Role In Treg Function Research Articles

The functional role of Helios in CD4+ T cells

Abstract The transcription factor Helios is expressed in the majority of Foxp3+ Regulatory T cells (Treg), where it plays an essential role in Treg function. Loss of Helios in the Treg population induces systemic autoimmunity, marked by CD4 and CD8 hyperproliferation, enhanced germinal center formation, and systemic immune activation. Helios is also expressed in a small subset of conventional CD4 +T cells. However, when Helios is deleted in all CD4 cells, conventional and Treg, by crossing the mice to CD4Cre, this phenotype is abrogated, indicating that Helios must also play a significant role in the function of conventional CD4 +T cells. In order to examine specific subsets of CD4 +memory cells, we performed single cell transcriptome analysis of CD44 himemory cells from WT mice and mice with the CD4 specific deletion of Helios. Characterization of the CD44 hiHelios +population showed that Helios expression changed dramatically with age, so cells from young (5–6 weeks) and old (5–6 months) mice were included in the analysis. Both longitudinal (young vs old) and cross-sectional (WT vs KO) comparisons were made, with emphasis placed on cross-sectional comparisons at old age. The sequencing results indicated significant transcriptomic and numeric shifts in multiple subpopulations of the analyzed splenocytes, including the almost complete loss of one cluster in both the young and old Helios deficient mice. Taken together, the observed transcriptomic and clustering changes may explain the role Helios plays in the abrogation of autoimmunity in our model and may provide a starting point for us to analyze its function in other biological pathways. This work was supported by the Intramural Research Program of NIAID, NIH This work was supported by the Intramural Research Program of NIAID, NIH

Characteristics of regulatory T-cell function in patients with chronic hepatitis B and C coinfection.

Regulatory T cells (Tregs) affect the pathogenesis and disease progression of chronic viral hepatitis. This study evaluated the frequency and function of Tregs in patients with chronic HBV/HCV coinfection. Seventy-four untreated HBV/HCV co-infected patients were enrolled in this study. These subjects were divided into four subgroups: HBV-active/HCV-active (BACA), HBV-inactive/HCV-active (BICA), HBV-active/HCV-inactive (BACI) and HBV-inactive/HCV-inactive (BICI). Treg frequency was calculated as the fraction of CD4+ Foxp3+ T cells among CD4+ T cells. Treg-mediated inhibition was measured as percent of inhibition of T-cell proliferation. The expression of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-10 with/without Treg inhibition was also studied. Among the patients, there were 8 cases of BACA (10.8%), 38 of BICA (51.4%), 14 of BACI (18.9%) and 14 of BICI (18.9%).The frequency of CD4+ Foxp3+ T cells was comparable between the four groups. The inhibitory function of Tregs among the patients in the BACA and BICA was higher than that in the BICI (BACA vs BICI, P=.0210; BICA vs BICI, P=.0301). Patients in the BACA and BICA had higher fibrosis-4 (FIB-4) scores and serum ALT levels and lower serum albumin levels than those of the other groups. ALT abnormality was significantly and independently associated with a higher Treg immunosuppressive ability. The IFN-γ expression of the effector T cells in the BACA was higher than that of the other groups. In conclusion, the inhibitory function of Tregs is higher among the HBV/HCV co-infected patients with active HCV infection. ALT abnormality plays a dominant role in Treg function.

Tbet provides survival advantage to Tregs during Type 1 inflammation

Abstract Regulatory T-cells (Tregs) comprise subgroups with transcriptional programs similar to helper T-cell lineages. To visualize Tbet expression by Tregs during a T helper 1 (Th1) driven inflammation induced by Complete Freund’s Adjuvant in vivo, we imaged Tregs in the lymph nodes of a Tbet.ZsGreen-Foxp3.RFP mouse. While the majority of conventional Foxp3+ Tregs, resided within the T-cell zone, Tbet+Foxp3+ Tregs localized to the interfollicular zone that also contained the recently activated Th1 cells. To understand whether Tbet serves merely as a marker of activation or plays an actual role in Treg function, we generated bone marrow chimeras from Tbet+/+ and Tbet−/− mice. The peripheral Treg pool in chimeric mice was equally constituted by the Tbet+/+ and Tbet−/− cells. However, when the mice were challenged with Plasmodium chabaudi, a parasite that initiates a strong systemic Th1 response, the Treg compartment was significantly dominated by Tbet+/+ Tregs that displayed increased Tbet expression over time. While Tbet+/+ and Tbet−/− Tregs displayed similar activation status and proliferation, Tbet−/− Treg were more prone to apoptosis. Furthermore, Tbet−/− Tregs expressed lower levels of the ATP ectonucleotidase CD39 that was more pronounced during the peak of infection. Pharmacological inhibition of the activity of CD39 increased apoptosis of Tbet+/+ but not Tbet−/− Tregs in an ATP enriched culture in-vitro, suggesting a positive correlation between the availability of Tbet, CD39 activity and viability. Taken together, Tbet may preserve the ability of Tregs to cope with elevated toxic extracellular ATP at the inflammation site by tuning their CD39 expression or activity.

