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IL-2-inducible T cell kinase (ITK) is a key signaling mediator downstream of TCR, mediating T cell positive selection, as well as innate T cell and CD4+ Th2/Th17 differentiation. In this article, we show that ITK also negatively tunes IL-2-induced expansion of conventional Foxp3-expressing regulatory T cells (Tregs). In vivo, Treg abundance is inversely correlated with ITK expression, and inducible Treg development is inversely dependent on ITK kinase activity. While Treg development normally requires both hematopoietic and thymic MHC class 2 (MHC2) expression, the absence of ITK allows Treg development with MHC2 expression in either compartment, with preference for selection by thymic MHC2, suggesting a gatekeeper role for ITK in ensuring that only Tregs selected by both thymic and hematopoietic MHC2 survive selection. Although ITK suppresses Treg development and is not required for maintenance of neuropilin-1-positive natural Tregs in the periphery, it is indispensable for Treg functional suppression of naive CD4+ T cell-induced colitis in Rag-/- recipients. ITK thus regulates the development and function of Tregs. [ABSTRACT FROM AUTHOR]

IL-2, a cytokine with pleiotropic effects, is critical for immune cell activation and peripheral tolerance. Although the therapeutic potential of IL-2 has been previously suggested in autoimmune diseases, the mechanisms whereby IL-2 mitigates autoimmunity and prevents organ damage remain unclear. Using an inducible recombinant adeno-associated virus vector, we investigated the effect of low systemic levels of IL-2 in lupus-prone MRL/Faslpr/lpr (MRL/lpr) mice. Treatment of mice after the onset of disease with IL-2-recombinant adeno-associated virus resulted in reduced mononuclear cell infiltration and pathology of various tissues, including skin, lungs, and kidneys. In parallel, we noted a significant decrease of IL-17-producing CD3+CD4-CD8- double-negative T cells and an increase in CD4+CD25+Foxp3+ immunoregulatory T cells (Treg) in the periphery. We also show that IL-2 can drive double-negative (DN) T cell death through an indirect mechanism. Notably, targeted delivery of IL-2 to CD122+ cytotoxic lymphocytes effectively reduced the number of DN T cells and lymphadenopathy, whereas selective expansion of Treg by IL-2 had no effect on DN T cells. Collectively, our data suggest that administration of IL-2 to lupus-prone mice protects against end-organ damage and suppresses inflammation by dually limiting IL-17-producing DN T cells and expanding Treg. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8+CD44high) T cells displaying a CD25−NKG2D+ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion. [ABSTRACT FROM AUTHOR]

Background: QuantiFERON-TB Gold In-Tube (QFT) is an IFNγ-release assay used in the diagnosis of Mycobacterium tuberculosis (MTB) infection. The risk of TB progression increases with the magnitude of the MTB-specific IFNγ-response. QFT reversion, also associated with low Tuberculin Skin Test responses, may therefore represent a transient immune response with control of M. tuberculosis infection. However, studies at the single cell level have suggested that the quality (polyfunctionality) of the T-cell response is more important than the quantity of cytokines produced. Objective: To explore the quality and/or magnitude of mycobacteria-specific T-cell responses associated with QFT reversion and persistent QFT-positivity. Methods: Multi-color flowcytometry on prospectively collected peripheral blood mononuclear cells was applied to assess mycobacteria-specific T-cell responses in 42 QFT positive Indian adolescents of whom 21 became QFT negative (reverters) within one year. Ten QFT consistent negatives were also included as controls. Results: There was no difference in the qualitative PPD-specific CD4+ T-cell response between QFT consistent positives and reverters. However, compared with QFT consistent positives, reverters displayed lower absolute frequencies of polyfunctional (IFNγ+IL2+TNFα+) CD4+ T-cells at baseline, which were further reduced to the point where they were not different to QFT negative controls one year later. Moreover, absolute frequencies of these cells correlated well with the magnitude of the QFT-response. Conclusion: Whereas specific polyfunctional CD4+ T-cells have been suggested to protect against TB progression, our data do not support that higher relative or absolute frequencies of PPD-specific polyfunctional CD4+ T-cells in peripheral blood can explain the reduced risk of TB progression observed in QFT reverters. On the contrary, absolute frequencies of these cells correlated with the QFT-response, suggesting that this readout reflects antigenic load. [ABSTRACT FROM AUTHOR]

Abstract: Background and methods: We showed previously that rabbit ATG induction induces a strong decrease of CD4+ T cells together with impaired in vitro IL-2 secretion up to 1year post-transplant. To further characterize long-term immunological effects of ATG induction 2 and 5years post-transplant, we used sensitive intracellular cytokine analysis in the same prospective study of 84 renal transplant recipients (ATG, n =44). Results: A significantly increased frequency of severe infectious disease (HR=2.0, p =0.027) as well as suppressed T cell functions were found within 2years after ATG induction but not beyond (logistic regression (logreg): CD4 cell IL-10 responses, p =0.064; T cell proliferation, p =0.038). Impaired T cell proliferation at 2years was associated with occurrence of severe infection (p =0.017). Importantly, a strong and persistent decrease of CD4 cell counts (p <0.0005 at 5years) was independently associated with ATG induction (logreg p =0.002) but not related to functional CD4 cell impairment (helper activity/cytokine production) or an increased risk of infection. Conclusions: Severe infection up to 2years after ATG induction was associated with impaired T cell proliferative capacity but not with the profound decline in CD4 cell counts that occurred after ATG induction and persisted up to 5years. [Copyright &y& Elsevier]

MiR-568 targets NFAT5 and inhibits human CD4+ T cells and Treg cellsCD4+ T cells play critical roles in orchestrating adaptive immune responses. Their activation and proliferation are critical steps that occur before they execute their biological functions. Despite the important role of this process, the underlying molecular events are not fully understood. MicroRNAs (miRNAs) have been shown to play important roles in lymphocyte development and function. However, the miRNAs that regulate T-cell differentiation, activation and proliferation are still largely unknown. In our previous study, using a miRNA array, we found that several miRNAs (including miR-202, 33b, 181c, 568 and 576) are differentially expressed between resting and activated CD4+ T cells. In this study, we focused on the function of miR-568 during CD4+ T-cell activation. We showed that the expression level of miR-568 decreased during the activation of T cells, including Jurkat cells and human peripheral blood CD4+ T cells. When Jurkat or human peripheral blood CD4+ T cells were transfected with miR-568 mimics, cell activation was significantly inhibited, as shown by the inhibited expression of activation markers such as CD25, CD69 and CD154; decreased IL-2 production; and inhibited cell proliferation. Using software predictions and confirmatory experiments, we demonstrated that nuclear factor of activated T cells 5 (NFAT5) is a target of miR-568. Treg cells are an important CD4+ T-cell subpopulation, so we also evaluated the function of miR-568 in Treg-cell activation and differentiation. We showed that the miR-568 level decreased, while the NFAT5 protein level increased during CD4+CD25+ Treg-cell activation, and the transfection of miR-568 mimics inhibited the NFAT5 expression, inhibited the production of both TGF-β and IL-10 and also inhibited the proliferation of Treg cells. Our further study showed that over-expression of miR-568 can inhibit Treg-cell differentiation and can inhibit the suppressive effect of these cells on effector cells. In addition, inhibition of NFAT5 by siRNA-mediated knockdown can inhibit the activation and differentiation of Treg cells. These findings reveal that miR-568 can inhibit the activation and function of both CD4+ T cells and Treg cells by targeting NFAT5. Since miR-568 plays an important role in both CD4+ T cells and Treg cells, these findings may provide leads for the development of novel treatments for human inflammatory and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Highlights: [•] MAP4K4 is required for the proliferation and activation of CD4+ T cells. [•] MAP4K4 deletion in CD4+ T cells suppresses the IL-2 and IFN-γ production. [•] MAP4K4 is essential for promoting the development of CD25+ CD4+ Tregs. [•] MAP4K4 deficiency prevents the phosphorylation of Erk, Jnk, p38 and PKC-θ. [ABSTRACT FROM AUTHOR]

