Natural Regulatory CD4 Research Articles

Prolongation of allograft survival by passenger donor regulatory T cells.

Tissue resident lymphocytes are present within many organs, and are presumably transferred at transplantation, but their impact on host immunity is unclear. Here, we examine whether transferred donor natural regulatory CD4 T cells (nT‐regs) inhibit host alloimmunity and prolong allograft survival. Transfer of donor‐strain lymphocytes was first assessed by identifying circulating donor‐derived CD4 T cells in 21 consecutive human lung transplant recipients, with 3 patterns of chimerism apparent: transient, intermediate, and persistent (detectable for up to 6 weeks, 6 months, and beyond 1 year, respectively). The potential for transfer of donor nT‐regs was then confirmed by analysis of leukocyte filters recovered from ex vivo normothermic perfusion circuits of human kidneys retrieved for transplantation. Finally, in a murine model of cardiac allograft vasculopathy, depletion of donor CD4 nT‐regs before organ recovery resulted in markedly accelerated heart allograft rejection and augmented host effector antibody responses. Conversely, adoptive transfer or purified donor‐strain nT‐regs inhibited host humoral immunity and prolonged allograft survival, and more effectively so than following administration of recipient nT‐regs. In summary, following transplantation, passenger donor‐strain nT‐regs can inhibit host adaptive immune responses and prolong allograft survival. Isolated donor‐derived nT‐regs may hold potential as a cellular therapy to improve transplant outcomes.

Distinct Roles for CD4+ Foxp3+ Regulatory T Cells and IL-10-Mediated Immunoregulatory Mechanisms during Experimental Visceral Leishmaniasis Caused by Leishmania donovani.

The outcome of intracellular parasitic infection can be determined by the immunoregulatory activities of natural regulatory CD4+ Foxp3+ T (Treg) cells and the anti-inflammatory cytokine IL-10. These mechanisms protect tissue but can also suppress antiparasitic CD4+ T cell responses. The specific contribution of these regulatory pathways during human parasitic diseases remains unclear. In this study, we investigated the roles of Treg cells and IL-10 during experimental visceral leishmaniasis caused by Leishmania donovani infection of C57BL/6 mice. We report only a limited contribution of Treg cells in suppressing antiparasitic immunity, but important roles in delaying the development of splenic pathology and restricting leukocyte expansion. We next employed a range of cell-specific, IL-10- and IL-10R-deficient mice and found these Treg cell functions were independent of IL-10. Instead, conventional CD4+ T cells and dendritic cells were the most important cellular sources of IL-10, and the absence of IL-10 in either cell population resulted in greater control of parasite growth but also caused accelerated breakdown in splenic microarchitecture. We also found that T cells, dendritic cells, and other myeloid cells were the main IL-10-responding cells because in the absence of IL-10R expression by these cell populations, there was greater expansion of parasite-specific CD4+ T cell responses associated with improved control of parasite growth. Again, however, there was also an accelerated breakdown in splenic microarchitecture in these animals. Together, these findings identify distinct, cell-specific, immunoregulatory networks established during experimental visceral leishmaniasis that could be manipulated for clinical advantage.

Strain differences in thymic atrophy in rats immunized for EAE correlate with the clinical outcome of immunization

