Are CD4+CD25-Foxp3+ cells in untreated new-onset lupus patients regulatory T cells?

Introduction  

Our previous study has reported that, in patients with untreated new-onset lupus (UNOL), there was an abnormal increase in the number of CD4⁺CD25^-Foxp3⁺ T cells that correlated with disease activity and significantly decreased after treatment. However, little is known about the nature of this cell entity. The aim of this study was to explore the nature of abnormally increased CD4⁺CD25^-Foxp3⁺ T cells in UNOL patients.     

A glucocorticoid amplifies IL-2-induced selective expansion of CD4^＋CD25^＋FOXP3^＋ regulatory T cells in vivo and suppresses graft-versus-host disease after allogeneic lymphocyte transplantation

Regulatory T （Treg） cells are a subpopulation of T cells that not only prevent autoimmunity, but also control a wide range of T cell-dependent immune responses. Glucocorticoid treatment （dexamethasone, or Dex） has been reported to amplify IL-2-mediated selective in vivo expansion of Treg cells. We simultaneously administered Dex and IL-2 to the donor in a murine allogeneic lymphocyte transplantation model to expand functional suppressive CD4＋CD25＋FOXP3＋ T cells in the graft and to raise the regulatory T cell/effector T cell （Treg/ Teff） ratio to prevent graft-versus-host disease （GVHD）. After combined treatment of the donor with Dex （5 mg/kg/day） and IL-2 （300,000 IU/mouse/day） for 3 days, grafts were subjected to flow cytometric analysis, and transplantation was carried out from male C57BL/6 mice to female BALB/c mice aged 8-12 weeks. Results showed that short-term simultaneous administration of Dex and IL-2 markedly expanded functional suppressive CD4＋CD25＋FOXP3＋ T cells in the murine spleen. In this murine allogeneic transplantation model, the grafts from donors with Dex and IL-2 pre-treatment led to a longer survival time for the recipients than for the control group （median survival time 〉 60 day vs. 12 day, P = 0.0002）. The ratio of Treg/Teff also increased remarkably （0.43 ±0.15 vs. 0.14 ± 0.01, P = 0.01）. This study demonstrated that co-stimulation with Dex and IL-2 selectively expanded functional CD4＋CD25＋FOXP3＋ T cells in vivo, and that grafts from donors pre-treated with Dex and IL-2 led to longer survival time and greater suppression of GVHD after allogeneic transplantation. Thus, GVHD can be suppressed by the specific expansion of regulatory T cells with Dex and IL-2 in graft donors.     

The activation, by antigen, of naïve TCR transgenic CD4 T cells cultured at physiological, rather than artificially high, frequencies more accurately reflects the in vivo activation of normal numbers of naïve CD4(+) T cells

The majority of in vitro studies investigating the activation of na?ve TCR transgenic T cells routinely employ an artificially high frequency of such cells. To assess whether employing high frequencies of TCR transgenic cells in vitro accurately reflects the in vivo activation of a normal number of T cells, we cultured between 300 and 3×10(6) Rag2(-/-) DO11.10 T cells per well under otherwise identical conditions. We find that those T cells cultured at low frequencies proliferate more and are more potently activated, as assessed by the expression of CD44 and CD62L, each giving rise to a much larger number of cytokine producing cells, comparable to the number generated in vivo when a normal number of CD4(+) T cells are activated. The effect of T cell frequency on the level of their activation was not due to differences in MHCII or CD80/86 expression by B cells, the major APC population present, nor to increased death of B cells in high frequency cultures. Taken together, our observations illustrate the necessity of culturing na?ve TCR transgenic CD4(+) T cells at a physiological frequency if one is to more accurately recapitulate the in vivo activation of na?ve CD4(+) T cells.     

Effective modulation of CD4+CD25+high regulatory T and NK cells in malignant patients by combination of interferon-α and interleukin-2