Background The primary aim of this pilot study was to determine the feasibility and safety of an adoptive transfer and in vivo expansion of human haploidentical γδ T lymphocytes. Methods Patients with advanced haematological malignancies who are not eligible for allogeneic transplantation received peripheral blood mononuclear cells from half-matched family donors. For that, a single unstimulated leukapheresis product was incubated with both the anti-CD4 and anti-CD8 antibodies conjugated to paramagnetic particles. The depletion procedure was performed on a fully automated CliniMACS ® device according to the manufacturer's instructions. On average, patients received 2.17x106/kg (range 0.9-3.48) γδ T cells with <1% CD4- or CD8-positive cells remaining in the product. All patients received prior lymphopenia-inducing chemotherapy (fludarabine 20-25 mg/m2 day -6 until day -2 and cyclophosphamide 30-60 mg/kg day -6 and -5) and were treated with 4 mg zoledronate on day 0 and 1.0x106 IU/m2 IL-2 on day +1 until day +6 for the induction of γδ T cell proliferation in vivo. Results This resulted in a marked in vivo expansion of donor γδ T cells and, to a lower extent, natural killer cells and double-negative αβ T cells (mean 68-fold, eight-fold, and eight-fold, respectively). Proliferation peaked by around day +8 and donor cells persisted up to 28 days. Although refractory to all prior therapies, three out of four patients achieved a complete remission, which lasted for 8 months in a patient with plasma cell leukaemia. One patient died from an infection 6 weeks after treatment. Conclusion This pilot study shows that adoptive transfer and in vivo expansion of haploidentical γδ T lymphocytes is feasible and suggests a potential role of these cells in the treatment of haematological diseases. [ABSTRACT FROM AUTHOR]

Highlights: [•] Myh is critically involved in remodeling the IL-2 Promoter for transcription. [•] Myh, not in naïve CD4+ T cells, can be transiently induced after T cell activation. [•] Myh−/− had blunted demethylation of the promoter and impaired IL-2 secretion. [•] Its glycosylase activity was specific for certain CpG sites in the IL-2 Promoter. [•] DNA demethylation of IL-2 is being selectively orchestrated in naïve CD4+ T cells. [ABSTRACT FROM AUTHOR]

Interactions of T cell antigen receptors (TCRs) with complexes of self peptide and major histocompatibility complex (MHC) are crucial to T cell development, but their role in peripheral T cell responses remains unclear. Specific and nonspecific stimulation of LLO56 and LLO118 T cells, which transgenically express a TCR specific for the same Listeria monocytogenes epitope, elicited distinct interleukin 2 (IL-2) and phosphorylated kinase Erk responses, the strength of which was set in the thymus and maintained in the periphery in proportion to the avidity of the binding of the TCR to the self peptide-MHC complex. Deprivation of self peptide-MHC substantially compromised the population expansion of LLO56 T cells in response to L. monocytogenes in vivo. Despite their very different self-reactivity, LLO56 T cells and LLO118 T cells bound cognate peptide-MHC with an identical affinity, which challenges associations made between these parameters. Our findings highlight a crucial role for selecting ligands encountered during thymic 'education' in determining the intrinsic functionality of CD4+ T cells. [ABSTRACT FROM AUTHOR]

Objective: Herpes simplex virus 1 (HSV-1) is regarded as an important underlying cause of Bell's palsy, but the immunologic mechanism remains unknown. Here, we employed a mouse facial paralysis model to investigate the expressions of CD4 T lymphocytes and interleukin (IL)-2 and -4 in the left draining cervical lymph nodes (LCLN) and spleen, as well as the inhibitory effects of glucocorticoids (GCs). Methods: HSV-1 was inoculated into the surface of the posterior auricle to generate the facial paralysis model. The paralyzed mice were divided into three groups; in one group without any treatment, mice were killed at different time points, and those in the other two groups were injected with methylprednisolone sodium succinate (MPSS) or with a combination of MPSS and GC receptor blocker (RU486). The expression levels of CD4 T lymphocytes and CD4-IL-2 and CD4-IL-4 cells in the LCLN and spleen were detected by fluorescence-activated cell sorting. Results: Expression levels of CD4, IL-2, and IL-4 first increased then decreased in LCLN and spleen and peaked 5 and 7 days, respectively, after the manifestation of facial paralysis. All the data at the peak points were significantly different compared with control ( p < 0.05), and these effects were inhibited by MPSS. Conclusion : Our results suggest that CD4, IL-2, and IL-4 participate in the HSV-1-induced facial paralysis immune response. MPSS can effectively attenuate HSV-1-mediated nervous system damage, which is associated with its inhibitory effect on expression of these inflammatory markers. [ABSTRACT FROM AUTHOR]

Cytolytic CD4 T cells (CD4 CTL) have been identified in vivo in response to viral infections; however, the factors necessary for driving the cytolytic phenotype have not been fully elucidated. Our previously published work suggests IL-2 may be the master regulator of perforin-mediated cytotoxicity in CD4 effectors. To further dissect the role of IL-2 in CD4 CTL generation, T cell receptor transgenic mice deficient in the ability to produce IL-2 or the high affinity IL-2 receptor (IL-2Rα, CD25) were used. Increasing concentrations of IL-2 were necessary to drive perforin (Prf) expression and maximal cytotoxicity. Granzyme B (GrB) expression and killing correlated with STAT5 activation and CD25 expression in vitro, suggesting that signaling through the high affinity IL-2R is critical for full cytotoxicity. IL-2 signaling was also necessary in vivo for inducing the Th1 phenotype and IFN-γ expression in CD4 T cells during influenza A (IAV) infection. In addition, GrB expression, as measured by mean fluorescent intensity, was decreased in CD25 deficient cells; however, the frequency of CD4 cells expressing GrB was unchanged. Similarly, analysis of cytolytic markers such as CD107a/b and Eomesodermin indicate high IL-2Rα expression is not necessary to drive the CD4 CTL phenotype during IAV infection. Thus, inflammatory signals induced by viral infection may overcome the need for strong IL-2 signals in driving cytotoxicity in CD4 cells. [ABSTRACT FROM AUTHOR]

Objective: Aging has been reported to be associated with changes in immune function. Although frequent infection and the development of malignancy suggest the decline of immune function with aging, changes toward proinflammatory conditions also develop at the same time. Th17 cells are well known CD4+ T cell subpopulation closely linked to chronic inflammation and autoimmunity. In this study, changes in the Th17 population were investigated to elucidate a possible mechanism for this response with aging. Methods: Splenocytes were isolated from 2-month-old (young) and 20-month-old (aged) mice. CD4+CD44+ memory T cells and CD4+CD62L+ naïve T cells were isolated and sorted using magnetic beads and flow cytometry. The frequency of IL-17-producing cells was measured using flow cytometry. The expression of IL-17 and Th17-related factors at the mRNA level was measured with RT-PCR. IL-17 and Il-1β expression in spleen tissues was additionally assessed using confocal microscopy. Results: The proportion of IL-17-producing CD4+ T cells was higher in the splenocytes among the old mice than those of the young mice. When splenocytes were cultured in Th17 polarizing conditions, the proportion of IL-17 producing CD4+ T cells was higher in aged mice as well. This was consistently observed when naïve and memory cells were isolated and differentiated into Th17 respectively. In addition, the expression of retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt) and other Th17-related factors (AhR, CCR6, and CCL20) increased in the splenocytes of aged mice compared to the young mice. The expression of IL-1β, showing to promote Th17 differentiation, was higher in the aged mice. Likewise, CD4+ T cell expression of IL-1R was higher in the aged mice, suggesting that the CD4+ T cells of the aged mice are readily prepared to differentiate into Th17 cells in response to IL-1β. Confocal microscopy showed that cells positive for IL-1R or IL-1β were more frequent in the spleens of the aged mice. When an anti-IL-2 antibody was applied, the proportion of IL-17-producing cells increased more prominently in the young mice. We observed that IL-2 production and IL-2R expression were reduced in the aged mice, respectively, explaining the blunted response to the anti-IL-2 antibody treatment and the consequent minimal change in the Th17 population. Conclusion: We demonstrated that the proportion of Th17 cells increased in the aged mice both in naïve and memory cell populations. Elevation of IL-1R and IL-1β expression and the reduction in IL-2 and IL-2R expression in aged mice seemed to promote Th17 differentiation. Our results suggest that enhanced Th17 differentiation in aging may have a pathogenic role in the development of Th17-mediated autoimmune diseases. [ABSTRACT FROM AUTHOR]

