Absence Of Treg Cells Research Articles

Phosphorylation and Enzymatic Hydrolysis with Alcalase and Papain Effectively Reduce Allergic Reactions to Gliadins in Normal Mice.

Gliadins are major allergens responsible for wheat allergies. Food processing is an effective strategy to reduce the allergenicity of gluten. In the present study, we determined the secondary and tertiary structures of gluten and gliadins treated by chemical, physical, and enzymatic means through FTIR, surface hydrophobicity, intrinsic fluorescence spectra, and UV absorption spectra. The results showed that the three treatments of phosphorylation and alcalase and papain hydrolyses significantly changed the conformational structures of gliadins, especially the secondary structure. Then, the potential allergenicity of the phosphorylated and alcalase and papain hydrolyzed gliadins were further characterized, and we observed a significant decrease in the allergenicity through the results of the index of spleen, serum total IgE, gliadin-specific IgE, histamine, and serum cytokine concentrations. An elevation of Th17 cells, the absence of Treg cells, and an imbalance in Treg/Th17 are associated with allergy. On the basis of the expression levels of related cytokines and key transcription factors, we also confirmed that phosphorylation and alcalase and papain hydrolysis could effectively reduce the allergenicity of gliadins by improving the imbalance of both Th1/Th2 and Treg/Th17 in the spleens of sensitized mice. This study suggested that the changes in conformational structure contribute to gliadin hyposensitization and that phosphorylation and alcalase and papain hydrolysis may be promising strategies for the production of wheat products with low allergenicity.

Decreased regulatory function of CD4+CD25+CD45RA+ T cells and impaired IL-2 signalling pathway in patients with type 2 diabetes mellitus.

In this study, the frequency and function of CD4+CD25+CD45RA+ regulatory T cells (Treg) and intracellular IL-2 signalling molecules in patients with type 2 diabetes mellitus (T2DM) were investigated. Tregs and responder T cells (Tresp, CD4+CD25- T cells) were sorted and suppression assays were performed using flow cytometry. Phosphorylation of signal transducer and activator of transcription-5 (pSTAT5) were assessed using flow cytometry. Gene expression of FOXP3 was performed with the SYBR green Real Time PCR method. Production of IL-2 from cultured cells was assessed using ELISA. We observed a functional defect of CD4+CD25+CD45RA+ Tregs in T2DM patients with higher proliferation of Tresp cells, in response to anti-CD3 and anti CD28 stimulation in the presence of Tregs invitro. The results showed that the proliferation of Tresps in the absence of Treg cells was higher in T2DM patients than in healthy controls. Decreased FOXP3 mRNA expression and pSTAT5 were observed within the Tregs of the patients, whereas the level of secreted IL-2 from PBMCs culture was not statically different between T2DM patients and healthy individuals. Changes in intracellular IL-2 pathways and FOXP3 gene expression may contribute to the defect of Tregs in T2DM patients. These findings indicating that the purified CD4+CD25+CD45RA+ Treg cells have reduced functional capacity together with impaired IL-2 pathway in T2DM, and the Tregs could be used for a potential novel therapeutic target.

Salivary-gland-protective regulatory T-cell dysfunction underlies female-specific sialadenitis in the non-obese diabetic mouse model of Sjögren syndrome.

Immune cell-mediated destruction of salivary glands is a hallmark feature of Sjögren syndrome. Similar to the female predominance in humans, female non-obese diabetic (NOD) mice develop spontaneous salivary gland autoimmunity. However, in both humans and mice it is unclear what factors contribute to the initial immune infiltration of the salivary glands. Here, we used an adoptive transfer model of Sjögren syndrome to determine if female mice harbor a sex-specific defect in salivary-gland-protective regulatory T (Treg) cells. Transfer of cervical lymph node (LN) cells from female NOD mice into sex-matched NOD-severe combined immunodeficient (SCID) recipients resulted in sialadenitis, regardless of the presence or absence of Treg cells. In contrast, transfer of cervical LN cells from male NOD mice into sex-matched NOD-SCID recipients only resulted in sialadenitis when Treg cells were depleted before transfer, suggesting that male NOD mice have functional salivary-gland-protective Treg cells. Notably, the host environment affected the ability of Treg cells to prevent sialadenitis with testosterone promoting salivary gland protection. Treg cells from male mice did not protect against sialadenitis in female recipients. Testosterone treatment of female recipients of bulk cervical LN cells decreased sialadenitis, and Treg cells from female mice were capable of protecting against development of sialadenitis in male recipients. Hence, our data demonstrate that female NOD mice develop sialadenitis through a defect in salivary-gland-protective Treg cells that can be reversed in the presence of testosterone.