The cytoplasmic domain of CTLA-4 control autoimmunity via inducing regulatory T cells

Abstract Regulatory T cells (Tregs) have critical roles for maintaining immune tolerance and homeostasis. Recently, it is revealed that CTLA-4 is an essential membrane protein for the suppressive function of Treg. However, the molecular mechanism of cytoplasmic domain of CTLA-4 (ctCTLA-4) and its role in Treg function and differentiation is not clearly understood. In this study, we utilized ctCTLA-4 peptide and its various mutant forms conjugated with dNP2, a cell-permeable peptide, to investigate the mechanism of ctCTLA-4 in Treg function and differentiation. We found that the ctCTLA-4 convert naïve T cells to Foxp3-positive cells in T helper 17 cell differentiation condition in vitro, while the lysine-rich motif mutant form could not. The dNP2-ctCTLA-4 also significantly inhibits experimental autoimmune encephalomyelitis (EAE) in vivo, by reducing CNS-infiltrating Th17 cells with significantly increased Treg proportion. As similar with in vitroresults, lysine-rich motif mutant form could not increase Treg proportion and inhibit EAE. To elucidate its molecular mechanisms, we conducted HuProt protein microarray to identify lysine-rich motif dependent binding partner of ctCTLA-4 related with increasing Tregs. With the results, we found that ctCTLA-4 decreased a phosphorylation of MAPK in T cell stimulation conditions resulting enhanced nuclear localization of Smad2/3, a key transcription factor for Foxp3 expression. These results suggest that ctCTLA-4 has a critical molecular mechanism of inducing Tregs to maintain peripheral immune tolerance to control autoimmunity.

Toll-like receptors blocking restores in vitro microbicidal activity in latent tuberculosis-infected subjects.

Latent tuberculous infection (LTBI) can function as a 'reservoir' for Mycobacterium tuberculosis. Given that T-regulatory cell (Treg) numbers are augmented in LTBI, it is likely that Toll-like receptors (TLRs) may have a role in Treg function. Elucidation of the immune mechanisms associated with tuberculosis (TB) development may help to control M. tuberculosis spread.OBJECTIVE: To investigate the role of TLR2, TLR4 and TLR9 in hindered in vitro microbicidal activity and increase Treg number during LTBI. Whole blood cell cultures from individuals with LTBI and healthy controls (HCs) infected with live M. tuberculosis H37Rv strain were used to investigate the effect of TLR2, TLR4 and TLR9 on Treg number, microbicidal activity, and interferon-gamma and interleukin (IL)10 production. LTBI subjects were characterised by increased Treg number and impaired microbicidal activity when compared with HCs. Specific blockade of TLR4 and TLR9 led to a significant reduction in Treg number, a decrease in IL-10 production and substantial upregulation of microbicidal activity. M. tuberculosis infection may activate TLR4 and TLR9 pathways to suppress M. tuberculosis-specific immune responses. Here, we show that activation of TLR4 and TLR9 hinder microbicidal activity during LTBI.

ASSOCIATION OF FOXP3 GENE VARIANTS WITH RISK OF HASHIMOTO'S THYROIDITIS AND CORRELATION WITH ANTI-TPO ANTIBODY LEVELS.