An accumulating body of evidence suggests that development of autoimmune pathologies leads to thymic dysfunction and changes in peripheral T-cell compartment, which, in turn, perpetuate their pathogenesis. To test this hypothesis, thymocyte differentiation/maturation in rats susceptible (Dark Agouti, DA) and relatively resistant (Albino Oxford, AO) to experimental autoimmune encephalomyelitis (EAE) induction was examined. Irrespective of strain, immunization for EAE (i) increased the circulating levels of IL-6, a cytokine causally linked with thymic atrophy, and (ii) led to thymic atrophy reflecting partly enhanced thymocyte apoptosis associated with downregulated thymic IL-7 expression. Additionally, immunization diminished the expression of Thy-1, a negative regulator of TCRαβ-mediated signaling and activation thresholds, on CD4+CD8+ TCRαβlo/hi thymocytes undergoing selection and thereby impaired thymocyte selection/survival. This diminished the generation of mature CD4+ and CD8+ single positive TCRαβhi thymocytes and, consequently, CD4+ and CD8+ recent thymic emigrants. In immunized rats, thymic differentiation of natural regulatory CD4+Foxp3+CD25+ T cells (nTregs) was particularly affected reflecting a diminished expression of IL-7, IL-2 and IL-15. The decline in the overall thymic T-cell output and nTreg generation was more pronounced in DA than AO rats. Additionally, differently from immunized AO rats, in DA ones the frequency of CD28- cells secreting cytolytic enzymes within peripheral blood CD4+ T lymphocytes increased, as a consequence of thymic atrophy-related replicative stress (mirrored in CD4+ cell memory pool expansion and p16INK4a accumulation). The higher circulating level of TNF-α in DA compared with AO rats could also contribute to this difference. Consistently, higher frequency of cytolytic CD4+ granzyme B+ cells (associated with greater tissue damage) was found in spinal cord of immunized DA rats compared with their AO counterparts. In conclusion, the study indicated that strain differences in immunization-induced changes in thymopoiesis and peripheral CD4+CD28- T-cell generation could contribute to rat strain-specific clinical outcomes of immunization for EAE.

Thymic hyperplasia after chemotherapy in adults with mature B cell lymphoma and its influence on thymic output and CD4+ T cells repopulation

ABSTRACTTo investigate the thymic regenerative potential in adults accepting chemotherapy for lymphoma. The dynamics of thymic activity in 54 adults from baseline to 12 mo post-chemotherapy was analyzed by assessing thymic structural changes with serial computed tomography (CT) scans, and correlating these with measurements of thymic output by concurrent analysis of single-joint (sj) T-cell receptor excision circles (sjTREC) and CD31+ recent thymic emigrants (RTE) in peripheral blood. Furthermore, the consequence of thymic renewal on peripheral CD4+ T cell recovery after chemotherapy was evaluated. Time-dependent changes of thymic size and thymic output assessed by both sjTREC levels and CD31+ RTE counts in peripheral blood were observed during and after chemotherapy. Enlargement of thymus over baseline following chemotherapy regarded as rebound thymic hyperplasia (TH) was identified in 20 patients aged 18−53 y (median 33 y). By general linear models repeated measure analysis, it was found that, patients with TH (n = 20) had a faster recovery of sjTREC levels and CD31+ RTE counts after chemotherapy than patients with comparable age, gender, diagnosis, disease stage, thymic volume and output function at baseline but without TH (n = 18) (p = 0.035, 0.047); besides, patients with TH had a faster repopulation of both naïve CD4+ T cell and natural regulatory CD4+ T cell subsets than those without TH (p = 0.042, 0.038). These data suggested that adult thymus retains the capacity of regeneration after chemotherapy, especially in young adults. The presence of TH could contribute to the renewal of thymopoiesis and the replenishment of peripheral CD4+ T cell pool following chemotherapy in adults.

MicroRNAs in CD4+ T cell subsets are markers of disease risk and T cell dysfunction in individuals at risk for type 1 diabetes

MicroRNAs (miRNAs) regulate T cell development and function and the disruption of miRNAs in natural regulatory CD4+ FOXP3+ T cells (nTreg) leads to autoimmune disease in mice. To investigate miRNA expression in relation to autoimmune disease risk in humans we sequenced them in purified CD4+ T cell subsets from individuals at high risk of type 1 diabetes (pre-T1D), as well as other healthy individuals. Differences in miRNA expression patterns were observed between specific T cell subsets and, within subsets, between pre-T1D and healthy individuals. Compared to healthy, naive CD4+ T cells in pre-T1D displayed 32 differentially expressed miRNAs, potentially a template for altered miRNA expression in effector memory T cells in T1D. Naive nTreg in pre-T1D displayed two differentially expressed miRNAs, Let-7c and miR-15a. In contrast, nTreg activated in vivo displayed a large number of differentially expressed miRNAs, revealing a pro-inflammatory and FOXP3-repressive signature. Differential expression of specific miRNAs was also a signpost to altered T cell function. For example, in pre-T1D, increased expression of miR-26a in nTreg activated in vivo or in vitro was associated with decreased expression of its target, the histone methyltransferase EZH2. Chemical inhibition of EZH2 decreased the number of activated naïve nTreg and their expression of nTreg signature genes FOXP3 and TIGIT. Our findings demonstrate that miRNAs differentially expressed in CD4+ T cell subsets are markers of risk and T cell dysfunction in T1D.