Overinduced CD4⁺CD25^+high regulatory T cells (Treg) and downregulated NK cells contribute to tumor-relevant immune tolerance and interfere with tumor immunity. In this study, we aimed to design a novel strategy with cytokine combination to correct the dysregulated Treg and NK cells in malignant patients. Initially, a total of 58 healthy individuals and 561 malignant patients were analyzed for their cellular immunity by flow cytometry. The average percentages of CD4⁺CD25^+high/lymphocyte were 1.30?±?1.19?% ( $ bar{x} $ ?±?SD) in normal adults and 3.274?±?4.835?% in malignant patients (p?<?0.001). The ratio of CD4⁺CD25^+high to CD4⁺ was 3.58?±?3.19?% in normal adults and 6.01?±?5.89?% to 13.50?±?23.60?% in different kinds of malignancies (p?<?0.001). Of normal adults, 15.52?% had >3?% Treg and 12.07?% had <10?% NK cells. In contrast, the Treg (>3?%) and NK (<10?%) percentages were 40.82 and 34.94?% in malignant patients, respectively. One hundred and ten patients received the immunomodulation therapy with IFN-?? and/or IL-2. The overinduced Treg in 86.3?% and the reduced NK cells in 71.17?% of the patients were successfully modulated. In comparison, other lymphocyte subpopulations in most patients were much less affected by this treatment. No other treatment-relevant complications except slight pyrexia, fatigue, headache, and myalgia were observed. In conclusion, dysregulated Treg and/or NK cells were common in malignant patients. Different from any regimens ever reported, this strategy was simple and effective without severe complications and will become a basic regimen for other cancer therapies.     

Signaling thresholds govern heterogeneity in IL-7-receptor-mediated responses of naïve CD8(+) T cells

Variable sensitivity to T-cell-receptor (TCR)- and IL-7-receptor (IL-7R)-mediated homeostatic signals among na?ve T cells has thus far been largely attributed to differences in TCR specificity. We show here that even when withdrawn from self-peptide-induced TCR stimulation, CD8(+) T cells exhibit heterogeneous responses to interleukin-7 (IL-7) that are mechanistically associated with IL-7R expression differences that correlate with relative CD5 expression. Whereas CD5(hi) and CD5(lo) T cells survive equivalently in the presence of saturating IL-7 levels in vitro, CD5(hi) T cells proliferate more robustly. Conversely, CD5(lo) T cells exhibit prolonged survival when withdrawn from homeostatic stimuli. Through quantitative experimental analysis of signaling downstream of IL-7R, we find that the enhanced IL-7 responsiveness of CD5(hi) T cells is directly related to their greater surface IL-7R expression. Further, we identify a quantitative threshold in IL-7R-mediated signaling capacity required for proliferation that lies well above an analogous threshold requirement for survival. These distinct thresholds allow subtle differences in IL-7R expression between CD5(lo) and CD5(hi) T cells to give rise to significant variations in their respective IL-7-induced proliferation, without altering survival. Heterogeneous IL-7 responsiveness is observed similarly in vivo, with CD5(hi) na?ve T cells proliferating preferentially in lymphopenic mice or lymphoreplete mice administered with exogenous IL-7. However, IL-7 in lymphoreplete mice appears to be maintained at an effective level for preserving homeostasis, such that neither CD5(hi) IL-7R(hi) nor CD5(lo) IL-7R(lo) T cells proliferate or survive preferentially. Our findings indicate that IL-7R-mediated signaling not only maintains the size but also impacts the diversity of the na?ve T-cell repertoire.     

Murine Peyer's patch dendritic cells prime naïve CD4+ T cells to produce interferon-γ

We investigated the role of Peyer's patch (PP) dendritic cells (DCs) in the production of interferon (IFN)-γ from naïve CD4⁺ T cells of T cell receptor transgenic mice. PP DCs were found to prime naïve CD4⁺ T cells for the production of higher levels of IFN-γ, when compared to spleen (SP) DCs. However, a similar level of interleukin-12 (IL-12) production was observed for PP and SP DCs stimulated via the CD40 molecule. In addition, PP DCs expressed slightly higher levels of B7.2 (CD86) compared to SP DCs. This data demonstrates that PP DCs have a distinct function in the induction of IFN-γs and suggests that PP DCs may enhance IFN-γ production via another cytokine or costimulatory molecule, in addition to IL-12.     

Selective depletion of FOXP3high cells by Fas–Fas-L–induced apoptosis occurs in CD4+CD25+-enriched populations during repeated expansion