Interleukin-2-regulatory T cell axis critically regulates maintenance of hematopoietic stem cells.

The role of IL-2 in HSC maintenance is unknown. Here we show that Il2−/− mice develop severe anomalies in HSC maintenance leading to defective hematopoiesis. Whereas, lack of IL-2 signaling was detrimental for lympho- and erythropoiesis, myelopoiesis was enhanced in Il2−/− mice. Investigation of the underlying mechanisms of dysregulated hematopoiesis in Il2−/− mice shows that the IL-2-Treg cell axis is indispensable for HSC maintenance and normal hematopoiesis. Lack of Treg activity resulted in increased IFN-? production by activated T cells and an expansion of the HSCs in the bone marrow (BM). Though, restoring Treg population successfully rescued HSC maintenance in Il2−/− mice, preventing IFN-? activity could do the same even in the absence of Treg cells. Our study suggests that equilibrium in IL-2 and IFN-? activity is critical for steady state hematopoiesis, and in clinical conditions of BM failure, IL-2 or anti-IFN-? treatment might help to restore hematopoiesis.

Reversible lacrimal gland-protective regulatory T-cell dysfunction underlies male-specific autoimmune dacryoadenitis in the non-obese diabetic mouse model of Sjögren syndrome.

CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells are required to maintain immunological tolerance; however, defects in specific organ-protective Treg cell functions have not been demonstrated in organ-specific autoimmunity. Non-obese diabetic (NOD) mice spontaneously develop lacrimal and salivary gland autoimmunity and are a well-characterized model of Sjögren syndrome. Lacrimal gland disease in NOD mice is male-specific, but the role of Treg cells in this sex-specificity is not known. This study aimed to determine if male-specific autoimmune dacryoadenitis in the NOD mouse model of Sjögren syndrome is the result of lacrimal gland-protective Treg cell dysfunction. An adoptive transfer model of Sjögren syndrome was developed by transferring cells from the lacrimal gland-draining cervical lymph nodes of NOD mice to lymphocyte-deficient NOD-SCID mice. Transfer of bulk cervical lymph node cells modelled the male-specific dacryoadenitis that spontaneously develops in NOD mice. Female to female transfers resulted in dacryoadenitis if the CD4(+) CD25(+) Treg-enriched population was depleted before transfer; however, male to male transfers resulted in comparable dacryoadenitis regardless of the presence or absence of Treg cells within the donor cell population. Hormone manipulation studies suggested that this Treg cell dysfunction was mediated at least in part by androgens. Surprisingly, male Treg cells were capable of preventing the transfer of dacryoadenitis to female recipients. These data suggest that male-specific factors promote reversible dysfunction of lacrimal gland-protective Treg cells and, to our knowledge, form the first evidence for reversible organ-protective Treg cell dysfunction in organ-specific autoimmunity.

Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity.

The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies.

Regulatory T Cells Prevent Liver Fibrosis During HIV Type 1 Infection in a Humanized Mouse Model

Human immunodeficiency virus type 1 (HIV-1) disease is associated with aberrant immune activation, and coinfection with hepatitis C virus (HCV) exacerbates hepatic inflammation and fibrosis. However, the role of HIV-1 infection or host immune modulation in liver pathogenesis is not clearly defined. Here, we report that regulatory T (Treg) cells prevent liver immunopathogenesis during HIV-1 infection in a humanized mouse model. In the absence of Treg cells, HIV-1 infection induced liver fibrosis associated with hepatic stellate cell activation, hepatitis, and liver injury. Our findings provide new insight linking Treg cells and liver immunopathogenesis during HIV-1 infection.

Jagged2‐signaling promotes IL‐6‐dependent transplant rejection

The Notch pathway is an important intercellular signaling pathway that plays a major role in controlling cell fate. Accumulating evidence indicates that Notch and its ligands present on antigen-presenting cells might be important mediators of T helper cell differentiation. In this study, we investigated the role of Jagged2 in murine cardiac transplantation by using a signaling Jagged2 mAb (Jag2) that activates recombinant signal-binding protein-Jκ. While administration of Jag2 mAb had little effect on graft survival in the fully allogeneic mismatched model BALB/c→B6, it hastened rejection in CD28-deficient recipients. Similarly, Jag2 precipitated rejection in the bm12→B6 model. In this MHC class II-mismatched model, allografts spontaneously survive for >56 days due to the emergence of Treg cells that inhibit the expansion of alloreactive T cells. The accelerated rejection was associated with upregulation of Th2 cytokines and proinflammatory cytokine IL-6, despite expansion of Treg cells. Incubation of Treg cells with recombinant IL-6 abrogated their inhibitory effects in vitro. Furthermore, neutralization of IL-6 in vivo protected Jag2-treated recipients from rejection and Jagged2 signaling was unable to further accelerate rejection in the absence of Treg cells. Our findings therefore suggest that Jagged2 signaling can affect graft acceptance by upregulation of IL-6 and consequent resistance to Treg-cell suppression.