Optimal T cell activation and expansion require binding of the common gamma-chain (γc) cytokine Interleukin-2 (IL-2) to its cognate receptor that in turn engages a γc/Janus tyrosine kinase (Jak)3 signaling pathway. Because of its restricted expression by antigen-activated T cells and its obligatory role in promoting their survival and proliferation, IL-2 has been considered as a selective therapeutic target for preventing T cell mediated diseases. However, in order to further explore IL-2 targeted therapy, it is critical to precisely understand its role during early events of T cell activation. In this study, we delineate the role of IL-2 and other γc cytokines in promoting the survival of CD4 and CD8 T cells during early phases of priming. Under IL-2 inhibitory conditions (by neutralizing anti-IL-2 mAbs), the survival of activated CD8+ T cells was reduced, whereas CD4+ T cells remained much more resistant. These results correlated with reduced Bcl-2 expression, and mitochondrial membrane potential in CD8+ T cells in comparison to CD4+ T cells. However, using transwell co-culture assays we have found that CD4+ T cells could rescue the survival of CD8+ T cells even under IL-2 deprived conditions via secretion of soluble factors. A cytokine screen performed on CD8+ T cells cultured alone revealed that IL-21, another γc cytokine, was capable of rescuing their survival under IL-2 deprivation. Indeed, blocking the IL-21 signaling pathway along with IL-2 neutralization resulted in significantly reduced survival of both CD4+ and CD8+ T cells. Taken together, we have shown that under IL-2 deprivation conditions, IL-21 may act as the major survival factor promoting T cell immune responses. Thus, investigation of IL-2 targeted therapies may need to be revisited to consider blockade of the IL-21 signaling pathways as an adjunct to provide more effective control of T cell immune responses. [ABSTRACT FROM AUTHOR]

Integration is a central event in the replication of retroviruses, yet≥90% of HIV-1 reverse transcripts fail to integrate, resulting in accumulation of unintegrated viral DNA in cells. However, understanding what role, if any, unintegrated viral DNA plays in the natural history of HIV-1 has remained elusive. Unintegrated HIV-1 DNA is reported to possess a limited capacity for gene expression restricted to early gene products and is considered a replicative dead end. Although the majority of peripheral blood CD4+ T cells are refractory to infection, nonactivated CD4 T cells present in lymphoid and mucosal tissues are major targets for infection. Treatment with cytokine interleukin-2 (IL-2), IL-4, IL-7, or IL-15 renders CD4+ T cells permissive to HIV-1 infection in the absence of cell activation and proliferation and provides a useful model for infection of resting CD4+ T cells. We found that infection of cytokine-treated resting CD4+ T cells in the presence of raltegravir or with integrase active-site mutant HIV-1 yielded de novo virus production following subsequent T cell activation. Infection with integration-competent HIV-1 naturally generated a population of cells generating virus from unintegrated DNA. Latent infection persisted for several weeks and could be activated to virus production by a combination of a histone deacetylase inhibitor and a protein kinase C activator or by T cell activation. HIV-1 Vpr was essential for unintegrated HIV-1 gene expression and de novo virus production in this system. Bypassing integration by this mechanism may allow the preservation of genetic information that otherwise would be lost. [ABSTRACT FROM AUTHOR]

T cell costimulation is a key component of adaptive immunity to viral infection but has also been associated with pathology because of excessive or altered T cell activity. We recently demonstrated that the TNFR family costimulatory molecule OX40 (CD134) is critically required to sustain antiviral T cell and Ab responses that enable control of viral replication in the context of chronic lymphocytic choriomeningitis virus (LCMV) infection. In this study, we investigated whether reinforcing OX40 stimulation through an agonist Ab had the potential to prevent LCMV persistence. We observed that anti-OX40 injection early after LCMV clone 13 infection increased CD8 T cell-mediated immunopathology. More strikingly, OX40 stimulation of virus-specific CD4 T cells promoted expression of the transcriptional repressor Blimp-1 and diverted the majority of cells away from follicular Th cell differentiation. This occurred in both acute and chronic infections, and resulted in dramatic reductions in germinal center and Ab responses to the viral infection. The effect of the OX40 agonist was dependent on IL-2 signaling and the timing of OX40 stimulation. Collectively, our data demonstrate that excessive OX40 signaling can result in deleterious consequences in the setting of LCMV infection. [ABSTRACT FROM AUTHOR]

Endurance, marathon-type exertion is known to induce adverse changes in the immune system. Increased airway hyper-responsiveness and airway inflammation are well documented in endurance athletes and endurance exercise is considered a major risk factor for asthma in elite athletes. Yet, the mechanisms underlying this phenomenon are still to be deduced. We studied the effect of strenuous endurance exercise (marathon and half-ironman triathlon) on CD4+ lymphocyte sub-populations and on the balance between effector and regulatory CD4+ lymphocytes in the peripheral blood of trained athletes, Endurance exercise induced a significant increase in Th17 cells and a sustained decrease in peripheral blood regulatory T cells (Tregs). While interleukin (IL)-2 levels remained undetectable, post-race serum IL-6 and transforming growth factor (TGF) β levels were significantly elevated. Treg levels in sedentary controls' decreased in vitro after incubation with athletes' post-exercise serum, an effect that was attenuated by supplements of IL-2 or anti IL-6 neutralizing antibodies. Our data suggest that exercise-induced changes in serum cytokine levels promote alterations in Tregs and Th17 cell populations, which may divert the subtle balance in the immune system towards inflammation. This may explain allergic and autoimmune phenomena previously reported in endurance athletes and contribute to our understanding of exercise-related asthma. [ABSTRACT FROM AUTHOR]

Highlights: [•] We compared CD4+ T cell responses in HIV-1 patients by those induced in uninfected HIV-1 vaccinees. [•] IL-2, IFN-γ, TNF-α and CD40L expression measured after in vitro stimulation with p17, p24, RT and Nef. [•] Viral controllers (VC) display polyfunctional CD4+ T cell responses to p17, p24, RT and Nef. [•] CD4+ responses in VC are similar to those induced in healthy volunteers by an HIV vaccine candidate. [Copyright &y& Elsevier]

Highlights: [•] Id1 expression enables naïve T cell proliferation without anti-CD28 co-stimulation. [•] Id1 expression facilitates T cells survival when stimulated with anti-CD3. [•] Elevation of IL-2 production by Id1 contributes increased proliferation and survival. [•] Id1 potentiates NF-κB activation by anti-CD3 stimulation. [ABSTRACT FROM AUTHOR]

The bacille Calmette-Guérin vaccine provides very efficient protection in standard animal models of Mycobacterium tuberculosis challenge. We show in this article that although bacille Calmette-Guérin controlled M. tuberculosis growth for 7 wk of infection, the protection was gradually lost as the infection entered the chronic phase. The regrowth of M. tuberculosis coincided with an almost complete disappearance of IL-2-producing CD4 T cells. Booster vaccination with a subunit vaccine (Ag85B-ESAT-6+CAF01) expanded IL-2+ CD4+ T cell coexpressing either TNF-α or TNF-α/IFN-γ, and the maintenance of this population in the late stage of infection was associated with enhanced control of bacterial growth. The IL-2+ CD4+ T cell subsets were KLRG1- (nonterminally differentiated), were found to be CD62Lhigh, and further maintained a pronounced proliferative and cytokine-producing potential in the draining lymph nodes, when the animals were challenged 2 y postvaccination. These results suggest that the CD4+ KLRG1- IL-2-secreting subsets are central memory T cells with the potential to continuously replenish the T cells at the site of infection and prevent attrition and functional exhaustion. [ABSTRACT FROM AUTHOR]

