated reduced functional capacity of suppressor cells and diminished IL‐10 secretion in IRD patients. Plasma levels of IL‐7 were increased in patients and, interestingly, exogenous IL‐7 and other cytokines strongly inhibited Treg suppression. These data suggest that despite substantial Treg expansion in IRD, their ability to induce suppression, and thereby downregulate aberrant immune responses, is compromised.

Differential Regulation of the Let-7 Family of MicroRNAs in CD4+ T Cells Alters IL-10 Expression

MicroRNAs (miRNAs) are ∼22-nt small RNAs that are important regulators of mRNA turnover and translation. Recent studies have shown the importance of the miRNA pathway in HIV-1 infection, particularly in maintaining latency. Our initial in vitro studies demonstrated that HIV-1–infected HUT78 cells expressed significantly higher IL-10 levels compared with uninfected cultures. IL-10 plays an important role in the dysregulated cytotoxic T cell response to HIV-1, and in silico algorithms suggested that let-7 miRNAs target IL10 mRNA. In a time course experiment, we demonstrated that let-7 miRNAs fall rapidly following HIV-1 infection in HUT78 cells with concomitant rises in IL-10. To show a direct link between let-7 and IL-10, forced overexpression of let-7 miRNAs resulted in significantly reduced IL-10 levels, whereas inhibition of the function of these miRNAs increased IL-10. To demonstrate the relevance of these results, we focused our attention on CD4+ T cells from uninfected healthy controls, chronic HIV-1–infected patients, and long-term nonprogressors. We characterized miRNA changes in CD4+ T cells from these three groups and demonstrated that let-7 miRNAs were highly expressed in CD4+ T cells from healthy controls and let-7 miRNAs were significantly decreased in chronic HIV-1 infected compared with both healthy controls and long-term nonprogressors. We describe a novel mechanism whereby IL-10 levels can be potentially modulated by changes to let-7 miRNAs. In HIV-1 infection, the decrease in let-7 miRNAs may result in an increase in IL-10 from CD4+ T cells and provide the virus with an important survival advantage by manipulating the host immune response.

High Levels of Human Antigen-Specific CD4+ T Cells in Peripheral Blood Revealed by Stimulated Coexpression of CD25 and CD134 (OX40)

Ag-specific human CD4+ memory T lymphocytes have mostly been studied using assays of proliferation in vitro. Intracellular cytokine and ELISPOT assays quantify effector cell populations but barely detect responses to certain recall Ags that elicit strong proliferative responses, e.g., tetanus toxoid, that comprise non-Th1 CD4+ cells. We have found that culturing whole blood with Ag for 40–48 h induces specific CD4+ T cells to simultaneously express CD25 and CD134. This new technique readily detects responses to well-described CD4+ T cell recall Ags, including preparations of mycobacteria, CMV, HSV-1, influenza, tetanus toxoid, Candida albicans, and streptokinase, as well as HIV-1 peptides, with high specificity. The assay detects much higher levels of Ag-specific cells than intracellular cytokine assays, plus the cells retain viability and can be sorted for in vitro expansion. Furthermore, current in vitro assays for human CD4+ memory T lymphocytes are too labor-intensive and difficult to standardize for routine diagnostic laboratories, whereas the whole-blood CD25+CD134+ assay combines simplicity of setup with a straightforward cell surface flow cytometry readout. In addition to revealing the true extent of Ag-specific human CD4+ memory T lymphocytes, its greatest use will be as a simple in vitro monitor of CD4+ T cell responses to Ags such as tuberculosis infection or vaccines.

Role of miR-155 in the regulation of lymphocyte immune function and disease

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression within cells. One particular miRNA, miR‐155, is highly expressed within lymphocytes (both B and T cells) and mediates a number of important roles. These include shaping the transcriptome of lymphoid cells that control diverse biological functions vital in adaptive immunity. The use of mice engineered to be deficient in miR‐155, as well as the identification of endogenous targets of miR‐155 in T cells by transcriptome‐wide analysis, has helped to unravel the crucial role that this miRNA plays in fine tuning the regulation of lymphocyte subsets such as B cells, CD8+ and CD4+ T cells ranging from T helper type 1 (Th1), Th2, Th17 and regulatory T cells. In this review, we summarize what we have learned about miR‐155 in the regulation of lymphocyte responses at the cellular and molecular levels and in particular, we focus on the recent findings showing that miR‐155 shapes the balance between tolerance and immunity.

Infection of CD127+ (Interleukin-7 Receptor+) CD4+ Cells and Overexpression of CTLA-4 Are Linked to Loss of Antigen-Specific CD4 T Cells during Primary Human Immunodeficiency Virus Type 1 Infection

ABSTRACT We recently found that human immunodeficiency virus (HIV)-specific CD4+ T cells express coreceptor CCR5 and activation antigen CD38 during early primary HIV-1 infection (PHI) but then rapidly disappear from the circulation. This cell loss may be due to susceptibility to infection with HIV-1 but could also be due to inappropriate apoptosis, an expansion of T regulatory cells, trafficking out of the circulation, or dysfunction. We purified CD38+++CD4+ T cells from peripheral blood mononuclear cells, measured their level of HIV-1 DNA by PCR, and found that about 10% of this population was infected. However, a small subset of HIV-specific CD4+ T cells also expressed CD127, a marker of long-term memory cells. Purified CD127+CD4+ lymphocytes contained fivefold more copies of HIV-1 DNA per cell than did CD127-negative CD4+ cells, suggesting preferential infection of long-term memory cells. We observed no apoptosis of antigen-specific CD4+ T cells in vitro and only a small increase in CD45RO+CD25+CD127dimCD4+ T regulatory cells during PHI. However, 40% of CCR5+CD38+++ CD4+ T cells expressed gut-homing integrins, suggesting trafficking through gut-associated lymphoid tissue (GALT). Furthermore, 80% of HIV-specific CD4+ T cells expressed high levels of the negative regulator CTLA-4 in response to antigen stimulation in vitro, which was probably contributing to their inability to produce interleukin-2 and proliferate. Taken together, the loss of HIV-specific CD4+ T cells is associated with a combination of an infection of CCR5+ CD127+ memory CD4+ T cells, possibly in GALT, and a high expression of the inhibitory receptor CTLA-4.