T-cell-based immunotherapy of autoimmune diseases

Dysregulated Th1/Th17 responses, frequently paralleled by a reduction or absence of Treg cells, are key features of autoimmune disease. Conventional therapeutic approaches for autoimmune disease treatment are largely based on nonspecific immunosuppression leading to substantial side effects. Recent developments in the area of CD4+ T-cell differentiation, together with experimental and preclinical findings with blockers of the IL-17 pathway and the use of Treg cell-based therapy, indicate that this CD4+ effector subset could represent effective targets to restore immune tolerance. Here, the authors summarize the recent progress in our understanding of the pathways and cues that drive CD4+ T-cell differentiation into specialized effectors, focusing on Th17 and Treg cells. The authors also discuss novel immunotherapeutic strategies based on targeting these T-cell subsets for autoimmune disease treatment.

Crosstalk of regulatory T cells and tolerogenic dendritic cells prevents contact allergy in subjects with low zone tolerance

Allergic contact dermatitis is one of the most common occupational diseases. Amain protective mechanism in those who do not develop allergic contact dermatitis is tolerance induction by repeated exposure to low doses of contact allergen, which is termed low zone tolerance (LZT). The mechanisms that determine the tolerance induction in subjects with LZT are still elusive. We performed analysis of the role of CD4(+)CD25(+) forkhead box protein 3 (FOXP3)-positive regulatory T (Treg) cells and dendritic cells (DCs) in mice with LZT. Mechanisms of tolerance induction were analyzed in a murine model of LZT by using FOXP3 and IL-10 reporter mice, as well as mice that allow the selective depletion of Treg cells or DCs. Depletion of CD4(+)CD25(+)FOXP3(+) Treg cells during tolerance induction completely abolishes the development of LZT, resulting in a pronounced contact hypersensitivity response. Adoptive transfer experiments, depletion studies, and use of cell type-specific deficient mice revealed that IL-10 production is critical for the suppressor function of Treg cells in mice with LZT and that tolerogenic CD8(+)CD11c(+) DCs located in the skin-draining lymph nodes are essential for LZT. In the absence of Treg cells, DCs did not develop tolerogenic functions, indicating that activated IL-10(+) Treg cells might imprint the tolerogenic DC phenotype. Cell communication analysis revealed that the education of tolerogenic DCs might involve a direct interaction with Treg cells mediated by gap junctions. Subsequently, induction of tolerogenic CD11c(+) DCs leads to the generation of hapten-specific CD8(+) Treg cells, which protect against contact hypersensitivity. Our data demonstrate critical interactions between CD4(+)CD25(+)FOXP3(+) Treg cells and tolerogenic CD8(+)CD11c(+) DCs during the induction of LZT.

Expansion of follicular helper T cells in the absence of Treg cells: Implications for loss of B‐cell anergy

The maintenance of B-cell anergy is essential to prevent the production of autoantibodies and autoimmunity. However, B-cell extrinsic mechanisms that regulate B-cell anergy remain poorly understood. We previously demonstrated that regulatory T (Treg) cells are necessary for the maintenance of B-cell anergy. We now show that in Treg-cell-deficient mice, helper T cells are necessary and sufficient for loss of B-cell tolerance/anergy. In addition, we show that the absence of Treg cells is associated with an increase in the proportion of CD4(+) cells that express GL7 and correlated with an increase in germinal center follicular helper T (GC-T(FH) ) cells. These GC-T(FH) cells, but not those from Treg-cell-sufficient hosts, were sufficient to drive antibody production by anergic B cells. We propose that a function of Treg cells is to prevent the expansion of T(FH) cells, especially GC-T(FH) cells, which support autoantibody production.

