Regulating the adaptive immune response to blood-stage malaria: role of dendritic cells and CD4⁺Foxp3⁺ regulatory T cells

Although a clearer understanding of the underlying mechanisms involved in protection and immunopathology during blood-stage malaria has emerged, the mechanisms involved in regulating the adaptive immune response especially those required to maintain a balance between beneficial and deleterious responses remain unclear. Recent evidence suggests the importance of CD11c⁺ dendritic cells (DC) and CD4⁺Foxp3⁺ regulatory T cells in regulating immune responses during infection and autoimmune disease, but information concerning the contribution of these cells to regulating immunity to malaria is limited. Here, we review recent findings from our laboratory and others in experimental models of malaria in mice and in Plasmodium-infected humans on the roles of DC and natural regulatory T cells in regulating adaptive immunity to blood-stage malaria.     

Partial depletion of natural CD4⁺CD25⁺ regulatory T cells with anti-CD25 antibody does not alter the course of acute influenza A virus infection

Foxp3⁺ CD4⁺ regulatory T cells represent a T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, and there is emerging evidence to suggest these cells temper pulmonary inflammation in response to acute viral infection. Recent studies have demonstrated treatment with PC61 CD25-depleting antibody potentiates inflammation in a murine model of RSV infection, while paradoxically delaying recruitment of CD8⁺ T cells to the site of inflammation. The present study therefore sought to examine the role of these cells in a murine model of acute influenza A virus infection through the administration of PC61 CD25-depleting antibody. PC61 antibody is able to partially deplete CD25⁺Foxp3⁺ regulatory T cells to a comparable degree as seen within previous work examining RSV, however this does not alter influenza A-virus induced mortality, weight loss, viral clearance and cellularity within the lung. Collectively, these data demonstrate that partial depletion of CD4⁺CD25⁺ regulatory T cells with PC61 antibody does not alter the course of influenza A virus infection.     

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.     

Downregulation of CD4＋CD25＋ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.     

HSP70 Enhances Immunosuppressive Function of CD4+CD25+FoxP3+ T Regulatory Cells and Cytotoxicity in CD4+CD25? T Cells

Human CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4⁺CD25⁻ target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4⁺CD25⁻ cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.     

Depletion of CD25⁺ cells during acute toxoplasmosis does not significantly increase mortality in Swiss OF1 mice

The interleukin (IL)-2R alpha chain (CD25) is expressed on regulatory T cells (Treg), which constitute more than 85% of the CD25+ T cell population in a na?ve mouse. CD25 is also expressed on effector T cells in mice suffering from an acute infection by the obligate intracellular protozoan parasite, Toxoplasma gondii. Lethal toxoplasmosis is accompanied by a significant loss of Treg in mice naturally susceptible to toxoplasmosis. The present study was done to explore the role of Treg cells using an anti-CD25 antibody-mediated depletion in mice naturally resistant to toxoplasmosis. Although a significant decrease in the percentage of Treg cells was observed following anti-CD25 monoclonal antibody injections, the depletion of CD25+ cells during acute toxoplasmosis did not significantly increase the mortality of Swiss OF1 mice and no significant difference was observed in the brain parasitic load between the mice in the depleted-infected and isotype-infected groups. We found no significant difference between the titres of total IgG in the sera of the mice from the two groups in the chronic phase. However, CD25+ cells depletion was followed by significantly higher levels of IL-12 in the serum of depleted mice than in that of mice injected with the isotype control antibody.     

Peroxisome proliferator-activated receptor α and γ agonists together with TGF-β convert human CD4+CD25- T cells into functional Foxp3+ regulatory T cells

Human peripheral CD4(+)CD25(-) T cells can be induced to express Foxp3 when activated in vitro by TCR stimulation with TGF-β and IL-2. However, these TGF-β-induced Foxp3(+) regulatory T cells (iTregs) lack a regulatory phenotype. From libraries of nuclear receptor ligands and bioactive lipids, we screened three peroxisome proliferator-activated receptor (PPAR)α (bezafibrate, GW7647, and 5,8,11,14-eicosatetraynoic acid) and two PPARγ agonists (ciglitazone and 15-deoxy-Δ-(12,14)-PG J(2)) as molecules that increased Foxp3 expression in human iTregs significantly compared with that in DMSO-treated iTregs (control). These PPARα and PPARγ agonist-treated iTregs maintained a high level of Foxp3 expression and had suppressive properties. There were no significant differences in the suppressive properties of iTregs treated with the three PPARα and two PPARγ agonists, and all of the treated iTregs increased demethylation levels of the Foxp3 promoter and intronic conserved noncoding sequence 3 regions. Furthermore, PPARα and PPARγ agonists, together with TGF-β, more strongly inhibited the expression of all three DNA methyltransferases (DNMTs) (DNMT1, DNMT3a, and DNMT3b) in activated CD4(+) T cells. These results demonstrate that PPARα and PPARγ agonists together with TGF-β elicit Foxp3 DNA demethylation through potent downregulation of DNMTs and induce potent and stable Foxp3 expression, resulting in the generation of functional iTregs. Moreover, trichostatin A and retinoic acid enhanced the generation of iTregs synergistically with PPARα and PPARγ agonists.     