Regulatory T cells (Tregs) have critical roles in preventing autoimmune diseases such as Hashimoto's thyroiditis (HT). Forkhead box P3 (Foxp3), the master transcription factor of Tregs, plays a pivotal role in Treg function. Herein, we investigated the association of two single nucleotide polymorphisms (SNPs) of the Foxp3 gene with HT development. A total of 129 HT patients and 127 healthy subjects were genotyped for rs3761548 (-3279 A/C) and rs3761549 (-2383 C/T) in the Foxp3 gene, using polymerase chain reaction-restriction fragment length polymorphism. Genotypic and allelic distribution of rs3761548 SNP showed a significant association with HT. The CC genotype was observed in 37.2% of patients versus 22.1% of the controls [P<0.008, odds ratio (OR): 2.1; 95% confidence interval (CI): 1.2-3.6] and the AC genotype in 41.1% of patients compared to 54.3% of the controls (P<0.025, OR: 2.1; CI: 1.2-3.6). In addition, higher frequency of C allele in patients compared to controls (P=0.05, OR: 1.4; 95% CI: 0.9-2) suggested that patients with the CC genotype and C allele had increased susceptibility to HT. There were significantly higher serum levels of anti-thyroid peroxidase (ATPO) antibody in patients with the rs3761548 CC genotype (1156±163 IU/mL) compared to the other genotypes (≈582-656 IU/mL; P<0.004). We observed a greater frequency of the AC genotype in patients who had decreased ATPO antibody levels (P=0.02). The association of the rs3761548 SNP with risk of HT and its influence on ATPO antibody levels suggested an important role for Foxp3 in the biology and pathogenesis of HT.

Telomerase Mediates Lymphocyte Proliferation but Not the Atherosclerosis-Suppressive Potential of Regulatory T-Cells.

Atherosclerosis is an age-related disease characterized by systemic oxidative stress and low-grade inflammation. The role of telomerase and telomere length in atherogenesis remains contentious. Short telomeres of peripheral leukocytes are predictive for coronary artery disease. Conversely, attenuated telomerase has been demonstrated to be protective for atherosclerosis. Hence, a potential causative role of telomerase in atherogenesis is critically debated. In this study, we used multiple mouse models to investigate the regulation of telomerase under oxidative stress as well as its impact on atherogenesis in vitro and in vivo. Using primary lymphocytes and myeloid cell cultures, we demonstrate that cultivation under hyperoxic conditions induced oxidative stress resulting in chronic activation of CD4+ cells and significantly reduced CD4+ T-cell proliferation. The latter was telomerase dependent because oxidative stress had no effect on the proliferation of primary lymphocytes isolated from telomerase knockout mice. In contrast, myeloid cell proliferation was unaffected by oxidative stress nor reliant on telomerase. Telomerase reverse transcriptase deficiency had no effect on regulatory T-cell (Treg) numbers in vivo or suppressive function ex vivo. Adoptive transfer of telomerase reverse transcriptase-/- Tregs into Rag2-/- ApoE-/- (recombination activating gene 2/apolipoprotein E) double knockout mice demonstrated that telomerase function was not required for the ability of Tregs to protect against atherosclerosis. However, telomere length was critical for Treg function. Telomerase contributes to lymphocyte proliferation but plays no major role in Treg function, provided that telomere length is not critically short. We suggest that oxidative stress may contribute to atherosclerosis via suppression of telomerase and acceleration of telomere attrition in Tregs.

Identification of key genes implicated in the suppressive function of human FOXP3+CD25+CD4+ regulatory T cells through the analysis of time‑series data.

Human forkhead box P3 (FOXP3)+ cluster of differentiation (CD)25+CD4+ regulatory T cells (Tregs) are a type of T cell that express CD4, CD25 and FOXP3, which are critical for maintaining immune homeostasis. The present study aimed to determine the mechanisms underlying Treg function. The GSE11292 dataset was downloaded from the Gene Expression Omnibus, which included data from Treg cells at 19 time points (0–360 min) with an equal interval of 20 min, and corresponding repeated samples. However, data for Treg cells at time point 120 min were missing. Using the Mfuzz package, the key genes were identified by clustering analysis. Subsequently, regulatory networks and protein-protein interaction (PPI) networks were constructed and merged into integrated networks using Cytoscape software. Using Database for Annotation, Visualization and Integrated Discover software, enrichment analyses were performed for the genes involved in the PPI networks. Cluster 1 (including 292 genes), cluster 2 (including 111 genes), cluster 3 (including 194 genes) and cluster 4 (including 103 genes) were obtained from the clustering analysis. GAPDH (degree, 40) in cluster 1, Janus kinase 2 (JAK2) (degree, 10) and signal transducer and activator of transcription 5A (STAT5A) (degree, 9) in cluster 3, and tumor necrosis factor (TNF) (degree, 26) and interleukin 2 (IL2) (degree, 22) in cluster 4 had higher degrees in the PPI networks. In addition, it was indicated that several genes may have a role in Treg function by targeting other genes [e.g. microRNA (miR)-146b-3p→TNF, miR-146b-5p→TNF, miR-142-5p→TNF and tripartite motif containing 28 (TRIM28)→GAPDH]. Enrichment analyses indicated that IL2 and TNF were enriched in the immune response and T cell receptor signaling pathway. In conclusion, GAPDH targeted by TRIM28, TNF targeted by miR-146b-3p, miR-146b-5p and miR-142-5p, in addition to JAK2, IL2, and STAT5A may serve important roles in Treg function.