Sarcoidosis pathogenesis is characterized by peripheral anergy and an exaggerated, pulmonary CD4+ Th1 response. In this study, we demonstrate that CD4+ anergic responses to polyclonal TCR stimulation are present peripherally and within the lungs of sarcoid patients. Consistent with prior observations, spontaneous release of IL-2 was noted in sarcoidosis bronchoalveolar lavage CD4+ T cells. However, in contrast to spontaneous hyperactive responses reported previously, the cells displayed anergic responses to polyclonal TCR stimulation. The anergic responses correlated with diminished expression of the Src kinase Lck, protein kinase C-θ, and NF-κB, key mediators of IL-2 transcription. Although T regulatory (Treg) cells were increased in sarcoid patients, Treg depletion from the CD4+ T cell population of sarcoidosis patients did not rescue IL-2 and IFN-γ production, whereas restoration of the IL-2 signaling cascade, via protein kinase C-θ overexpression, did. Furthermore, sarcoidosis Treg cells displayed poor suppressive capacity indicating that T cell dysfunction was a global CD4+ manifestation. Analyses of patients with spontaneous clinical resolution revealed that restoration of CD4+ Th1 and Treg cell function was associated with resolution. Conversely, disease progression exhibited decreased Th1 cytokine secretion and proliferative capacity, and reduced Lck expression. These findings implicate normalized CD4+ T cell function as a potential therapeutic target for sarcoidosis resolution. [ABSTRACT FROM AUTHOR]

The role of surface-bound TGF-β on regulatory T cells (Tregs) and the mechanisms that mediate its functions are not well defined. We recently identified a cell-surface molecule called Glycoprotein A Repetitions Predominant (GARP), which is expressed specifically on activated Tregs and was found to bind latent TGF-β and mediate a portion of Treg suppressive activity in vitro. In this article, we address the role of GARP in regulating Treg and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. We found that, despite forced expression of GARP on all T cells, stimulation through the TCR was required for efficient localization of GARP to the cell surface. In addition, IL-2 signals enhanced GARP cell surface expression specifically on Tregs. GARP-transgenic CD4+ T cells and Tregs, especially those expressing higher levels of GARP, were significantly reduced in the periphery. Mature Tregs, but not conventional CD4+ T cells, were also reduced in the thymus. CD4+ T cell reduction was more pronounced within the effector/memory subset, especially as the mouse aged. In addition, GARP-overexpressing CD4+ T cells stimulated through the TCR displayed reduced proliferative capacity, which was restored by inhibiting TGF-β signaling. Furthermore, inhibiting TGF-β signals greatly enhanced surface expression of GARP on Tregs and blocked the induction of Foxp3 in activated CD4+ T cells overexpressing GARP. These findings suggest a role for GARP in natural and induced Treg development through activation of bound latent TGF-β and signaling, which negatively regulates GARP expression on Tregs. [ABSTRACT FROM AUTHOR]

The frequency and function of regulatory T cells (Tregs) were studied in stage II-III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4 T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8 T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8 T cell effectors when used in the inflammatory environment of vaccination. [ABSTRACT FROM AUTHOR]

Abstract: Recently, LSECtin was identified as a co-inhibitory molecule involved in the control of T cell-mediated acute liver injury. However, it is not known whether LSECtin also suppresses the function of different CD4+ T-cell subsets. In this study, we demonstrate that LSECtin-mediated signaling potently inhibits the proliferation and IL-2 production of total, naive and pre-activated CD4+ T cells. However, exogenous IL-2 does not overcome the inhibitory effect on the proliferation of naive CD4+ T cells. Based on the LSECtin-mediated inhibition of pre-activated CD4+ T cells, we further analyzed the role of LSECtin in different effector CD4+ T-cell subsets including Th1, Th2 and Th17. We observed that LSECtin significantly suppressed the production of their signature cytokines suggesting that LSECtin may also play an inhibitory role in the effector phase. LSECtin did not inhibit the CD4+ T-cell proliferation induced by PMA (phorbol myristate acetate) and ionomycin suggesting that the LSECtin-mediated inhibition reduces the activation of the proximal TCR signalsome. The inhibition of CD4+ T-cell activation mediated by LSECtin is associated with the up-regulation of Cbl-b and has no effect on GRAIL, Itch and SHP-2 phosphatase. Furthermore, LSECtin-initiated inhibition is compromised in Cbl-b mutant CD4+ T cells. Taken together, these data demonstrate that LSECtin-mediated signaling up-regulates the threshold of CD4+ T cell activation via tuning the expression of Cbl-b. [Copyright &y& Elsevier]

The molecular mechanism of the extrathymic generation of adaptive, or inducible, CD4+Foxp3+ regulatory T cells (iTregs) remains incompletely defined. We show that exposure of splenic CD4+CD25+Foxp3- cells to IL-2, but not other common -chain cytokines, resulted in Stat5 phosphorylation and induced Foxp3 expression in ~10% of the cells. Thus, IL-2/Stat5 signaling may be critical for Foxp3 induction in peripheral CD4+CD25-+Foxp3- iTreg precursors. In this study, to further define the role of IL-2 in the formation of iTreg precursors as well as their subsequent Foxp3 expression, we designed a two-step iTreg differentiation model. During the initial "conditioning" step, CD4+CD25-Foxp3- naive T cells were activated by TCR stimulation. Inhibition of IL-2 signaling via Jak3-Stat5 was required during this step to generate CD4+CD25+Foxp3- cells containing iTreg precursors. During the subsequent Foxp3-induction step driven by cytokines, IL-2 was the most potent cytokine to induce Foxp3 expression in these iTreg precursors. This two-step method generated a large number of iTregs with relatively stable expression of Foxp3, which were able to prevent CD4+CD45RBhigh cell-mediated colitis in Rag1-/- mice. In consideration of this information, whereas initial inhibition of IL-2 signaling upon T cell priming generates iTreg precursors, subsequent activation of IL-2 signaling in these precursors induces the expression of Foxp3. These findings advance the understanding of iTreg differentiation and may facilitate the therapeutic use of iTregs in immune disorders. [ABSTRACT FROM AUTHOR]

Human CD4 T cell recall responses to influenza virus are strongly biased towards Type 1 cytokines, producing IFNγ, IL-2 and TNFα. We have now examined the effector phenotypes of CD4 T cells in more detail, particularly focusing on differences between recent versus long-term, multiply-boosted responses. Peptides spanning the proteome of temporally distinct influenza viruses were distributed into pools enriched for cross-reactivity to different influenza strains, and used to stimulate antigen-specific CD4 T cells representing recent or long-term memory. In the general population, peptides unique to the long-circulating influenza A/New Caledonia/20/99 (H1N1) induced Th1-like responses biased toward the expression of IFNγ+TNFα+ CD4 T cells. In contrast, peptide pools enriched for non-cross-reactive peptides of the pandemic influenza A/California/04/09 (H1N1) induced more IFNγ−IL-2+TNFα+ T cells, similar to the IFNγ−IL-2+ non-polarized, primed precursor T cells (Thpp) that are a predominant response to protein vaccination. These results were confirmed in a second study that compared samples taken before the 2009 pandemic to samples taken one month after PCR-confirmed A/California/04/09 infection. There were striking increases in influenza-specific TNFα+, IFNγ+, and IL-2+ cells in the post-infection samples. Importantly, peptides enriched for non-cross-reactive A/California/04/09 specificities induced a higher proportion of Thpp-like IFNγ−IL-2+TNFα+ CD4 T cells than peptide pools cross-reactive with previous influenza strains, which induced more Th1 (IFNγ+TNFα+) responses. These IFNγ−IL-2+TNFα+ CD4 T cells may be an important target population for vaccination regimens, as these cells are induced upon infection, may have high proliferative potential, and may play a role in providing future effector cells during subsequent infections. [ABSTRACT FROM AUTHOR]