Prophylactic use of low-dose interleukin-2 early post-transplantation: a randomised study

BackgroundEvidence suggests that natural killer cells and regulatory CD4+CD25+FoxP3+ T (Treg) cells, which are expanded and stimulated by the application of interleukin-2, could mediate protection against graft-versus-host disease (GVHD) while maintaining graft anti-tumour activity. The aim of this study was to confirm that a low dose of interleukin-2 can prevent relapse while reducing chronic GVHD. This study has been presented in part as an oral presentation at the 56th American Society of Hematology Annual Meeting and Exposition (Dec 6, 2014) in San Francisco, CA, USA. MethodsIn this open-label, prospective, randomised controlled trial in a single centre in China, donor–recipient pairs were randomly assigned (1:1) into the interleukin-2 group and control group by the sub-investigator (treating physician of our transplant centre) according to a computer-generated randomisation system. We enrolled patients (aged ≥15 years) with standard-risk leukaemia (except Ph+ ALL and T-ALL) who had undergone unmanipulated allogeneic stem cell transplantation. The transplantation procedure for control group patients was the same as that of the interleukin-2 group, except for interleukin-2 treatment. Interleukin-2 treatment (daily 1×106 IU/m2 body-surface area) was repeated every 14 days for six courses until disease progression or unacceptable toxicity. The primary endpoint was the cumulative incidence of haematological relapse. The primary efficacy end point was a between-group comparison of the time from transplantation to relapse in the intention-to-treat population (all randomly assigned patients). 360 subjects were estimated to be needed to detect a 10% difference between groups in cumulative incidence of haematological relapse based on our previous results (from 20% to 10%) with a one-sided α of 0·05 and a power of 80%. Futility analyses after a quarter, half, and three-quarters of 360 cases were specified. This study was approved by the Institutional Review Board of Peking University, and written informed consent was obtained from all patients in accordance with the Declaration of Helsinki. This trial is registered with ClinicalTrials.gov, number NCT01517347. FindingsSince this study began in January, 2012, we have randomly assigned 43 patients to the interleukin-2 group and 47 patients to the control group. First interim analysis was done when 90 cases were enrolled. The cumulative incidence of haematological relapse of the interleukin-2 and control cohorts were 27% (95% CI 18–35%) and 9% (5–12%), respecitively; p=0·12). Low dose of interleukin-2 augmented reconstituted Treg cells and natural killer cells. The suppressive functions of interleukin-2-expanded Treg cells in inhibiting T-cell activation were comparable to healthy donors. Cytotoxicity of interleukin-2-expanded natural killer cells against K562 cells as well as against resting or activated conventional T cells (Tcon) was significantly enhanced compared to control group. Patients with severe chronic GVHD had a lower ratio of Treg to Tcon cells at 2 months and 3 months, and a lower ratio of CD56dim to CD56bri NK cells from 2 months to 5 months after allogeneic stem cell transplantation. Interleukin-2 therapy was well tolerated; the most common adverse event was fever. InterpretationLow dose of interleukin-2 given post-transplant did not decrease the cumulative incidence of haematological relapse and was associated with a higher frequency of an MRD-positive test and a lower frequency of chronic GVHD, which might be through expansion of Treg cells. Thus, the prophylactic application of interleukin-2 should be cautious and its effect on leukaemia relapse needs to be further explored. FundingThe National Natural Science Foundation of China, the Major State Basic Research Development Program of China, and Milstein Medical Asian American Partnership (MMAAP) Foundation Research Project Award in Hematology.

Neuropilin-1+ regulatory T cells promote skin allograft survival and modulate effector CD4+ T cells phenotypic signature.