In vivo expansion of naïve CD4+ CD25(high) FOXP3+ regulatory T cells in patients with colorectal carcinoma after IL-2 administration

Regulatory T cells (T_reg cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of T_reg cells was established. In IL-2 treated cancer patients a further T_reg-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional T_reg cells of a naïve phenotype - as determined by CCR7 and CD45RA expression - are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve T_reg-cell pool. Higher frequencies of T-cell receptor excision circles in naïve T_reg cells indicate IL-2 dependent thymic generation of naïve T_reg cells as a mechanism leading to increased frequencies of T_reg cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine T_reg cells after IL-2 administration. These results point to a more complex regulation of T_reg cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve T_reg 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.     

Induction of IL-10 and TGFβ from CD4+CD25+FoxP3+ T Cells Correlates with Parasite Load in Indian Kala-azar Patients Infected with Leishmania donovani

Background

Visceral leishmaniasis (VL) is distinguished by a complex interplay of immune response and parasite multiplication inside host cells. However, the direct association between different immunological correlates and parasite numbers remains largely unknown.

Methodology/Principal Findings

We examined the plasma levels of different disease promoting/protective as well as Th17 cytokines and found IL-10, TGFβ and IL-17 to be significantly correlated with parasite load in VL patients (r = 0.52, 0.53 and 0.51 for IL-10, TGFβ and IL-17, respectively). We then extended our investigation to a more antigen-specific response and found leishmanial antigen stimulated levels of both IL-10 and TGFβ to be significantly associated with parasite load (r = 0.71 and 0.72 for IL-10 and TGFβ respectively). In addition to cytokines we also looked for different cellular subtypes that could contribute to cytokine secretion and parasite persistence. Our observations manifested an association between different Treg cell markers and disease progression as absolute numbers of CD4⁺CD25⁺ (r = 0.55), CD4⁺CD25^hi (r = 0.61) as well as percentages of CD4⁺CD25⁺FoxP3⁺ T cells (r = 0.68) all correlated with parasite load. Encouraged by these results, we investigated a link between these immunological components and interestingly found both CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ Treg cells to secrete significantly (p<0.05) higher amounts of not only IL-10 but also TGFβ in comparison to corresponding CD25^- T cells.

Conclusions/Significance

Our findings shed some light on source(s) of TGFβ and suggest an association between these disease promoting cytokines and Treg cells with parasite load during active disease. Moreover, the direct evidence of CD4⁺CD25⁺FoxP3⁺ Treg cells as a source of IL-10 and TGFβ during active VL could open new avenues for immunotherapy towards cure of this potentially fatal disease.     

Effects of acute and chronic endurance exercise on intracellular nitric oxide and superoxide in circulating CD34⁺ and CD34⁻ cells

We investigated the influence of acute and chronic endurance exercise on levels of intracellular nitric oxide (NO), superoxide (O?·?), and expression of genes regulating the balance between these free radicals in CD34? and CD34? peripheral blood mononuclear cells (PBMCs; isolated by immunomagnetic cell separation). Blood samples were obtained from age- and body mass index (BMI)-matched endurance-trained (n = 10) and sedentary (n = 10) men before and after 30 min of exercise at 75% maximal oxygen uptake (·VO(?max)). Baseline levels of intracellular NO (measured by DAF-FM diacetate) and O?·? (measured by dihydroethidium) were 26% (P < 0.05) and 10% (P < 0.05) higher, respectively, in CD34? PBMCs from the sedentary group compared with the endurance-trained group. CD34? PBMCs from the sedentary group at baseline had twofold greater inducible nitric oxide synthase (iNOS) mRNA and 50% lower endothelial NOS (eNOS) mRNA levels compared with the trained group (P < 0.05). The baseline group difference in O?·? was eliminated by acute exercise. Experiments with apocynin indicated that the training-related difference in O?·? levels was explained by increased NADPH oxidase activity in the sedentary state. mRNA levels of additional angiogenic and antioxidant genes were consistent with a more angiogenic profile in CD34? cells of trained subjects. CD34? PBMCs, examined for exploratory purposes, also displayed a more angiogenic mRNA profile in trained subjects, with vascular endothelial growth factor (VEGF) and eNOS being more highly expressed in trained subjects. Overall, our data suggest an association between the sedentary state and increased nitro-oxidative stress in CD34? cells.     

