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As one specialized subset of regulatory T cells (Tregs), follicular regulatory T cells (TFR) could suppress follicular helper T cells (TFH) and B cells in germinal centers to maintain immune homeostasis. The unbalance of TFR and TFH cells could result in abnormal germinal center responses and contribute to pathogenesis of autoimmune diseases. However, the role of TFR cells in systemic lupus erythematosus (SLE) remains unclear. This study revealed a significant increase of CD4 + CXCR5 + FOXP3 + TFR cells in peripheral blood of SLE patients compared with healthy controls. Meanwhile, the suppression ability of circulating TFR cells was not altered. The ratios of TFR/TFH were increased in SLE patients and the frequency of TFR was positively correlated with auto-antibodies and SLEDAI scores of SLE patients. Our results demonstrated that circulating TFR cells were increased during SLE, which suggested that elevated TFR might be a response to the pathogenesis of SLE to suppress TFH function and may provide novel insight for the pathogenesis of SLE. [ABSTRACT FROM AUTHOR]

CD4 T cells in human infants and adults differ in the initiation and strength of their responses. The molecular basis for these differences is not yet understood. To address this the principle key molecular events of TCR- and CD28-induced signaling in naive CD4 T cells, such as Ca2+ influx, NFAT expression, phosphorylation and translocation into the nucleus, ERK activation and IL-2 response, were analyzed over at least the first 3 years of life. We report dramatically reduced IL-2 and TNFα responses in naive CD31+ T cells during infancy. Looking at the obligatory Ca2+ influx required to induce T cell activation and proliferation, we demonstrate characteristic patterns of impairment for each stage of infancy that are partly due to the differential usage of Ca2+ stores. Consistent with those findings, translocation of NFATc2 is limited, but still dependent on Ca2+ influx as demonstrated by sensitivity to cyclosporin A (CsA) treatment. Thus weak Ca2+ influx functions as a catalyst for the implementation of restricted IL-2 response in T cells during infancy. Our studies also define limited mobilization of Ca2+ ions as a characteristic property of T cells during infancy. This work adds to our understanding of infants’ poor T cell responsiveness against pathogens. [ABSTRACT FROM AUTHOR]

Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance. Therapeutic manipulation of Tregs is subject to numerous clinical investigations including trials for adoptive Treg transfer. Since the number of naturally occurring Tregs (nTregs) is minute, it is highly desirable to develop a complementary approach of inducing Tregs (iTregs) from naïve T cells. Mouse studies exemplify the importance of peripherally induced Tregs as well as the applicability of iTreg transfer in different disease models. Yet, procedures to generate iTregs are currently controversial, particularly for human cells. Here we therefore comprehensively compare different established and define novel protocols of human iTreg generation using TGF-β in combination with other compounds. We found that human iTregs expressed several Treg signature molecules, such as Foxp3, CTLA-4 and EOS, while exhibiting low expression of the cytokines Interferon-γ, IL-10 and IL-17. Importantly, we identified a novel combination of TGF-β, retinoic acid and rapamycin as a robust protocol to induce human iTregs with superior suppressive activity in vitro compared to currently established induction protocols. However, iTregs generated by these protocols did not stably retain Foxp3 expression and did not suppress in vivo in a humanized graft-versus-host-disease mouse model, highlighting the need for further research to attain stable, suppressive iTregs. These results advance our understanding of the conditions enabling human iTreg generation and may have important implications for the development of adoptive transfer strategies targeting autoimmune and inflammatory diseases. [ABSTRACT FROM AUTHOR]

The vast majority of Foxp3 regulatory T cells (Treg) exhibits constitutive expression of CD25 (IL-2Rα), which allows the constitution of the high affinity IL-2Rαβγ receptor, ensuring efficient IL-2 binding by Treg. Maintenance of CD25 expression at Treg surface depends on both cell intrinsic factors and environmental stimuli such as IL-2 itself. Whether other factors can participate to maintenance of CD25 expression in vivo is at present unknown. In the present work we demonstrated that IL-7, a gamma-chain cytokine exerting a crucial role in T cell development and homeostasis, is able and necessary to sustain the expression of high levels of CD25 at Treg surface. We demonstrated that, during in vitro cultures performed in the absence of IL-2, IL-7 is able to sustain CD25 expression at Treg surface through a transcriptional mechanism. By studying mice in which IL-7 signaling is either genetically impaired or increased and by employing adoptive transfer murine models, we demonstrated that IL-7 is necessary for sustained expression of CD25 at Treg surface in vivo. To ascertain the biological impact of IL-7 mediated modulation of CD25 expression, we demonstrated that IL-7 modulation of CD25 expression at Treg surface affected their ability to efficiently bind IL-2 and transduce IL-2 signaling. Finally, we demonstrated that IL-7 dependent modulation of CD25 associated with potentiated IL-2 induced expansion of Treg in vivo. Collectively, our results identify IL-7 as a necessary factor contributing to sustained CD25 expression at Treg surface in vivo thereby affecting their ability to efficiently react to IL-2. [ABSTRACT FROM AUTHOR]