Determination of antigen-specific T cell repertoires in human blood has been a challenge. Here, we show a novel integrated approach that permits determination of multiple parameters of antigen-specific T cell repertoires. The approach consists of two assays: the Direct assay and the Cytokine-driven assay. Briefly, human PBMCs are first stimulated with overlapping peptides encoding a given antigen for 48 hours to measure cytokine secretion (Direct assay). Peptide-reactive T cells are further expanded by IL-2 for 5 days; and after overnight starvation, expanded cells are stimulated with the same peptides from the initial culture to analyze cytokine secretion (Cytokine-driven assay). We first applied this integrated approach to determine the type of islet-antigen-specific T cells in healthy adults. Out of ten donors, the Direct assay identified GAD65-specific CD4+ T cells in three adults and zinc transporter 8 (ZnT8)-specific CD4+ T cells in five adults. The intracytoplasmic cytokine staining assay showed that these islet-antigen-specific CD4+ T cells belonged to the CD45RO+ memory compartment. The Cytokine-driven assay further revealed that islet-antigen-specific CD4+ T cells in healthy adults were capable of secreting various types of cytokines including type 1 and type 2 cytokines as well as IL-10. We next applied our integrated assay to determine whether the type of ZnT8-specific CD4+ T cells is different between Type 1 diabetes patients and age/gender/HLA-matched healthy adults. We found that ZnT8-specific CD4+ T cells were skewed towards Th1 cells in T1D patients, while Th2 and IL-10-producing cells were prevalent in healthy adults. In conclusion, the Direct assay and the Cytokine-driven assay complement each other, and the combination of the two assays provides information of antigen-specific T cell repertoires on the breadth, type, and avidity. This strategy is applicable to determine the differences in the quality of antigen-specific T cells between health and disease. [ABSTRACT FROM AUTHOR]

Summary: Knowledge of human T cells derives chiefly from studies of peripheral blood, whereas their distribution and function in tissues remains largely unknown. Here, we present a unique analysis of human T cells in lymphoid and mucosal tissues obtained from individual organ donors, revealing tissue-intrinsic compartmentalization of naive, effector, and memory subsets conserved between diverse individuals. Effector memory CD4+ T cells producing IL-2 predominated in mucosal tissues and accumulated as central memory subsets in lymphoid tissue, whereas CD8+ T cells were maintained as naive subsets in lymphoid tissues and IFN-γ-producing effector memory CD8+ T cells in mucosal sites. The T cell activation marker CD69 was constitutively expressed by memory T cells in all tissues, distinguishing them from circulating subsets, with mucosal memory T cells exhibiting additional distinct phenotypic and functional properties. Our results provide an assessment of human T cell compartmentalization as a new baseline for understanding human adaptive immunity. [ABSTRACT FROM AUTHOR]

Abstract: The phorbol ester Prostratin may either stimulate or inhibit human immunodeficiency virus-1 (HIV-1) replication. Here we report that Prostratin also exhibits a similar dual action upon feline immunodeficiency virus (FIV) replication in an IL-2-dependent feline CD4+ T-cell line (MYA-1). While withdrawal of IL-2 halted FIV spread, Prostratin rescued virus production and cell viability, mimicking the functions of the cytokine. Conversely, FIV grew rapidly in the presence of IL-2 and this was inhibited by Prostratin. In contrast to HIV-1, Prostratin mediated inhibition of FIV through means other than blocking virus entry. Co-application of the protein kinase C (PKC) inhibitor Gö6850 with Prostratin reversed both the inhibitory and stimulatory effects, suggesting that PKC is crucial for FIV replication. [Copyright &y& Elsevier]

Abstract: The impact of in vivo regulatory T cells (Treg) expansion using short-term injections of interleukin-2 (IL-2) coupled to a specific anti–IL-2 antibody was examined in various allogeneic combinations of murine skin transplantations. In a model of a single major histocompatibility complex (MHC) class II disparity, the IL-2–expanded Tregs infiltrated the transplanted skin, inhibited Th1 alloreactivity, and prevented acute graft rejection. However, in the presence of increased load of CD4-recognized alloantigens, exogenous IL-2 only moderately prolonged graft survival as attested by CD8 T cell-depletion in full minor plus major mismatched recipients treated with IL-2. If direct CD8 alloreactivity remained intact, the IL-2/anti-IL-2–mediated Tregs expansion failed to delay allograft rejection. This observation was confirmed by the inability of expanded Tregs to delay rejection of multiple minor disparate (MHC matched) skin allografts. Altogether, these results warn that cross-reactive CD8+ T cells represent an important hurdle to Treg-based tolerance induction. [Copyright &y& Elsevier]

Appropriate expression of IL-2 plays a central role during the priming and differentiation of T cells. A tight balance between IL-2 and the effector cytokine IL-17A is essential for immune homeostasis. Epigenetic mechanisms have been documented as a key component of cytokine regulation during lineage commitment. The molecular mechanisms that induce chromatin remodeling are less well understood. We investigated epigenetic regulators that mediate the diametric expression of IL-2 and IL-17A in naive, central memory, and effector memory CD4+ T cells. We demonstrate that cAMP response modulator (CREM)α contributes to epigenetic remodeling of IL2 in effector memory T cells through the recruitment of DNMT3a. CREMa also reduces CpG-DNA methylation of the IL17A promoter. CREMa expression is regulated at the epigenetic level by CpG-DNA methylation, which allows increased CREMa expression in effector memory CD4+ T cells. T cells from patients with systemic lupus erythematosus (SLE) express increased levels of CREMa and exhibit a phenotype that is similar to effector memory CD4+ T cells with epigenetically predetermined expression patterns of IL-2 and IL-17A. We conclude that CREMa mediates epigenetic remodeling of the 1L2 and IL17A gene during T-cell differentiation in favor of effector memory T cells in health and disease. [ABSTRACT FROM AUTHOR]

Abstract: Background: Several correlates of HIV control have been described; however their predictive values remain unclear, since most studies have been performed in cross-sectional settings. Objectives: We evaluated the cause and consequence relationship between quality of HIV-specific T-cell response and viral load dynamic in a temporal perspective. Study design: HIV-1-specific T-cell responses were monitored over 7 years in a patient that following treatment interruption maintained a stable/low viral set point for 3.1 years before control of viral replication was lost and antiretroviral therapy restarted. Results: We observed that high frequencies of HIV-1-specific CD4 and CD8 T cells were unable to prevent loss of viral control. Gradual loss of functionality was observed in these responses, characterized by early loss of IL-2, viral load-dependent decrease of IFN-γ and CD154 expression as well as increase of MIP-1β production. Terminally differentiated HIV-1-specific CD8 T cells expressing CD45RA were lost independently of viral load and preceded the loss-of-control phase of HIV infection. Conclusion: By describing qualitative changes in HIV-1-specific T-cell responses that coincide with loss of viral control, we identified specific correlates of disease progression and putative markers of viral control. Our findings suggest including the markers IL-2, IFN-γ, MIP-1β, CD154 and CD45RA into monitoring of HIV-specific T-cell-responses to prospectively determine correlates of protection from disease-progression. [Copyright &y& Elsevier]

Human CMV establishes lifelong persistence after primary infection. Chronic CMV infection is associated with intermittent viral reactivation inducing high frequencies of CD4+ T lymphocytes with potent antiviral and helper properties. Primary CMV infection is characterized by an intense viral replication lasting for several months. The impact of this prolonged exposure to high Ag loads on the functionality of CD4+ T cells remains incompletely understood. In pregnant women with primary CMV infection, we observed that CMV-speciflc CD4+ T lymphocytes had a decreased capacity to proliferate and to produce IL-2. A very large proportion of CMV-speciflc CD4+ T cells had downregulated the expression of CD28, a costimulatory molecule centrally involved in the production of IL-2. Unexpectedly, both CD28- and CD28+CD4+ T cells produced low levels of IL-2. This defective production of IL-2 was part of a larger downregulation of cytokine production. Indeed, CMV-speciflc CD4+ T cells produced lower amounts of IFN-γ and TNF-&agr; and showed lower functional avidity during primary as compared with chronic infection. Increased programmed death-1 expression was observed in CD28+ CMV-speciflc CD4+ T cells, and programmed death-1 inhibition increased proliferative responses. These results indicate that primary CMV infection is associated with the exhaustion of CMV-speciflc CD4+ T cells displaying low functional avidity for viral Ags. [ABSTRACT FROM AUTHOR]