Background aimsExpansion of anti-CD25 bead-isolated human Tregs culture has paradoxically resulted in reduced suppressive activity, but the mechanism(s) responsible for these observations are poorly defined.MethodsMagnetic-bead isolated human CD25⁺ cells were expanded with anti-CD3/CD28 beads and high doses of rhIL-2. Detection of Fas and Fas ligand (Fas-L) expression, activation of Caspase 8, cell proliferation and cytokine production was evaluated by multi-color fluorescence-activated cell sorting analysis. The role of Fas–Fas-L–mediated cell death was dissected through the use of agonist or antagonist monoclonal antibodies directed at Fas and Fas-L.ResultsRepeated expansion of bead-enriched CD4⁺CD25⁺ cells generated a cellular product with markedly reduced suppressive activity and with significantly increased CD8⁺ T cells and CD4⁺ T cells producing interferon-γ and/or interleukin-2. We showed that Fas–Fas-L–mediated apoptosis of CD4⁺FOXP3^high cells and rapid cell-cycling of CD8⁺ T cells were collectively responsible for the reduced proportion of CD4⁺FOXP3^high cells in expanded cultures. The depletion of CD4⁺FOXP3^high cells and activation of Caspase 8 in CD4⁺FOXP3^high cells was attenuated by Fas antagonist antibody, ZB4, in short-term culture. However, the loss of CD4⁺FOXP3^high cells during expansion was not prevented by either Fas or Fas-L antagonist antibodies.ConclusionsTaken together, the data show that Fas–Fas-L–mediated apoptosis may limit the expansion of anti-CD25 bead-isolated cells in vitro.     

Interleukin-21 maintains the expression of CD16 on monocytes via the production of IL-10 by human naïve CD4+ T cells

Interleukin 21 exerts a variety of regulatory effects on both innate and adaptive immune cells. Although the suppressive effect of IL-21 via the induction of IL-10 in mouse model has been defined, the inhibitory effect of IL-21 in humans is not well understood. In the present study, we showed that IL-21 induced IL-10 production by human naive CD4⁺ T cells. Most of the IL-10-producing CD4⁺ T cells did not co-express IFN-γ. IL-21 increased the expression of IL-21R on activated naïve CD4⁺ T cells. Further analysis indicated that IL-21 induced phosphorylation of STAT1, STAT3 and STAT5 in activated naïve CD4⁺ T cells. In addition, IL-21 maintained the expression of CD16 on monocytes via the production of IL-10 by human naïve CD4⁺ T cells. Taken together, our data indicated that IL-21 had a modulating effect on monocytes at least in part by inducing IL-10 production.     

IL-4 inducibility in NKT cells,naïve CD4<Superscript>+</Superscript> T cells and TCR-γ δ T cells

NKT cells, na?ve CD4(+) T cells, and TCR-gammadelta T cells belong to distinct T cell lineages but all express T cell receptors generated through random combinatorial joining of V-(D)-J genes. These distinct lineage T cells also possess the property of promptly activating the IL-4 gene upon T cell receptor stimulation. A comparative accounting of features as they pertain to IL-4 inducibility in these three distinct lineage T cells is provided here.     

miRNA profiling of naïve, effector and memory CD8 T cells

microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific na?ve, effector and memory CD8+ T cells using 3 different methods--small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f) alone accounted for approximately 60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs) was observed in effector T cells compared to na?ve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to na?ve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3'end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function.     

Peripheral residence of naïve CD4 T cells induces MHC class II-dependent alterations in phenotype and function

Background

As individual naïve CD4 T lymphocytes circulate in the body after emerging from the thymus, they are likely to have individually varying microenvironmental interactions even in the absence of stimulation via specific target recognition. It is not clear if these interactions result in alterations in their activation, survival and effector programming. Naïve CD4 T cells show unimodal distribution for many phenotypic properties, suggesting that the variation is caused by intrinsic stochasticity, although underlying variation due to subsets created by different histories of microenvironmental interactions remains possible. To explore this possibility, we began examining the phenotype and functionality of naïve CD4 T cells differing in a basic unimodally distributed property, the CD4 levels, as well as the causal origin of these differences.

Results

We examined separated CD4hi and CD4lo subsets of mouse naïve CD4 cells. CD4lo cells were smaller with higher CD5 levels and lower levels of the dual-specific phosphatase (DUSP)6-suppressing micro-RNA miR181a, and responded poorly with more Th2-skewed outcomes. Human naïve CD4lo and CD4hi cells showed similar differences. Naïve CD4lo and CD4hi subsets of thymic single-positive CD4 T cells did not show differences whereas peripheral naïve CD4lo and CD4hi subsets of T cell receptor (TCR)-transgenic T cells did. Adoptive transfer-mediated parking of naïve CD4 cells in vivo lowered CD4 levels, increased CD5 and reactive oxygen species (ROS) levels and induced hyporesponsiveness in them, dependent, at least in part, on availability of major histocompatibility complex class II (MHCII) molecules. ROS scavenging or DUSP inhibition ameliorated hyporesponsiveness. Naïve CD4 cells from aged mice showed lower CD4 levels and cell sizes, higher CD5 levels, and hyporesponsiveness and Th2-skewing reversed by DUSP inhibition.