Lymphocyte Activation Gene – 3 (LAG-3) is an immune checkpoint molecule that regulates both T-cell activation and homeostasis. However, the molecular mechanisms underlying LAG-3’s function are generally unknown. Using a model in which LAG-3 blockade or absence reliably augmented homeostatic proliferation in vivo, we found that IL-2 and STAT5 are critical for LAG-3 function. Similarly, LAG-3 blockade was ineffective in the absence of regulatory T-cells (Treg), suggesting an important role for LAG-3 in either the responsiveness of conventional T-cells (Tconv) to regulation, or a relative defect in the ability of LAG-3 KO regulatory T-cells (Treg) to suppress the proliferation of Tconv. In this model, LAG-3 KO Treg suppressed proliferation in a manner fairly similar to wild-type (WT) Treg, but LAG-3 KO Tconv were relatively resistant to suppression. Further studies also identified a role for LAG-3 in the induction/expansion of Treg. Finally, we found that LAG-3 blockade (or knockout) led to a relative skewing of naïve CD4 T-cells toward a TH1 phenotype both in vitro and in in vivo. Together, these data suggest that LAG-3 expression on Tconv cells makes them more susceptible to Treg based suppression, and also regulates the development of a TH1 T-cell response. [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]

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]

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]

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]

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]

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]

Abstract: Chronic immune activation is the leading event in the pathogenesis of HIV-1 infection and is associated with depletion of CD4+ T cells and Th1/Th2 imbalance. The role of Tregs in HIV infection is still controversial as these cells may control both immune activation and HIV-specific T cell responses. Aim of the present study was to correlate the degree of immune activation with FOXP3 expression and production of IL-2 and IL-10. Peripheral blood was obtained from 10 HIV-1-infected subjects with sustained response to HAART. Four cellular fractions were purified and simultaneously used in further experiments: PBMCs, CD4+CD45RO+, CD4+CD45RO+CD25− and CD4+CD45RO+CD25+. The level of immune activation was measured both by flow cytometry and radiometry as short-term 1-hour spontaneous lymphocyte proliferation; IL-2 and IL-10 production were determined by ELISA in supernatants of cultures non-stimulated or stimulated with PHA and p24 antigen. FOXP3 mRNA expression was detected by RT-PCR. A group of healthy subjects was used as control. Statistical analysis was performed by SPSS. The level of immune activation (total CD25 expression) was found correlated to both CD4+ and Tregs (CD4+CD25high+; FOXP3mRNA) and it was found higher in HIV-1 infected subjects in comparison to the healthy control group. The highest SLP-1 h was measured in the CD4+CD4+CD45RO+ CD25− population (T reg depleted), suggesting a role of Tregs in controlling immune activation during HIV-1 infection. In addition, CD4+ cells from HIV-infected individuals remained responsive to the negative regulation by Tregs (measured as IL-10 production) both spontaneously and upon stimulation. It is worth noting, however, that total PBMCs of these patients also responded well to specific antigen (p24) stimulation even though IL-2 release was impaired. The results obtained suggest that Tregs significantly influence the level of immune activation. At the same time, their function appears to be dependent on IL-2 production by the remaining T cells. These findings suggest that the control of Th1/Th2 balance could be an approach to immune-based therapy for HIV infection. [Copyright &y& Elsevier]

The article evaluates three cycles of interleukin-2 (IL) on clinical and immunologic outcomes, utilizing a CD4 T-cell count of <350 cells/mm³ after treatment interruption (TI) following antiretroviral therapy. IL-2 recipients have higher CD4 counts during TI for 48 weeks with a waning of the CD4 effect by 72 weeks. After TI, a higher viral set point and drug resistance mutations at virologic rebound were related with a shorter time to CD4 T-cell count of less than 350 cells/mm³.

The CD31 antigen (PECAM-1) has been reported to be a stable marker for a human CD4 T-cell subpopulation unable to produce interleukin-4 (IL-4). We show here that CD31 expression is not stable inasmuch as CD4 T-cell lines and clones derived from cell-sorted neonatal CD31+ cells lose CD31 upon repetitive cycles of stimulation and IL-2 expansion. Moreover, various cytokines (IL-1α, IL-4, IL-6, transforming growth factor-β) fail to reinduce CD31 on CD31- clones. Whereas all CD31+ CD4 T cells rapidly express high levels of the CD45RO antigen and downregulate the L-selectin antigen after priming, CD31 disappears more slowly because only part of the cells lose CD31 expression upon each cycle of stimulation. Loss of CD31 reflects a functional maturation of CD45RO+ cells since, in a system which favours the development of Th2 effectors, IL-4 is produced by CD31- but not CD31+ effector T cells, whereas interferon-), is produced by both types of cells. However, CD31 is not a Th1 marker since it is not expressed on several Th1 antigen-specific clones. We conclude that CD31 is a maturation marker expressed on the great majority of naive CD45RO- CD4 T cells and on a subset of CD45RO+ CD4 T cells that are at an intermediate stage of maturation. [ABSTRACT FROM AUTHOR]

This article reports on interleukin-2 therapy and CD4+ T cells in HIV-1 infection. Since the HIV epidemic began, interleukin 2 has been at the forefront of immunology for this disease, with phase 1 studies of exogenous interleukin 2 being started as early as 1983. A strong rationale for such attempts was provided by the observation that the functional defects, including reduced proliferative responses to both antigens and mitogens, of CD4+ T cells in HIV-1-infected patients are associated with reduced production of interleukin 2, and that the integrity of interleukin-2-secreting CD4+ T lymphocytes is associated with favorable clinical outcome.