During allograft rejection, several immune cell types, including dendritic cells, CD4(+) and CD8(+) T cells among others, recirculate between the graft and the nearest draining lymph node, resulting in immunity against the 'foreign' tissue. Regulatory CD4(+) T cells are critical for controlling the magnitude of the immune response and may act to promote or maintain tolerance. They are characterized by the expression of CD25 and Foxp3, and more recently, Neuropilin-1 (Nrp1). The role of these suppressor cells during allograft rejection is not well understood. Our work shows that during graft rejection, there is an increase in the frequency of total CD4(+) T cells expressing Nrp1, but the expression of this molecule is downregulated in the regulatory CD4(+) T-cell compartment. Interestingly, the expression of the transcription factor Eos, which renders cell function stability, is also reduced. In adoptive transfer experiments, we observed that during allograft rejection: (i) natural regulatory CD4(+) T cells maintain high levels of Nrp1 expression, (ii) effector CD4(+) T cells (Nrp1(-)) become Nrp1(+)Eos(+) and (iii) the transfer of regulatory CD4(+) T cells (Nrp1(+)) can promote allograft survival, and also enhance the gain of Nrp1 and Eos on T-effector cells. Together, these data suggest that rejection occurs, at least in part, through the loss of Nrp1 expression on regulatory CD4(+) T cells, their stability or both. Additionally, the transfer of regulatory CD4(+) T cells (based on Nrp1 expression) permits the acceptance of the allograft, placing Nrp1 as a new target for immune therapy.

Foxp3-independent loss of regulatory CD4+T-cell suppressive capacities induced by self-deprivation

In the periphery, Foxp3 expression is considered sufficient to maintain natural regulatory CD4(+) T-cell suppressive function. In this study, we challenge this model. Indeed, in mouse chimeras in which major histocompatibility complex (MHC) class II expression is restricted to the thymus, peripheral regulatory CD4(+) T cells lack suppressive activity. In addition, regulatory CD4(+) T cells recovered 5 days after transfer into recipient mice lacking expression of MHC class II molecules (self-deprived) are unable to inhibit the proliferative response of conventional CD4(+) T cells both in vitro and in vivo. Disruption of TCR/MHC class II interactions rapidly leads to alterations in the regulatory CD4(+) T-cell phenotype, the ability to respond to stimulation and to produce interleukin-10, and the transcriptional signature. Interestingly, self-deprivation does not affect Foxp3 expression indicating that in regulatory CD4(+) T cells, self-recognition induces unique transcriptional and functional features that do not rely on Foxp3 expression.

Haematological parameters, natural regulatory CD4 + CD25 + FOXP3+ T cells and γδ T cells among two sympatric ethnic groups having different susceptibility to malaria in Burkina Faso

BackgroundFulani ethnic group individuals are less susceptible than sympatric Mossi ethnic group, in term of malaria infection severity, and differ in antibody production against malaria antigens. The differences in susceptibility to malaria between Fulani and Mossi ethnic groups are thought to be regulated by different genetic backgrounds and offer the opportunity to compare haematological parameters, Tregs and γδT cell profiles in seasonal and stable malaria transmission settings in Burkina Faso. The study was conducted at two different time points i.e. during the high and low malaria transmission period.ResultsTwo cross-sectional surveys were undertaken in adults above 20 years belonging either to the Fulani or the Mossi ethnic groups 1) at the peak of the malaria transmission season and 2) during the middle of the low malaria transmission season. Full blood counts, proportions of Tregs and γδ T cells were measured at both time-points.As previously shown the Fulani and Mossi ethnic groups showed a consistent difference in P. falciparum infection rates and parasite load. Differential white blood cell counts showed that the absolute lymphocyte counts were higher in the Mossi than in the Fulani ethnic group at both time points. While the proportion of CD4+CD25high was higher in the Fulani ethnic group at the peak of malaria transmission season (p = 0.03), no clear pattern emerged for T regulatory cells expressing FoxP3+ and CD127low. However CD3+γδ+ subpopulations were found to be higher in the Fulani compared to the Mossi ethnic group, and this difference was statistically significant at both time-points (p = 0.004 at low transmission season and p = 0.04 at peak of transmission).ConclusionOur findings on regulatory T cell phenotypes suggest an interesting role for immune regulatory mechanisms in response to malaria. The study also suggests that TCRγδ + cells might contribute to the protection against malaria in the Fulani ethnic group involving their reported parasite inhibitory activities.

The role of foxp3-expressing regulatory T cells and T helpers in immunopathogenesis of multidrug resistant pulmonary tuberculosis.