CD4+CD25+FoxP3+ regulatory T cells enhance the allogeneic activity of endothelial-specific CD8+/CD28-CTL

Numerous data indicate that CD4+CD25+FoxP3+ regulatory T cells (Treg cells) can attenuate alloresponses of conventional T lymphocytes against professional antigen-presenting cells and thus qualify for clinical use in various transplant settings. However, it is unknown whether Treg cells also influence T cell-endothelial cell interactions. CD8+ PBMC (CD8+ PBMC, CTL) from healthy human donors were stimulated for 7 days with an allogeneic microvascular endothelial cell line (CDC/EU. HMEC-1, an immortalized human microvascular endothelial cell line, further referred to as HMEC) and additional endothelial cell types and analysed for their lytic activity against these target cells in the presence or absence of Treg cells. Addition of Treg cells (1:1:1) to the CTL/HMEC co-cultures in the efferent immune phase (day -1 prior to the assay) led to an increased cytotoxicity against HMEC. In contrast, Treg cells alone did not lyse HMEC. Treg cell-mediated enhancement of CTL activity was endothelial cell specific since lysis of HLA-matched Epstein-Barr virus-transformed B lymphoblastoid cells (B-LCL) was not influenced by the addition of Treg cells. Further analysis of CD28-positive and CD28-negative CTL sub-populations revealed that only the CD28-negative CTL showed an increased activity against HMEC after Treg cell co-culture. Although there is no doubt about the potential therapeutic efficacy of Treg cells to ameliorate outcome of allogeneic transplants, the endothelium might require additional protective interventions to prevent endothelial cell type-specific alloreactivity.

ICOS expression by effector T cells influences the ability of regulatory T cells to inhibit anti-chromatin B cell responses in recipient mice

T regulatory cells are critical for the prevention of autoimmunity. Specifically, Treg cells can control anti-chromatin antibody production in vivo, and this correlates with decreased ICOS expression on CD4 + T helper cells. Here we test the significance of high ICOS expression by T effector cells, firstly in terms of the anti-chromatin B cell response, and secondly on the ability of Treg cells to suppress T cell help. We bred CD4 + T cell receptor transgenic mice with mice that carry the Roquin san/san mutation. The Roquin gene functions to limit ICOS mRNA such that CD4 T cells from mutant mice express elevated ICOS. Using an in vivo model, TS1.Roquin san/san Th cells were compared with wild-type TS1 Th cells with regard to their ability to help anti-chromatin B cells in the presence or absence of Treg cells. Both TS1 and TS1.Roquin san/san Th cells induced anti-chromatin IgM a antibodies, but the TS1.Roquin san/san Th cells resulted in the recovery of more class-switched and germinal center B cells. Neither source of Th cells were capable of inducing long-lived autoantibodies. Treg cells completely suppressed anti-chromatin IgM a antibody production and reduced anti-chromatin B cell recovery induced by TS1 Th cells. Importantly, this suppression was less effective when TS1.Roquin san/san Th cells were used. Thus, high ICOS levels on effector T cells results in autoimmunity by augmenting the autoreactive B cell response and by dampening the effect of Treg cell suppression.

SHP-1 influences resistance of CD4+ T cells to regulatory T cell suppression (89.25)

Abstract The mechanistic and molecular understanding about factors that regulate the susceptibility and/or resistance of T cells to suppression by regulatory T cells (Treg) is very limited. The protein tyrosine phosphatase SHP-1 is a negative regulator of TCR-mediated signaling. SHP-1-deficient T cells hyper-proliferate in response to antigen stimulation. We therefore asked whether SHP-1 affects the susceptibility of CD4+ T cells to suppression by Treg cells. Our in vitro suppression assays showed that heterozygous (me/+) and homozygous (me/me) SHP-1-deficient CD4+ T cells were more resistant to Treg cell-mediated suppression than +/+ CD+ T cells, indicating that SHP-1 modulates the resistance to Treg cell suppression. In order to gain a better understanding of the mechanism that controls the resistance to suppression, we examined the conjugate formation between CD4+ T cells and the antigen presenting cells (APCs). Using flow cytometric and ImageStream100 (Amnis) analyses, we observed that me/me and me/+ T cells form more conjugates with APCs than +/+ T cells both in the presence or absence of Treg cells. Moreover, +/+ T cells treated with an inhibitor of SHP-1 showed a comparable increased conjugate formation as SHP-1-deficient T cells indicating that the observed phenotype is T cell intrinsic and not due to developmental differences. Furthermore, SHP-1-deficient T cells exhibited elevated surface levels of ICAM-1. Ongoing studies are addressing whether ICAM-1 directly affects the resistance to suppression. Taken together, the increased resistance to suppression displayed by SHP-1-deficient T cells and the enhanced capability of forming conjugates with APCs compared to wild type T cells suggest that the ability of forming T cell-APC conjugates influences to the susceptibility and/or resistance of a T cells to suppression. Source: NIH, RO1 AI 048672