Chromatin-associated protein kinase C-θ regulates an inducible gene expression program and microRNAs in human T lymphocytes.

Studies in yeast demonstrate that signaling kinases have a surprisingly active role in the nucleus, where they tether to chromatin and modulate gene expression programs. Despite these seminal studies, the nuclear mechanism of how signaling kinases control transcription of mammalian genes is in its infancy. Here, we provide evidence for a hitherto unknown function of protein kinase C-theta (PKC-θ), which physically associates with the regulatory regions of inducible immune response genes in human T cells. Chromatin-anchored PKC-θ forms an active nuclear complex by interacting with RNA polymerase II, the histone kinase MSK-1, and the adaptor molecule 14-3-3ζ. ChIP-on-chip reveals that PKC-θ binds to promoters and transcribed regions of genes, as well as to microRNA promoters that are crucial for cytokine regulation. Our results provide a molecular explanation for the role of PKC-θ not only in normal T cell function, but also in circumstances of its ectopic expression in cancer.

Regulatory T Cells Negatively Affect IL-2 Production of Effector T Cells through CD39/Adenosine Pathway in HIV Infection

The mechanisms by which Regulatory T cells suppress IL-2 production of effector CD4+ T cells in pathological conditions are unclear. A subpopulation of human Treg expresses the ectoenzyme CD39, which in association with CD73 converts ATP/ADP/AMP to adenosine. We show here that Treg/CD39+ suppress IL-2 expression of activated CD4+ T-cells more efficiently than Treg/CD39−. This inhibition is due to the demethylation of an essential CpG site of the il-2 gene promoter, which was reversed by an anti-CD39 mAb. By recapitulating the events downstream CD39/adenosine receptor (A2AR) axis, we show that A2AR agonist and soluble cAMP inhibit CpG site demethylation of the il-2 gene promoter. A high frequency of Treg/CD39+ is associated with a low clinical outcome in HIV infection. We show here that CD4+ T-cells from HIV-1 infected individuals express high levels of A2AR and intracellular cAMP. Following in vitro stimulation, these cells exhibit a lower degree of demethylation of il-2 gene promoter associated with a lower expression of IL-2, compared to healthy individuals. These results extend previous data on the role of Treg in HIV infection by filling the gap between expansion of Treg/CD39+ in HIV infection and the suppression of CD4+ T-cell function through inhibition of IL-2 production.

Accelerated age-dependent transition of human regulatory T cells to effector memory phenotype

We and others recently described a method for isolating viable forkhead boxp3 (FoxP3(+)) T regulatory cells (Tregs) by means of the surface phenotype CD4(+)CD127(lo)CD25(+). In this study, we used the new strategy to measure Treg numbers, phenotype and function at different ages. Mean percentages of CD4(+)CD127(lo)CD25(+) Tregs increased only slightly throughout life, from 6.10% in cord blood to 7.22% in PBMC from adults between 20 and 25 years and 7.50% in PBMC from adults over the age of 60. In all age groups, a higher proportion of Tregs had acquired a CD45RA(-) memory phenotype compared with CD4(+)Foxp3(-) conventional T cells. This increase was entirely attributable to increased Tregs with an effector memory phenotype, whereas central memory phenotype cells were comparably represented within the Treg and conventional CD4(+) T-cell populations. Expression of CD95 also differed between Tregs and conventional CD4(+) T cells at all ages. However there was no difference in the suppressive capacity of the different naive and memory Treg subsets. These results suggest that, compared with their conventional CD4(+) T-cell counterparts, Tregs undergo preferential differentiation from a naive to an effector memory phenotype, driven by their specificity for self- rather than foreign antigen. However, number and function are remarkably stable throughout life.

Immunopathogenesis of immune reconstitution disease in HIV patients responding to antiretroviral therapy.

Purpose of reviewThe aim of this article is to review the most recent literature regarding the immunopathogenesis of pathogen-associated immune reconstitution disease and to discuss the role of immune activation and various effector molecules and cells such as macrophages, effector and regulatory T cells, and natural killer cells in immune reconstitution disease. Recent findingsMany HIV patients receiving antiretroviral treatment develop immune reconstitution disease, which is characterized by exaggerated inflammatory immune responses to replicating or dead pathogens. In the majority of these cases, immune reconstitution disease is associated with restoration of pathogen-specific cellular immune responses involving CD4+ or CD8+ effector T cells. The precise conditions that trigger immune reconstitution disease have not yet been identified. Immune reconstitution disease patients have overt immune activation, which may be due to poor homeostatic control after the fast initial immune recovery in patients receiving antiretroviral therapy. Poor homeostatic control in immune reconstitution disease patients may be linked to unbalanced restoration of effector and regulatory T cells. SummaryAlthough the precise mechanism of immune reconstitution disease is not well understood, it is probably related to rapid restoration of pathogen-specific immune responses and poor homeostatic control that promote exaggerated immunopathological responses, especially if viable pathogens or pathogen debris are present at high concentrations.