Subpopulation structure of regulatory T cells and T helpers of peripheral blood in patients with newly diagnosed pulmonary tuberculosis depending on the clinical form of disease and sensitivity of Mycobacterium tuberculosis to antituberculosis drugs has been analyzed in this work. It has been shown that the leading part in immune suppression at infiltrative, dissemination, and fibrosis-cavity pulmonary tuberculosis is played by natural regulatory CD4+CD25+Foxp3+-T lymphocytes. Thus we estimate increase of their number in blood by drug-resistance and drug-susceptible patients. It has been demonstrated that in patients with fibrocavernous and infiltrative form of the disease and drug-resistant pulmonary tuberculosis the number of CD4+CD25−Foxp3+-regulatory T cells was increasing. In patients with infiltrative pulmonary tuberculosis, including multidrug-resistant M. tuberculosis, an increased number of CD3+CD4+CD25− T helpers is determined by the pathogenic features of the development of the tuberculosis infection and is connected with the activation of Th1-dependent immune response. Reduction in the number of T-helpers in the blood of patients with dissemination and fibrosis-cavity pulmonary tuberculosis mediates inefficient implementation of cell-mediated protective immunity.

Subpopulation structure of regulatory Т-cells of blood in patients with multiple drugresistant pulmonary tuberculosis

Subpopulation structure of regulatory T-cells of peripheral blood in patients with newly diagnosed pulmonary tuberculosis depending on the clinical form of disease and sensitivity of Mycobacterium tuberculosis to anti-tuberculosis drugs have been analyzed in this work. It has been shown that in formation immune suppression at infiltrative, dissemination and fibrosis-cavity pulmonary tuberculosis the leading part play natural regulatory CD4+ CD25+ Foxр3+ -T-lymphocytes. Thus increase of their number in blood at drug-resistance and drugsusceptible patients. It has been demonstrated that in patients with fibro-cavernous and infiltrative form of the disease and drug-resistance pulmonary tuberculosis the number of CD4+ CD25- Foxр3+ -regulatory T-cells were increasing.

LAG-3 Expression Defines a Subset of CD4+CD25highFoxp3+ Regulatory T Cells That Are Expanded at Tumor Sites

Human natural regulatory CD4(+) T cells comprise 5-10% of peripheral CD4(+)T cells. They constitutively express the IL-2Ralpha-chain (CD25) and the nuclear transcription Foxp3. These cells are heterogeneous and contain discrete subsets with distinct phenotypes and functions. Studies in mice report that LAG-3 has a complex role in T cell homeostasis and is expressed in CD4(+)CD25(+) T regulatory cells. In this study, we explored the expression of LAG-3 in human CD4(+) T cells and found that LAG-3 identifies a discrete subset of CD4(+)CD25(high)Foxp3(+) T cells. This CD4(+)CD25(high)Foxp3(+)LAG-3(+) population is preferentially expanded in the PBMCs of patients with cancer, in lymphocytes of tumor-invaded lymph nodes and in lymphocytes infiltrating visceral metastasis. Ex vivo analysis showed that CD4(+)CD25(high)Foxp3(+)LAG-3(+) T cells are functionally active cells that release the immunosuppressive cytokines IL-10 and TGF-beta1, but not IL-2. An in vitro suppression assay using CD4(+)CD25(high)LAG-3(+) T cells sorted from in vitro expanded CD4(+)CD25(high) regulatory T cells showed that this subset of cells is endowed with potent suppressor activity that requires cell-to-cell contact. Our data show that LAG-3 defines an active CD4(+)CD25(high)Foxp3(+) regulatory T cell subset whose frequency is enhanced in the PBMCs of patients with cancer and is expanded at tumor sites.

In vivo expansion of naive and activated CD4 + CD25 + FOXP3 + regulatory T cell populations in interleukin-2–treated HIV patients