Conclusions

Our data show that, underlying a unimodally distributed property, the CD4 level, there are subsets of naïve CD4 cells that vary in the time spent in the periphery receiving MHCII-mediated signals and show resultant alteration of phenotype and functionality via ROS and DUSP activity. Our findings also suggest the feasibility of potential pharmacological interventions for improved CD4 T cell responses during vaccination of older people via either anti-oxidant or DUSP inhibitor small molecules.

     

Dendritic cells transduced with lentiviral vector targeting RelB gene using RNA interference induce hyporesponsiveness in memory CD4(+) T cells and naïve CD4(+) T cells

The ability of DCs to induce immune tolerance depends on its maturation status. RelB plays a pivotal role in DCs differentiation. A therapeutic protocol of DCs-based not only induces hyporesponsiveness in T(N)s, but also in alloreactive T(M)s is required. Thus, it is urgent to assess modulatory effects of RelB-silenced DCs on T(M)s and T(N)s. In this study, we constructed lentiviral vector which could efficiently silenced the RelB in DCs (DCs-miR RelB) to keep them immature. These DCs induced antigen-specific hyporesponsiveness in CD4(+) T(N)s. In contrast, upon re-stimulation with mature DCs, CD4(+) T(M)s primed by DCs-miR RelB maintained hyporesponsiveness in terms of proliferation and cytokine production. And these may be associated with micro155 and micro181a expression levels in T(M)s and T(N)s. These results may help developing the DCs-based therapeutical protocols by inducing hyporesponsiveness in CD4(+) T(N)s and T(M)s.     

Murine thymic stromal lymphopoietin promotes the differentiation of regulatory T cells from thymic CD4(+)CD8(-)CD25(-) naïve cells in a dendritic cell-independent manner

Human thymic stromal lymphopoietin (TSLP) activates dendritic cells (DCs), which promote the proliferation and differentiation of CD4+ T cells. However, murine TSLP (mTSLP) can act directly on CD4+ T cells and bring about their differentiation. We studied the role of mTSLP in the generation of CD4+CD25+FoxP3+ T cells from thymocytes. mTSLP promoted the differentiation of CD4+ single-positive thymocytes into CD4+CD25+FoxP3+ T cells. Although we cannot exclude an effect of TSLP mediated through DCs due to co-stimulatory effects, mTSLP appears to act directly on thymocytes. T-cell receptor and TSLP receptor signaling act synergistically on thymocytes to generate CD4+CD25+FoxP3+ T cells. mTSLP may play an important role in maintaining immune tolerance by promoting the conversion of thymocytes into natural regulatory T cells via escape from negative selection.     

SIDE effects associated with use of nevirapine in HIV treatment naïve patients with respect to baseline CD4 count

The rapid growth of bioscience in China is considered.     

Choice of resident costimulatory molecule can influence cell fate in human naïve CD4+ T cell differentiation

With antigen stimulation, naïve CD4+ T cells differentiate to several effector or memory cell populations, and cytokines contribute to differentiation outcome. Several proteins on these cells receive costimulatory signals, but a systematic comparison of their differential effects on naïve T cell differentiation has not been conducted. Two costimulatory proteins, CD28 and ICAM-1, resident on human naïve CD4+ T cells were compared for participation in differentiation. Under controlled conditions, and with no added cytokines, costimulation through either CD3+CD28 or CD3+CAM-1 induced differentiation to T effector and T memory cells. In contrast, costimulation through CD3+ICAM-1 induced differentiation to Treg cells whereas costimulation through CD3+CD28 did not.     

CD8 T cell activation after intravenous administration of CD3×CD19 bispecific antibody in patients with non-Hodgkin lymphoma

A bispecific antibody directed to T and B cells (CD3×CD19 bsAb) was daily infused intravenously in escalating doses from 10 g up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed at _1/2 of 10.5 h with peak levels of 200–300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon , IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1ß in serum). No gross changess in histology or number of IL-2R⁺, CD4⁺ or CD8⁺ cells were found in the lymph nodes after therapy, but one patient showed activated CD8⁺ T cells within the tumour nodules. In conclusion, after intravenously administered CD3×CD19 bsAb only moderate toxicity was found, probably due to CD8⁺ T cell activation and cytokine release, without CD4⁺ T cell activation.