Potential Inhibitory Influence of miRNA 210 on Regulatory T Cells during Epicutaneous Chemical Sensitization

Toluene diisocyanate (TDI) is a potent low molecular weight chemical sensitizer and a leading cause of chemical-induced occupational asthma. The regulatory potential of microRNAs (miRNAs) has been recognized in a variety of disease states, including allergic disease; however, the roles of miRNAs in chemical sensitization are largely unknown. In a previous work, increased expression of multiple miRNAs during TDI sensitization was observed and several putative mRNA targets identified for these miRNAs were directly related to regulatory T-cell (Treg) differentiation and function including Foxp3 and Runx3. In this work, we show that miR-210 expression is increased in the mouse draining lymph node (dLN) and Treg subsets following dermal TDI sensitization. Alterations in dLN mRNA and protein expression of Treg related genes/putative miR-210 targets (foxp3, runx3, ctla4, and cd25) were observed at multiple time points following TDI exposure and in ex vivo systems. A Treg suppression assay, including a miR-210 mimic, was utilized to investigate the suppressive ability of Tregs. Cells derived from TDI sensitized mice treated with miR-210 mimic had less expression of miR-210 compared to the acetone control suggesting other factors, such as additional miRNAs, might be involved in the regulation of the functional capabilities of these cells. These novel findings indicate that miR-210 may have an inhibitory role in Treg function during TDI sensitization. Because the functional roles of miRNAs have not been previously elucidated in a model of chemical sensitization, these data contribute to the understanding of the potential immunologic mechanisms of chemical induced allergic disease.

Helios Controls a Limited Subset of Regulatory T Cell Functions.

A subpopulation (60-70%) of Foxp3(+) regulatory T cells (Tregs) in both mouse and man expresses the transcription factor Helios, but its role in Treg function is still unknown. We generated Treg-specific Helios-deficient mice to examine the function of Helios in Tregs. We show that the selective deletion of Helios in Tregs leads to slow, progressive systemic immune activation, hypergammaglobulinemia, and enhanced germinal center formation in the absence of organ-specific autoimmunity. Helios-deficient Treg suppressor function was normal in vitro, as well as in an in vivo inflammatory bowel disease model. However, Helios-deficient Tregs failed to control the expansion of pathogenic T cells derived from scurfy mice, failed to mediate T follicular regulatory cell function, and failed to control both T follicular helper cell and Th1 effector cell responses. In competitive settings, Helios-deficient Tregs, particularly effector Tregs, were at a disadvantage, indicating that Helios regulates effector Treg fitness. Thus, we demonstrate that Helios controls certain aspects of Treg-suppressive function, differentiation, and survival.

In vivo anti-LAP mAb enhances IL-17/IFN-γ responses and abrogates anti-CD3-induced oral tolerance.

Regulatory T cells (Tregs) play a critical role in the maintenance of immunological tolerance. The best-characterized Tregs are those expressing the transcription factor Foxp3 and in vivo modulation of Foxp3 Tregs has been employed to study their role in immune homeostasis. Latency-associated peptide (LAP) is a membrane-bound TGF-β complex that has also been shown to play a role in Treg function and oral tolerance. We developed a novel anti-mouse LAP mAb that allowed us to investigate the effect of targeting LAP in vivo on immune function and on anti-CD3-induced oral tolerance. We found that in vivo anti-LAP mAb administration led to a decrease in the number of CD4+LAP+ Tregs in spleen and lymph nodes without affecting CD4+Foxp3+ Tregs. Spleen cells from anti-LAP-injected mice proliferated more in vitro and produced increased amounts of IL-2, IL-17 and IFN-γ. Moreover, injection of anti-LAP antibody abrogated the protective effect of oral anti-CD3 on experimental autoimmune encephalomyelitis (EAE). Finally, in vivo anti-LAP administration prior to myelin oligodendrocyte glycoprotein immunization resulted in severe EAE in the absence of pertussis toxin, which is used for EAE induction. Our findings demonstrate the importance of CD4+LAP+ T cells in the control of immune homeostasis and autoimmunity and provides a new tool for the in vivo investigation of murine LAP+ Tregs on immune function.