HIV-1 infection is characterized by a progressive decline in CD4(+) T cells leading to a state of profound immunodeficiency. IL-2 therapy has been shown to improve CD4(+) counts beyond that observed with antiretroviral therapy. Recent phase III trials revealed that despite a sustained increase in CD4(+) counts, IL-2-treated patients did not experience a better clinical outcome [Abrams D, et al. (2009) N Engl J Med 361(16):1548-1559]. To explain these disappointing results, we have studied phenotypic, functional, and molecular characteristics of CD4(+) T cell populations in IL-2-treated patients. We found that the principal effect of long-term IL-2 therapy was the expansion of two distinct CD4(+)CD25(+) T cell populations (CD4(+)CD25(lo)CD127(lo)FOXP3(+) and CD4(+)CD25(hi)CD127(lo)FOXP3(hi)) that shared phenotypic markers of Treg but could be distinguished by the levels of CD25 and FOXP3 expression. IL-2-expanded CD4(+)CD25(+) T cells suppressed proliferation of effector cells in vitro and had gene expression profiles similar to those of natural regulatory CD4(+)CD25(hi)FOXP3(+) T cells (Treg) from healthy donors, an immunosuppressive T cell subset critically important for the maintenance of self-tolerance. We propose that the sustained increase of the peripheral Treg pool in IL-2-treated HIV patients may account for the unexpected clinical observation that patients with the greatest expansion of CD4(+) T cells had a higher relative risk of clinical progression to AIDS.

Conserved anti-proliferative effect and poor inhibition of TNFα secretion by regulatory CD4+CD25+ T cells in patients with systemic lupus erythematosus

A role of natural regulatory CD4+CD25+ T cells in peripheral tolerance has been established, but a causative role of these cells has not been proved in human autoimmune diseases. Functional alterations have been reported in arthritis and contradictory results have been published in systemic lupus erythematosus (SLE). In this study, the CD4+CD25+ T cell function was studied in 12 SLE patients (6 with only antimalarials and 6 also with corticoids treatment) in parallel to 12 healthy controls. CD4+CD25- T cells were cocultured with allogenic monocyte derived dendritic cells (MDDC) and CD4+CD25+ T cells were added. Proliferation was measured by [3H]-thymidine incorporation and TNFalpha secretion was assessed by ELISA. A better anti-proliferative function of CD4+CD25+ T cells was found in patients than in controls (p=0.009), whereas higher TNFalpha secretion was found in patients (p=0.028). There were no significant differences regarding treatment. In summary, CD4+CD25+ T cells from SLE patients showed an undamaged anti-proliferative function. Our results and other authors' have not proved a SLE causative role for CD4+CD25+ T cells. The reduced inhibitive function on TNFalpha secretion found in patients could have some implications in lupus pathogenesis.

Non-obese diabetic mice select a low-diversity repertoire of natural regulatory T cells

Thymus-derived Foxp3(+) natural regulatory CD4 T cells (nTregs) prevent autoimmunity through control of pathogenic, autoreactive T cells and other immune effector cells. Using T cell receptor (TCR) transgenic models, diversity within this lineage has been found to be similar to that of conventional CD4 T cells. To determine whether balanced TCR diversity may be perturbed in autoimmunity, we have analyzed receptor composition in C57BL/6 and autoimmune non-obese diabetic (NOD) mice. The natural regulatory and conventional CD4 repertoires of C57BL/6 had similar diversities. Despite the apparently normal thymic development of the NOD nTreg lineage, TCR diversity within the selected repertoire was markedly restricted. Detailed analysis of TCRalpha and -beta chain composition is consistent with positive selection into the natural regulatory lineage being under stringent audition for interaction with MHC class II/self-peptide. The NOD MHC region, including the unique H2-A(g7) class II molecule, partly accounts for the reduction in diversity, but additional NOD genetic contribution(s) are required for complete repertoire compaction. Mechanistic links between MHC, autoimmunity, and nTreg diversity identified in this study are discussed.

Functional Characterization and Gene Expression Analysis of CD4+CD25+ Regulatory T Cells Generated in Mice Treated with 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Although the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated through binding and activation of the aryl hydrocarbon receptor (AhR), the subsequent biochemical and molecular changes that confer immune suppression are not well understood. Mice exposed to TCDD during an acute B6-into-B6D2F1 graft-vs-host response do not develop disease, and recently this has been shown to correlate with the generation of CD4(+) T cells that express CD25 and demonstrate in vitro suppressive function. The purpose of this study was to further characterize these CD4(+) cells (TCDD-CD4(+) cells) by comparing and contrasting them with both natural regulatory CD4(+) T cells (T-regs) and vehicle-treated cells. Cellular anergy, suppressive functions, and cytokine production were examined. We found that TCDD-CD4(+) cells actively proliferate in response to various stimuli but suppress IL-2 production and the proliferation of effector T cells. Like natural T-regs, TCDD-CD4(+) cells do not produce IL-2 and their suppressive function is contact dependent but abrogated by costimulation through glucocorticoid-induced TNFR (GITR). TCDD-CD4(+) cells also secrete significant amounts of IL-10 in response to both polyclonal and alloantigen stimuli. Several genes were significantly up-regulated in TCDD-CD4(+) cells including TGF-beta3, Blimp-1, and granzyme B, as well as genes associated with the IL12-Rb2 signaling pathway. TCDD-CD4(+) cells demonstrated an increased responsiveness to IL-12 as indicated by the phosphorylation levels of STAT4. Only 2% of TCDD-CD4(+) cells express Foxp3, suggesting that the AhR does not rely on Foxp3 for suppressive activity. The generation of CD4(+) cells with regulatory function mediated through activation of the AhR by TCDD may represent a novel pathway for the induction of T-regs.