CD4+CD25+FOXP3+ T cells,Foxp3 gene and protein expression contribute to antiasthmatic effects of San’ao decoction in mice model of asthma

ObjectiveSan’ao decoction (SAD) is a commonly used traditional combinatorial formula composed of Herba Ephedrae, Radix Glycyrrhizae and Amygdalus Communis Vas. Early studies showed that in the OVA sensitization asthmatic mice model its compatibility could lower airway reactivity and airway inflammatory cell infiltration. Based on the above results, this study mainly discussed San’ao decoction's immunomodulatory effects on Tregs.MethodsUPLC–PDA–TOF-MS was applied to analyze chemicals of SAD, and under the optimized chromatographic and MS condition, the major components in SAD were well separated and detected within 22 min. An asthma model was established in BALB/c mice that were sensitized and challenged with ovalbumin. After 2 weeks’ treatment, peripheral blood and bronchoalveolar lavage fluid (BALF) were assessed for inflammatory cell counts; histological change of lung tissue were detected; flow cytometry detection of splenic CD4⁺CD25⁺Foxp3⁺ Treg cells of the mice were counted; Foxp3 expression in lung tissues were examined as well.Results22 Peaks signal chemical components in SAD were identified by UPLC–QTO-MS method. In terms of the percentage of eosinophile in peripheral blood and bronchoalveolar lavage fluid (BALF), SAD groups were significantly lower (p < 0.01) than model group. Compared with model group, lung histological changes of SAD groups were reduced; the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in CD4⁺ cells of asthmatic mice also decreased; SAD significantly increased the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells and promoted Foxp3 expression in a mouse model of asthma.ConclusionThese results suggest that the antiasthmatic effects of SAD are at least partially associated with CD4⁺CD25⁺Foxp3⁺ Treg cells.     

Transmission of clonal hepatitis C virus genomes reveals the dominant but transitory role of CD8⁺ T cells in early viral evolution

The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8(+) T cells is a dominant force driving early amino acid replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8(+) T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute-phase nonsynonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome. Moreover, the ratio of nonsynonymous to synonymous mutations, a measure of positive selection pressure, was increased 50-fold in class I epitopes compared with the rest of the HCV genome. Finally, some mutation of the clonal H77C genome toward a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the majority of these amino acid substitutions occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8(+) T cells. This influence of immune pressure on viral evolution appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.     

The capacity of CD4+ Vγ9Vδ2 T cells to kill cancer cells correlates with co-expression of CD56

Human Vγ9Vδ2 T cells are a unique T-cell type, and data from recent studies of Vγ9Vδ2 T cells emphasize their potential relevance to cancer immunotherapy. Vγ9Vδ2 T cells exhibit dual properties since they are both antigen-presenting cells and cytotoxic toward cancer cells. The majority of Vγ9Vδ2 T cells are double-negative for the co-receptors CD4 and CD8, and only 20–30% express CD8. Even though they are mostly neglected, a small fraction of Vγ9Vδ2 T cells also express the co-receptor CD4. Here the authors show that CD4⁺ Vγ9Vδ2 T cells comprise 0.1–7% of peripheral blood Vγ9Vδ2 T cells. These cells can be expanded in vitro using zoledronic acid, pamidronic acid or CD3 antibodies combined with IL-2 and feeder cells. Unlike most conventional CD4⁺ αβ T cells, CD4⁺ Vγ9Vδ2 T cells are potently cytotoxic and can kill cancer cells, which is here shown by the killing of cancer cell lines of different histological origins, including breast cancer, prostate cancer and melanoma cell lines, upon treatment with zoledronic acid. Notably, the killing capacity of CD4⁺ Vγ9Vδ2 T cells correlates with co-expression of CD56.