Transforming growth factor beta (TGF-β)- and Interleukin-2 (IL-2)-mediated signaling enables the generation and expansion of induced regulatory T (iTreg) cells that carry high hopes for the treatment of chronic inflammatory and autoimmune diseases. Knowledge about factors stabilizing their lineage commitment and lifespan, however, is limited. Here, we investigated the behavior of iTreg cells, derived from apoptosis-defective mouse mutants, during activated cell autonomous cell death, triggered by cytokine-deprivation, or activation-induced cell death (AICD) after restimulation of the T-cell receptor, and compared these responses with those of effector T cells. We observed that iTreg cells were much more sensitive to IL-2-deprivation but poorly susceptible to AICD. In fact, when apoptosis was compromised, T-cell receptor (TCR)-religation resulted in methylation-independent, ERK- and PI3K/mTOR-mediated loss of Foxp3 expression, impaired suppressive capacity and effector cytokine production. Although iTreg cells prevented colitis induction they rapidly lost Foxp3-GFP expression and gained ability to produce effector cytokines thereby imposing Th1 cell fate on resident effector cells. Surprisingly, iTreg cell conversion itself was limited by TGF-β-mediated Bim/Bcl2L11-dependent apoptosis. Hence, the very same cytokine that drives the generation of iTreg cells can trigger their demise. Our results provide novel insights in iTreg cell biology that will assist optimization of iTreg-based therapy. [ABSTRACT FROM AUTHOR]

Several reports in the literature have drawn a complex picture of the effect of treatments aiming to modulate IL2 activity in vivo. They seem to promote indistinctly immunity or tolerance, probably depending on the specific context, dose and timing of their application. Such complexity might derives from the dual role of IL2 on T-cell dynamics. To theoretically address the latter possibility, we develop a mathematical model for helper, regulatory and memory T-cells dynamics, which account for most well-known facts relative to their relationship with IL2. We simulate the effect of three types of therapies: IL2 injections, IL2 depletion using anti-IL2 antibodies and IL2/anti-IL2 immune complexes injection. We focus in the qualitative and quantitative conditions of dose and timing for these treatments which allow them to potentate either immunity or tolerance. Our results provide reasonable explanations for the existent pre-clinical and clinical data and further provide interesting practical guidelines to optimize the future application of these types of treatments. Particularly, our results predict that: (i) Immune complexes IL2/anti-IL2 mAbs, using mAbs which block the interaction of IL2 and CD25 (the alpha chain of IL2 receptor), is the best option to potentate immunity alone or in combination with vaccines. These complexes are optimal when a 1:2 molar ratio of mAb:IL2 is used and the mAbs have the largest possible affinity; (ii) Immune complexes IL2/anti-IL2 mAbs, using mAbs which block the interaction of IL2 and CD122 (the beta chain of IL2 receptor), are the best option to reinforce preexistent natural tolerance, for instance to prevent allograft rejection. These complexes are optimal when a 1:2 molar ratio of mAb:IL2 is used and the mAbs have intermediate affinities; (iii) mAbs anti-IL2 can be successfully used alone to treat an ongoing autoimmune disorder, promoting the re-induction of tolerance. The best strategy in this therapy is to start treatment with an initially high dose of the mAbs (one capable to induce some immune suppression) and then scales down slowly the dose of mAb in subsequent applications. [ABSTRACT FROM PUBLISHER]

CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H INI influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in the draining lymph node (DLN) and gamma interferon (IFN-γ) and IL-10 in the lung. ΙΡΝ-γ-deficient CD4 cells produced larger amounts of IL-17 and similar levels of TNF-α, IL-10, and IL-2 compared to wild-type (WT) CD4 cells. Both WT and IFN-γ - / ∼ CD4 cells exhibit influenza virus-specific cytotoxicity; however, IFN-deficient CD4 cells did not promote recovery after lethal infection as effectively as WT CD4 cells. PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung but not the DLN. These cells expressed granzyme Β (GrB) and required perforin to lyse peptide-pulsed targets. Lethally infected mice given influenza virus-specific CD4 cells deficient in perforin showed greater weight loss and a slower time to recovery than mice given WT influenza virus-specific CD4 cells. Taken together, these data strengthen the concept that CD4 Τ cell effectors are broadly multifunctional with direct roles in promoting protection against lethal influenza virus infection. [ABSTRACT FROM AUTHOR]

Murine and human mesothelioma tumors are susceptible to immunotherapy, particularly when immune adjuvants are delivered locally. We have shown that direct injection of IL-2 plus agonist anti-CD40 antibody induces regression of large mesothelioma tumors. These studies aimed to determine if NK cells contribute to IL-2/CD40 antibody-driven tumor eradication. We show that NK cells infiltrate developing mesothelioma tumors; however, their absence (in beige mice or in asialo GM1 antibody-depleted C57BL/6J mice) does not alter tumor growth rates suggesting that they cannot function as effector cells in this microenvironment. Anti-CD40 antibody treatment did not alter the percent of NK cells in treated tumors or in draining lymph nodes (dLNs), and tumor resolution occurred in the absence of NK cells. However, a two-tumor model showed that NK cells contributed to CD40-driven systemic immunity leading to resolution of untreated distal tumors. IL-2 treatment led to increased proportions of NK cells in tumors and dLNs, and in the absence of NK cells, IL-2 lost its therapeutic effect. In contrast, the absence of NK cells did not reduce the anti-tumor activity of the IL-2/anti-CD40 antibody combination yet tumors recurred in NK-deficient mice and > 37% of tumor cell re-challenged mice were unable to provide protection, implying insufficient memory. Furthermore, untreated distal tumors in NK-depleted mice were less readily cured than in immunologically intact mice. These data show that NK cells infiltrate mesothelioma tumors, which, after local IL-2 and/or anti-CD40 antibody treatment, provide help for the acquisition and/or maintenance of systemic immunity and long-term effector/memory responses. [ABSTRACT FROM PUBLISHER]

The transcription factors T-bet and Bcl-6 are required for the establishment of a T helper type 1 cell (TH1 cell) and follicular helper T cell (TFH cell) gene-expression profile, respectively. Here we found that high concentrations of interleukin 2 (IL-2) inhibited Bcl-6 expression in polarized TH1 cells. Mechanistically, the low concentrations of Bcl-6 normally found in effector TH1 cells did not repress its target genes because a T-bet-Bcl-6 complex masked the Bcl-6 DNA-binding domain. TH1 cells increased their Bcl-6/T-bet ratio in response to limiting IL-2 conditions, which allowed excess Bcl-6 to repress its direct target Prdm1 (which encodes the transcriptional repressor Blimp-1). The Bcl-6-dependent repression of Blimp-1 effectively induced a partial TFH profile because Blimp-1 directly repressed a subset of TFH signature genes, including Cxcr5. Thus, IL-2-signaling regulates the Bcl-6-Blimp-1 axis in TH1 cells to maintain flexibility with a TFH cell-like gene profile. [ABSTRACT FROM AUTHOR]

CD4-unhelped CD8+ T cells are functionally defective T cells primed in the absence of CD4+ T cell help. Given the co-stimulatory role of natural-killer group 2, member D protein (NKG2D) on CD8+ T cells, we investigated its ability to rescue these immunologically impotent cells. We demonstrate that augmented co-stimulation through NKG2D during priming paradoxically rescues memory, but not effector, CD8+ T cell responses. NKG2D-mediated rescue is characterized by reversal of elevated transcription factor T-box expressed in T cells (T-bet) expression and recovery of interleukin-2 and interferon-? production and cytolytic responses. Rescue is abrogated in CD8+ T cells lacking NKG2D. Augmented co-stimulation through NKG2D confers a high rate of survival to mice lacking CD4+ T cells in a CD4-dependent influenza model and rescues HIV-specific CD8+ T cell responses from CD4-deficient HIV-positive donors. These findings demonstrate that augmented co-stimulation through NKG2D is effective in rescuing CD4-unhelped CD8+ T cells from their pathophysiological fate and may provide therapeutic benefits. [ABSTRACT FROM AUTHOR]