The Proportion of Natural Regulatory T-Cells CD4+CD25+Foxp3+ in Chronic Phase Chronic Myelogenous Leukemia Patients Correlates with Sokal Score at Diagnosis and Is Increased during Imatinib Mesylate Treatment.

Background. Natural regulatory T cells (nTregs) are key players in immune homeostasis and protection towards autoimmunity. There is also strong evidence that Tregs are involved in the immune recognition of tumor-related antigens. However, little is known about the role of nTregs in Chronic Myelogenous leukaemia (CML), an immunogenic tumor.Aims. The aim of this study is to determine if the proportions of nTregs are significantly modified in CML patients at diagnosis or during imatinib mesylate (IM) treatment when compared to healthy counterparts, and if they correlate with prognosis factors and/or tumor burden.Patients. Fourty-three CML patients were enrolled, 12 at diagnosis (median age 55 (range 32–77) years) and 31 during IM treatment (median age 56 (29–84) years) alone (n=21) or in combination with cytarabine (n=4) or Peg-interferon (n=6). Four patients received interferon before IM. They were compared to 13 healthy donors (median age 42 (21–56) years). We also analysed if the proportion of nTregs correlates with diagnosis (Sokal was low for 6, intermediate for 6 for the 12 patients analysed at diagnosis) or on-going prognosis factors (normalized bcr-abl/abl ratio using PB RQ-PCR).Methods. Mononuclear cells were separated from PB by density gradient centrifugation and subsequently frozen until flow cytometry analysis. Before use, the CD4+ T-cell population was positively purified with immunomagnetic microbeads. Using a sequential gating strategy, nTregs were identified as CD4+CD25+Foxp3+ T-cells and proportions were expressed as a percentage of total CD4+ T-cells. Statistical analyses are based on at least 30,000 events gated on CD4+CD25+ T-cells. All comparisons were adjusted with patients or donors age at sample collection.Results. We confirm that nTregs percentages increase with age both in patients and healthy donors (p=0.023). At diagnosis, whilst there is no significant difference in nTregs between patients and healthy donors, the percentage of nTregs is found to correlate with Sokal score with a marked increase of nTregs for patients with aggressive disease compared to patients with less aggressive disease at diagnosis (p<0.001). In patients under IM, a higher nTregs median percentage is found (6.88% +/− 2.56%) compared both with healthy donors (4.32% +/− 1.88%, p=0.035) or patients at diagnosis (4.06% +/− 2.23%, p=0.002). Lastly, while we do not find a correlation between nTregs and response to treatment assessed by RQ-PCR, we show that the percentage of nTregs decreases along the IM-treatment duration (p=0.024). No significant correlations were found between proportion of nTregs and association with cytarabine or peg-interferon, or with interferon prior IM. CML patients CD4+CD25+ nTregs functional analysis is pending.Conclusions. In summary, we show that a high proportion of nTregs in CML patients PB at diagnosis is significantly associated with higher Sokal scores which predicts poorer outcome. However, we do not demonstrate an increase in nTregs at CML diagnosis compared to healthy donors, as found in other malignant diseases. On the opposite, patients with IM present a significant increase in nTregs, which slightly decrease over time regardless of their response to treatment. Further experiments and incremental numbers of patients with high risk Sokal score are needed to confirm these preliminary data.