Premature CD4+ T Cell Aging and Its Contribution to Lymphopenia-Induced Proliferation of Memory Cells in Autoimmune-Prone Non-Obese Diabetic Mice

Lymphopenia-induced proliferation (LIP), a mechanism to maintain a constant number of T cells in circulation, occurs in both normal aging and autoimmune disease. The incidence of most autoimmune diseases increases with age, and premature CD4+ T cell aging has been reported in several autoimmune diseases. In this study, we tested the hypothesis that premature CD4+ T cell aging can cause autoimmune disease by examining whether premature CD4+ T cell aging exists and causes LIP in our mouse model. Non-obese diabetic (NOD) mice were used because, in addition to Treg defects, the LIP of T cells has been shown to plays a causative role in the development of insulin-dependent diabetes mellitus (IDDM) in these mice. We found that with advancing age, NOD mice exhibited an accelerated decrease in the number of CD4+ T cells due to the loss of naïve cells. This was accompanied by an increase in the percentage of memory cells, leading to a reduced naïve/memory ratio. In addition, both the percentage of CD28+ cells in CD4+ T cells and IL-2 production decreased, while the percentage of FAS+CD44+ increased, suggesting that NOD mice exhibit premature CD4+ T cell aging. This process preferentially contributed to LIP of memory cells. Therefore, our results suggest that premature CD4+ T cell aging underlies the development of IDDM in NOD mice. Given that CD28 and IL-2 play important roles in Treg function, the relationships between premature CD4+ T cell aging and lymphopenia as well as Treg defects in autoimmune-prone NOD mice are proposed.

An obligate cell-intrinsic function for CD28 in Tregs

Tregs expressing the transcription factor FOXP3 are critical for immune homeostasis. The costimulatory molecule CD28 is required for optimal activation and function of naive T cells; however, its role in Treg function has been difficult to dissect, as CD28 is required for thymic Treg development, and blockade of CD28-ligand interactions has confounding effects in trans on nonregulatory cells. To address this question, we created Treg-specific Cd28 conditional knockout mice. Despite the presence of normal numbers of FOXP3+ cells, these animals accumulated large numbers of activated T cells, developed severe autoimmunity that primarily affected the skin and lungs, and failed to appropriately resolve induced experimental allergic encephalomyelitis. This in vivo functional impairment was accompanied by dampened expression of CTLA-4, PD-1, and CCR6. Disease occurrence was not due to subversion of Cd28-deficient Tregs into pathogenic cells, as complementation with normal Tregs prevented disease occurrence. Interestingly, in these "competitive" environments, Cd28-deficient Tregs exhibited a pronounced proliferative/survival disadvantage. These data demonstrate clear postmaturational roles for CD28 in FOXP3+ Tregs and provide mechanisms which we believe to be novel to explain how interruption of CD28-ligand interactions may enhance immune responses independent of effects on thymic development or on other cell types.

Prolactin down-regulates CD4+CD25hiCD127low/− regulatory T cell function in humans

Among its many functions, prolactin (PRL) participates in immune responses and promotes the activation, differentiation and proliferation of T cells. However, the mechanisms by which PRL regulates regulatory T (T(reg)) cells are still unknown. Our goal was to determine whether PRL plays a role in T(reg) function. We measured the expression of PRL and its receptor in T(reg) and effector T (T(eff)) cells from 15 healthy individuals. We also evaluated the functional activity of T(reg) cells by examining proliferation and cytokine secretion in cells activated with anti-CD3/CD28 in the presence or absence of PRL. We report that T(reg) cells constitutively expressed PRL receptor, whereas T(eff) cells required stimulation with anti-CD3/CD28 to induce PRL receptor expression. Expression of PRL was constitutive in both populations. We found that the addition of PRL inhibited the suppressor effect (proliferation) mediated by T(reg) cells in vitro, reducing suppression from 37.4 to 13% when PRL was added to co-cultures of T(reg) and T(eff) cells (P<0.05). Cultures treated with PRL favoured a Th1 cytokine profile, with increased production of TNF and IFNγ. We report for the first time that PRL receptor expression was constitutive in T(reg) cells but not in T(eff) cells, which require stimulation to induce PRL receptor expression. PRL inhibited the suppressive function of T(reg) cells, apparently through the induced secretion of Th1 cytokines.