Recent development in antiretroviral treatment against HIV can help AIDS patients to fight against HIV. But the question that whether the disease is to be partially or totally eradicated from HIV infected individuals still remains unsolved. Usually, the most effective treatment for the disease is HAART which can only control the disease progression. But as the immune system becomes weak, the patients can not fight against other diseases. Immune cells are activated and proliferated by IL-2 after the identification of antigen. IL-2 production is impaired in HIV positive patients and intermitted administration of immune activator IL-2 together with HAART which is a more effective treatment to fight against the disease. Thus, its expediency is essential and is yet to be explored. In this article we anticipated a mathematical model of the effect of IL-2 together with RTIs therapy in HIV positive patients. Our analytical as well as numerical study shows that the optimal schedule of treatment for best result is to be obtained by systematic drug therapy. But at the last stage of treatment, the infection level raises again due to minimisation of drug dosage. Thus we study the perfect adherence of the drugs and found out if RTIs are taken with sufficient interval then for fixed interval of IL-2 therapy, certain amount of drug dosages may be able to sustain the immune system at pre-infection stage and the infected CD4+T cells are going towards extinction. [ABSTRACT FROM AUTHOR]

Background: Despite inducing a sustained increase in CD4+ T cell counts, intermittent recombinant IL-2 (rIL-2) therapy did not confer a better clinical outcome in HIV-infected patients enrolled in large phase III clinical trials ESPRIT and SILCAAT. Several hypotheses were evoked to explain these discrepancies. Here, we investigated the impact of low and high doses of IL-2 in Rhesus macaques of Chinese origin infected with SIVmac251 in the absence of antiretroviral therapy (ART). Results: We demonstrated that rIL-2 induced a dose dependent expansion of CD4+ and CD8+ T cells without affecting viral load. rIL-2 increased CD4 and CD8 Treg cells as defined by the expression of CD25highFoxP3+CD127low. We also showed that rIL-2 modulated spontaneous and Fas-mediated CD4+ and CD8+ T cell apoptosis. The higher dose exhibited a dramatic pro-apoptotic effect on both CD4+ and CD8+ T cell populations. Finally, all the animals treated with rIL-2 developed a wasting syndrome in the month following treatment simultaneously to a dramatic decrease of circulating effector T cells. Conclusion: These data contribute to the understanding of the homeostatic and dosage effects of IL-2 in the context of SIV/HIV infection. [ABSTRACT FROM AUTHOR]

Abstract: Obliterative bronchiolitis (OB) is the major obstacle for long-term allograft survival in lung transplantation, and the underlying mechanism is still not well understood. Regulatory T cells (Tregs) have been shown to be essential in the maintenance of immune tolerance. In this study we investigated the role of Tregs in protecting OB in rat. We show that the combination of TGF-β, Interleukin (IL)-2, and all-trans retinoic acid (atRA) could induce naïve rat CD4+CD25− T cells to differentiate into CD4+CD25+Foxp3+ T cells in vitro, and they acquired suppressive function. In a rat orthotopic tracheal transplantation OB model, the adoptive transfer of the induced Tregs reduced symptoms of airway obliteration and fibrication of grafts when compared with adoptive transfer of control cells without suppressive property. Moreover, recipients treated with the induced Tregs secreted high level of immunosuppressive cytokine TGF-β and IL-10, and low level of pro-inflammatory cytokines IL-17, IFN-γ, IL-6, and MCP-1, and had fewer effector T cells including Th17 cells and Th1 cells in the graft. Taken together, these findings suggest that in vitro induced Tregs by the combination of TGF-β, IL-2, and atRA are effective in protecting rat trachea allograft rejection through the inhibition of effector T cells and their function. These datas implicate new therapies to prevent OB and allograft rejection in human lung transplantation. [Copyright &y& Elsevier]

Background aims. Several studies have demonstrated that the immunogenicity of chronic lymphocytic leukemia (CLL) cells can be increased by manipulation of the CD40/CD40-ligand (CD40L) pathway. Although immunologic, and perhaps clinical, benefits have been obtained with an autologous CLL tumor vaccine obtained by transgenic expression of CD40L and interleukin (IL)-2, there is little information about the optimal gene transfer strategies. Methods. We compared two different CLL vaccines prepared by adenoviral gene transfer and plasmid electroporation, analyzing their phenotype and immunostimulatory activity. Results. We found that higher expression of transgenic CD40L was mediated by adenoviral gene transfer than by plasmid transduction, and that adenoviral transfer of CD40L was associated with up-regulation of the co-stimulatory molecules CD80 and CD86 and adhesion molecule CD54. In contrast, transgenic IL-2 secretion was greater following plasmid transduction. These phenotypic differences in the vaccines were associated with different functionality, both ex vivo and following administration to patients. Thus adenoviral vaccines induced greater activation of leukemia-reactive T cells ex vivo than plasmid vaccines. In treated patients, specific T-cell (T helper 1 (Th1) and T helper 2 (Th2)) and humoral anti-leukemia responses were detected following administration of the adenoviral vaccine ( n == 15), while recipients of the plasmid vaccine ( n == 9) manifested only a low-level Th2 response. Progression-free survival at 2 years was 46.7% in the adenoviral vaccine recipients, versus 11.1 % in those receiving plasmid vaccine. Conclusions. CLL vaccines expressing the same transgenes but produced by distinct methods of gene transfer may differ in the polarity of the immune response they induce in patients. [ABSTRACT FROM AUTHOR]

CD28 costimulation is a critical event in the full activation of CD4+ T cells that augments cytokine gene transcription, promotes cytokine mRNA stability, prevents induction of anergy, increases cellular metabolism, and increases cell survival. However, despite extensive biochemical analysis of the signaling events downstream of CD28, molecular pathways sufficient to functionally replace the diverse aspects of CD28-mediated costimulation in normal T cells have not been identified. Ras/MAPK signaling is a critical pathway downstream of T cell receptor stimulation, but its role in CD28-mediated costimulation has been controversial. We observed that physiologic CD28 costimulation caused a relocalization of the RasGEF RasGRP to the T cell-APC interface by confocal microscopy. In whole cell biochemical analysis, CD28 cross-linking with either anti-CD28 antibody or B7.1-Ig augmented TCR-induced Ras activation. To determine whether Ras signaling was sufficient to functionally mimic CD28 costimulation, we utilized an adenoviral vector encoding constitutively active H-Ras (61L) to transduce normal, Coxsackie-Adenovirus Receptor (CAR) transgenic CD4+ T cells. Like costimulation via CD28, active Ras induced AKT, JNK and ERK phosphorylation. In addition, constitutive Ras signaling mimicked the ability of CD28 to costimulate IL-2 protein secretion, prevent anergy induction, increase glucose uptake, and promote cell survival. Importantly, we also found that active Ras mimicked the mechanism by which CD28 costimulates IL-2 production: by increasing IL-2 gene transcription, and promoting IL-2 mRNA stability. Finally, active Ras was able to induce IL-2 production when combined with ionomycin stimulation in a MEK-1-dependent fashion. Our results are consistent with a central role for Ras signaling in CD28-mediated costimulation. [ABSTRACT FROM AUTHOR]