Natural regulatory (CD4 +CD25 +FOXP +) T cells control the production of pro-inflammatory cytokines during Plasmodium chabaudi adami infection and do not contribute to immune evasion

Different functions have been attributed to natural regulatory CD4 +CD25 +FOXP + (Treg) cells during malaria infection. Herein, we assessed the role for Treg cells during infections with lethal (DS) and non-lethal (DK) Plasmodium chabaudi adami parasites, comparing the levels of parasitemia, inflammation and anaemia. Independent of parasite virulence, the population of splenic Treg cells expanded during infection, and the absolute numbers of activated CD69 + Treg cells were higher in DS-infected mice. In vivo depletion of CD25 + T cells, which eliminated 80% of CD4 +FOXP3 +CD25 + T cells and 60–70% of CD4 +FOXP3 + T cells, significantly decreased the number of CD69 + Treg cells in mice with lethal malaria. As a result, higher parasite burden and morbidity were measured in the latter, whereas the kinetics of infection with non-lethal parasites remained unaffected. In the absence of Treg cells, parasite-specific IFN-γ responses by CD4 + T cells increased significantly, both in mice with lethal and non-lethal infections, whereas IL-2 production was only stimulated in mice with non-lethal malaria. Following the depletion of CD25 + T cells, the production of IL-10 by CD90 − cells was also enhanced in infected mice. Interestingly, a potent induction of TNF-α and IFN-γ production by CD4 + and CD90 − lymphocytes was measured in DS-infected mice, which also suffered severe anaemia earlier than non-depleted infected controls. Taken together, our data suggest that the expansion and activation of natural Treg cells represent a counter-regulatory response to the overwhelming inflammation associated with lethal P.c. adami. This response to infection involves TH1 lymphocytes as well as cells from the innate immune system.

Age‐related changes in the occurrence and characteristics of thymic CD4+ CD25+ T cells in mice

Natural regulatory CD4(+) CD25(+) T cells play an important role in preventing autoimmunity by maintaining self-tolerance. They express CD25 constitutively and are produced in the thymus as a functionally mature T-cell population. Changes in the potential of these cells to regulate the activity of conventional effector lymphocytes may contribute to an increased susceptibility to infection, cancer and age-associated autoimmune diseases. In this study we demonstrated that the thymi of aged mice are populated by a higher percentage of CD4(+) CD25(+) thymocytes than in young animals. The expression of several surface markers (CD69, CD5, CD28, CTLA-4, CD122, FOXP3), usually used to characterize the phenotype of CD4(+) CD25(+) T regulatory cells, was compared between young and aged mice. We also examined the ability of sorted thymus-deriving regulatory T cells of young and aged BALB/c mice to inhibit the proliferation of lymph node lymphocytes activated in vitro. Natural regulatory T cells isolated from the thymi of young mice suppress the proliferation of responder lymph node cells. We demonstrated that thymus-deriving CD4(+) CD25(+) T cells of old mice maintain their potential to suppress the proliferation of activated responder lymphocytes of young mice. However, their potential to inhibit the proliferation of old responder T cells is abrogated. Differences in the occurrence and activity of CD4(+) CD25(+) thymocytes between young and old animals are discussed in relation to the expression of these surface markers.

Antibody blocking of MHC II on human activated regulatory T cells abrogates their suppressive potential

Natural regulatory CD4(+)CD25(+)Foxp3(+) T cells control peripheral immune responses. Freshly isolated regulatory T-cell populations are regarded as being unable to suppress the proliferation of strongly stimulated effector T cells. We now provide evidence that it is not the strength of the proliferative signal to effector T cells but activation and accessibility of regulatory T cells that determine whether suppression may occur. Human regulatory T cells were initially cocultured with allogeneic monocyte-derived dendritic cells for a short time and were then rendered accessible for effector T cells by removal of the dendritic cells. That way activated regulatory T cells effectively suppressed the proliferation of autologous effector T cells which was strongly driven by cell-sized Dynabeads coated with CD3/CD28 antibodies. Although regulatory T cells are known to display MHC II molecules and to upregulate their expression along with activation, a role of MHC II molecules in forming the contact to effector T cells was not yet envisaged. However, blocking of MHC II on activated regulatory T cells abrogated their suppressive potential. It should not be excluded that self-MHC molecules on physically accessible activated regulatory T cells arrange the contact to effector T cells.