CD4+CD25lowGITR+ cells: A novel human CD4+ T‐cell population with regulatory activity

Treg subsets play a role in sustaining peripheral tolerance, are characterized by markers such as forkhead winged-helix transcription factor (FOXP3) and CD25, and produce suppressive cytokines, such as IL-10 and TGF-β. Glucocorticoid-induced TNF receptor family-related (GITR) protein has been suggested to regulate Treg activity in mice. The aim of our study was to investigate GITR expression in human CD4(+) T lymphocytes and its possible role in Treg function. Results indicate that a subset of CD4(+) T cells in the peripheral blood expresses GITR and low levels of CD25 (CD4(+) CD25(low) GITR(+) ). These cells show Treg features as they express FOXP3, IL-10, TGF-β and are anergic but, as opposed to natural Tregs, express low levels of CTLA-4 and are CD127(high) . CD4(+) CD25(low) GITR(+) cells represent a low percentage of the CD4(+) T-cell population (0.32-1.74%) and are mostly memory cells. Functional experiments demonstrated that CD4(+) CD25(low) GITR(+) cells have relevant suppressive activity that depends on TGF-β. Moreover, an anti-GITR Ab inhibited their suppressive activity, as observed in CD4(+) CD25(+) murine Tregs. Taken together, these data indicate that human CD4(+) CD25(low) GITR(+) cells represent a distinct Treg subpopulation.

Identification of the E3 deubiquitinase USP21 as a positive regulator of GATA3 (152.25)

Abstract TCR signaling induces GATA3 expression in FOXP3+ Treg, but the underlying mechanism for GATA3 accumulation and its role in Treg function remains unclear. Here we identify the E3 deubiquitinase USP21 as a positive regulator of GATA3 in FOXP3+ T cells. By screening for regulators which may affect the function of key transcription factors essential for T cell lineage differentiation and function, we found that USP21 dramatically increases the cellular level of GATA3. Reciprocal co-immunoprecipitation studies indicated that USP21 can interact with GATA3 in vivo. Overexpression of wild type USP21, but not the enzymatically inactive mutant C221A, specifically promotes the up-regulation of GATA3 protein expression, suggesting that the deubiquitinase activity of USP21 is essential for the upregulation of GATA3. Moreover, we found that T cell activation via TCR and CD28 costimulation may upregulate the level of USP21 and GATA3 in FOXP3 expressing T cells. shRNA mediated knockdown of USP21 can prevent GATA3 accumulation after T cell activation, suggesting that the mechanism by which USP21 stablizes GATA3 may occur through preventing GATA3 degradation. Functionally, overexpression of wild-type USP21 promotes GATA3 mediated upregulation of IL-4. Our studies have identified a unique pathway to upregulate GATA3 activity at posttranslational level by reversing ubiqutination-mediated protein degradation.

Functional plasticity of regulatory T cells (168.7)

Abstract Regulatory T cells (Tregs) play a pivotal role in maintaining immune homeostasis, preventing autoimmunity, controlling exaggerated immune responses against infections and suppressing anti-tumor immunity. Tregs utilize multiple mechanisms to mediate their function. However, the number of mechanisms critical to exert maximal suppressive activity is unclear. It is conceivable that suppressive mechanisms could be tightly regulated by cross-regulatory networks. It has been shown that interleukin 10 (IL10) and IL35 have critical role in Treg function. Therefore, we assessed the functional capacity of Tregs lacking the ability to secrete IL10 and IL35. Surprisingly, our study revealed that Tregs lacking both IL10 and IL35 (Ebi3-/-Il10-/- or Il12a-/-Il10-/-) were fully functional and were able to suppress Tconv cells both in vitro and in vivo . Absence of IL10 and IL35 was compensated by an increase in cathepsin E (CTSE), influencing the release of soluble TRAIL and thereby resulting in suppression of target cells in a TRAIL-dependent manner. Inhibiting TRAIL activity disrupted the function of Ebi3-/-Il10-/- Tregs. The loss of suppressive function was maximal in Tregs lacking IL10, IL35 and TRAIL (Ebi3-/-Il10-/-Tnfsf10-/-Tregs). These data suggests the existence of cross-regulatory networks which controls the utilization of suppressive factors, thereby maintaining Treg functional plasticity.