Nuclear factor of activated T cells (NFAT) is a family of transcription factors composed of five proteins. Among them, NFAT1 is a predominant NFAT protein in CD4+ T cells. NFAT1 positively regulates transcription of a large number of inducible cytokine genes including IL-2, IL-4, IL-5 and other cytokines. However, disruption of NFAT1 results in an unexpected increase of IL-4. In this study, we have investigated the role of NFAT1 in regulation of IL-4 gene expression in T helper 2 cells (Th2) from an epigenetic viewpoint. NFAT1 deficient Th2 cells showed a sustained IL-4 expression while wild type (WT) cells reduced its expression. We tested whether epigenetic maintenance and changes in the chromatin architecture of IL-4 promoter locus play a role in differential IL-4 transcription between in WT and NFAT1 deficient Th2 cells. Compared with WT, NFAT1 deficient CD4+ Th2 cells exhibited enhanced chromatin accessibility with permissive histone modification and DNA demethylation in the IL-4 promoter region. Transcription factors bound to IL-4 promoter region in the absence of NFAT1 were identified by Micro-LC/LC-MS/MS analysis. Among the candidates, preferential recruitment of JUNB to the IL-4 promoter was confirmed by chromatin immunoprecipitation analysis. Overexpression of JUNB together with SATB1 synergistically upregulated IL-4 promoter activity, while knockdown JUNB significantly reduced IL-4 expression. Our results suggest that the prolonged IL-4 expression in NFAT1 deficient Th2 cells is mediated by preferential binding of JUNB/SATB1 to the IL-4 promoter with permissive chromatin architecture. [ABSTRACT FROM AUTHOR]

To investigate the role of CD4+CD25+Foxp3+ regulatory T (Treg) cells in blood-stage malaria, we compared Plasmodium chabaudi AS infection in wild-type (WT) C57BL/6 and transgenic mice overexpressing the transcription factor Foxp3 (Foxp3Tg) and observed that Foxp3Tg mice experienced lethal infection and deficient malaria-specific immune responses. Adoptive transfer of total CD4+ T cells from Foxp3Tg mice or CD4+CD25+ T cells from WT mice to naive WT recipients confirmed that high numbers of Treg cells compromised immune control of malaria. Transfer of GFP+CD4+CD25+ T cells to naive WT recipients together with immunohistochemical staining of spleens from infected WT mice demonstrated that Foxp3+ Treg cells localized in the T cell area of the spleen. Determination of CD4+Foxp3+ Treg cell responses in the spleen of infected WT mice revealed a significant but transient increase in CD4+Foxp3+ Treg cells early in infection. This was followed by a significant and sustained decrease due to reduced proliferation and apoptosis of CD4+Foxp3+ Treg cells. Importantly, the kinetics of IL-2 secretion by effector CD4+Foxp3- T cells coincided with changes in CD4+Foxp3+ cells and the differentiation of CD4+T-bet+IFN-γ+ cells required for immune control of infection. Administration of the IL-2/anti-IL-2 mAb (clone JES6-1) complex to infected WT mice increased the severity of P. chabaudi AS infection and promoted expansion of Foxp3+ Treg cells. Collectively, these data demonstrate that the ability to control and eliminate P. chabaudi AS infection is due to a tight balance between natural Treg cells and effector CD4+ Th1 cells, a balance regulated in part by IL-2. [ABSTRACT FROM AUTHOR]

The IL-2/IL-2R signaling pathway has an important role in autoimmunity. Several genes identified in genome-wide association (GWA) studies encode proteins in the IL-2/IL-2R signaling cascade that are associated with autoimmune diseases. One of these, PTPN2, encodes a protein tyrosine phosphatase that is highly expressed in T cells and regulates cytokine signaling. An intronic risk allele in PTPN2, rs1893217(C), correlated with decreased IL-2R signaling in CD4+ T cells as measured by phosphorylation of STAT5 (phosphorylated STAT5 (pSTAT5)). We modeled an additive single nucleotide polymorphism (SNP) genotype, in which each copy of the risk allele conferred a decrease in IL-2R signaling (P=4.4 × 10−8). Decreased pSTAT5 impacted IL-2Rβ chain signaling resulting in reduced FOXP3 expression in activated cells. This phenotype was not due to overt differences in expression of the IL-2R, molecules in the IL-2R signaling cascade or defects in STAT5. However, the rs1893217(C) risk variant did correlate with decreased PTPN2 expression in CD4+CD45RO T cells (P=0.0002). Thus, the PTPN2rs1893217(C) risk allele associated with reduced pSTAT5 in response to IL-2 and reduced PTPN2 expression. Together, these data suggest that decreased expression of PTPN2 may indirectly modulate IL-2 responsiveness. These findings, identified through genotype/phenotype relationships, may lead to identification of novel mechanisms underlying dysregulation of cytokine signaling in autoimmunity. [ABSTRACT FROM AUTHOR]

Background: After the recent discovery of Th17 cells, it was proposed that Th17 responses are involved in the pathogenesis of inflammatory bowel diseases (IBD). CD4(+)CD25(+) regulatory T cells (Treg) are considered to be an attractive tool for the treatment of IBD. Here, we investigated whether Treg are capable of suppressing Th17-mediated colitis.Methods: Naive CD4(+) T cells were transferred into SCID mice with or without Treg. In some experiments, Treg were transferred into recipient mice with established colitis. Mice treated with Treg were injected with an anti-transforming growth factor (TGF)-β mAb or control IgG. Clinical symptoms of colitis, histological changes and cytokine expressions were investigated.Results: SCID mice transferred with naive CD4(+) T cells developed chronic colitis with significant increases in Th1 and Th17 cytokine expressions in the colon. When Treg were co-transferred with naive CD4(+) T cells, development of colitis was prevented, and Th17 cytokine expressions were markedly reduced. Similarly, when Treg were transferred into mice with established colitis, the colitis was significantly ameliorated in association with dramatic reductions in Th17 cytokine expressions. Injection of anti-TGF-β mAb abolished the Treg-mediated suppression with significant elevations in Th17 cytokine productions.Conclusion: This adoptive transfer model of colitis was associated with augmented Th1 and Th17 responses, and Treg were capable of suppressing colonic inflammation by downregulating Th17 responses as well as Th1 responses via TGF-β. Consequently, Treg transfer therapy is expected to be efficacious for IBD even if Th17 is involved in the pathogenesis. [ABSTRACT FROM AUTHOR]

The article offers information on a study conducted on treatment response to different antiretroviral combination strategies for patients with advanced disease at HIV-1 diagnosis. It states that the response rate was different and better in baseline cluster of differentiation (CD) 4+ T cell levels. It mentions that the median time on the first-line antiretroviral therapy was 24 months, and the overall mortality was low.

Relatively little is known about regulatory T (Treg) cells and their functional responses in dogs. We have used the cross-reactive anti-mouse/rat Foxp3 antibody clone FJK-16s to identify a population of canine CD4 FOXP3 T cells in both the peripheral blood (PB) and popliteal lymph node (LN). FOXP3 cells in both PB and LN yielded positive staining with the newly developed anti-murine/human Helios antibody clone 22F6, consistent with the notion that they were naturally occurring Treg cells. Stimulation of mononuclear cells of LN origin with concanavalin A (Con A) in vitro yielded increased proportions and median fluorescence intensity of FOXP3 expression by both CD4 and CD8 T cells. Removal of the Con A and continued culture disclosed a CD4 FOXP3 population, distinct from the CD4 FOXP3 T cells; very few CD8 FOXP3 T cells were observed, though CD8 FOXP3 cells were present in equal abundance to CD4 FOXP3 cells. The CD4 FOXP3 T cells were thought to represent activated Treg cells, in contrast to the FOXP3 cells, which were thought to be a more heterogeneous population comprising predominantly activated conventional T cells. Co-staining with interferon-γ (IFN-γ) supported this notion, because the FOXP3 T cells were almost exclusively IFN-γ, whereas the FOXP3 cells expressed a more heterogeneous IFN-γ phenotype. Following activation of mononuclear cells with Con A and interleukin-2, the 5% of CD4 T cells showing the highest CD25 expression (CD4 CD25) were enriched in cells expressing FOXP3. These cells were anergic in vitro, in contrast to the 20% of CD4 T cells with the lowest CD25 expression (CD4 CD25), which proliferated readily. The CD4 CD25 FOXP3 T cells were able to suppress the proliferation of responder CD4 T cells in vitro, in contrast to the CD4 CD25 cells, which showed no regulatory properties. [ABSTRACT FROM AUTHOR]