CTLA‐4 is required by CD4+CD25+ Treg to control CD4+ T‐cell lymphopenia‐induced proliferation

CTLA-4 is constitutively expressed by CD4(+)CD25(+)Foxp3(+) Treg but its precise role in Treg function is not clear. Although blockade of CTLA-4 interferes with Treg function, studies using CTLA-4-deficient Treg have failed to reveal an essential requirement for CTLA-4 in Treg suppression in vivo. Conditional deletion of CTLA-4 in Foxp3(+) T cells disrupts immune homeostasis in vivo but the immune processes disrupted by CTLA-4 deletion have not been determined. We demonstrate that Treg expression of CTLA-4 is essential for Treg control of lymphopenia-induced CD4 T-cell expansion. Despite IL-10 expression, CTLA-4-deficient Treg were unable to control the expansion of CD4(+) target cells in a lymphopenic environment. Moreover, unlike their WT counterparts, CTLA-4-deficient Treg failed to inhibit cytokine production associated with homeostatic expansion and were unable to prevent colitis. Thus, while Treg developing in the absence of CTLA-4 appear to acquire some compensatory suppressive mechanisms in vitro, we identify a non-redundant role for CTLA-4 in Treg function in vivo.

Combination of CpG-ODN+anti-4-1BB but not CpG-ODN+anti-OX40 enhances antitumor immune responses in neu mice modulating the inhibitory function of Tregs (40.7)

Abstract Previous studies have established that 4-1BB and OX40, members of the TNFR family, enhance antitumor immune responses. Recent studies from our laboratory indicate that intratumoral injections of CpG-ODN induce antitumor responses in Her-2/neu tolerant mice. However, this treatment alone is not sufficient to completely eradicate tumors. Here we investigate whether the combination of CpG+anti-OX40 or anti-4-1BB enhance the antitumor responses in neu mice. Our results indicate that CpG+anti-4-1BB but not CpG+anti-OX40 eradicate tumor in neu mice. CpG+anti-4-1BB treatment significantly increased the number of CD8 cells within lymphoid organs as compared to control or anti-OX40 treated animals. More importantly, we observed that anti-4-1BB but not anti-OX40 inhibits the suppressive function of Tregs. Microarray analysis of Tregs revealed specific transcriptional differences brought about by treatment with anti-OX40 or anti-4-1BB. 4-1BB modulates differential gene expression in Tregs of over 300 genes that remain unaffected by OX40. Potential gene candidates include interleukin-9 and protein-tyrosine phosphatase receptor-type zeta-1, which are transcriptionally down and up regulated in the presence of anti-4-1BB. These proteins may play a role in Treg function, and could explain the enhanced anti-tumor effect observed in the presence of anti-4-1BB.

SHP‐1 plays a role in both conventional and CD4+CD25+Foxp3+ regulatory T cell function

While the importance of regulatory T (Treg) cells for immune tolerance has been increasingly appreciated, our mechanistic and molecular understanding of their function is still very limited. We have previously shown that mice lacking the SHP‐1, so‐called motheaten (me/me) mice, have increased numbers of Treg cells. SHP‐1 is a well‐recognized negative regulator of TCR‐mediated signaling. We therefore asked whether SHP‐1 also plays a role in Treg function. A comparison of the suppressive capabilities of wild type and me/me Treg cells demonstrated increased suppressive activity of me/me Treg cells in in vitro suppression assays. While this suppression is dependent on stimulation via the TCR, our data indicate that additional factors, which are critical for an effective Treg function, are regulated by SHP‐1. Interestingly when comparing wild type and me/me conventional T cells, we observed that SHP‐1 deficient CD4+CD25‐ T cells are more resistant to Treg‐mediated suppression than wild type T cells, indicating a role for SHP‐1 both in suppression as well as in resistance to suppression. We are currently addressing at the molecular level how SHP‐1 regulates these processes. We expect from these studies to not only better understand the regulatory role of SHP‐1 in conventional and Treg T cells but to also gain further insights into the mechanism of suppression by Treg cells.This work was supported by the NIH grant RO1 AI48672.