Regulatory T Cells Modulate Staphylococcal Enterotoxin B-Induced Effector T-Cell Activation and Acceleration of Colitis

Oral administration of bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) activates mucosal T cells but does not cause mucosal inflammation. We examined the effect of oral SEB on the development of mucosal inflammation in mice in the absence of regulatory T (Treg) cells. SCID mice were fed SEB 3 and 7 days after reconstitution with CD4(+) CD45RB(high) or CD4(+) CD45RB(high) plus CD4(+) CD45RB(low) T cells. Mice were sacrificed at different time points to examine changes in tissue damage and in T-cell phenotypes. Feeding SEB failed to produce any clinical effect on SCID mice reconstituted with CD4(+) CD45RB(high) and CD4(+) CD45RB(low) T cells, but feeding SEB accelerated the development of colitis in SCID mice reconstituted with CD4(+) CD45RB(high) T cells alone. The latter was associated with an increase in the number of CD4(+) Vbeta8(+) T cells expressing CD69 and a significantly lower number of CD4(+) CD25(+) Foxp3(+) T cells. These changes were not observed in SCID mice reconstituted with both CD45RB(high) and CD45RB(low) T cells. In addition, SEB impaired the development of Treg cells in the SCID mice reconstituted with CD4(+) CD45RB(high) T cells alone but had no direct effect on Treg cells. In the absence of Treg cells, feeding SEB induced activation of mucosal T cells and accelerated the development of colitis. This suggests that Treg cells prevent SEB-induced mucosal inflammation through modulation of SEB-induced T-cell activation.

In vivo expansion of CD4 +Foxp3 + regulatory T cells mediated by GITR molecules

CD4 +Foxp3 + regulatory T cells (Treg cells) play an important role in maintaining self-tolerance as suppressive/regulatory CD4 T cells. In vitro analyses have revealed the characteristics of Treg cells, that is, hyporesponsiveness when stimulated via TCR in the presence of splenic APC. In this study, we report a new mAb, G3c, which can induce the expansion of Treg cells stimulated with anti-CD3 Ab along with splenic APC, the culture conditions in which Treg cells exhibit hyporesponsiveness. Surprisingly, G3c mAb recognized glucocorticoid-induced TNFR family-related proteins (GITR). G3c mAb had stronger co-stimulatory activity for both Treg cells and responder T cells than another anti-GITR Ab (DTA1) in vitro. The in vivo administration of G3c increased the number of Treg cells and had less effect in inducing anti-tumor immunity in normal mice, although G3c had some anti-tumor effect on non-Treg cells in the absence of Treg cells in vivo, in contrast to the anti-tumor therapeutic effect of DTA1 in normal mice. Therefore, we need to know that the manipulation of immune responses with the use of anti-GITR Abs results from a balance between co-stimulatory effects on Treg cells and on responder cells, and we must aim at a narrow window leading to the therapeutic effects.

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

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

Only Naive CD45RA+CD4+CD25high T Cells from Human Peripheral Blood Give Rise to Homogeneous Regulatory T Cell Lines.

The adoptive transfer of donor CD4+CD25high regulatory T (Treg) cells has been suggested for the prevention of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). In preparation of such trials we previously described protocols for the efficient in vitro expansion of human Treg cells (Hoffmann et al. 2004, Blood 104:895). Strong costimulation provided by immobilized anti-CD3 and anti-CD28 antibodies together with high-dose IL-2 resulted in a more than 3-log polyclonal expansion of Treg cells with strong suppressive activity. In contrast to CD4+CD25− T cells, the majority of the CD4+CD25high Treg cells maintained expression of the lymph node homing receptors CD62L and CCR7 during in vitro culture.Detailed examination of sorted subpopulations from Treg cell lines now revealed that only CD62L+CCR7+ cells combined all phenotypic and functional characteristics of natural Treg cells, such as FOXP3 expression, lack of cytokine secretion and potent suppression of responder T (Tresp) cell proliferation. To elucidate the origin of this cell population, we initiated cultures from CD45RA+ naive as well as CD45RA− effector/memory-type CD4+CD25high Treg cells. The CD45RA+ population initially contained 95 ± 2.5% CD62L+CCR7+ cells and maintained this phenotype with still over 90% CD62L+CCR7+ cells after 2 weeks and appox.70% after 3 weeks in culture (n=10). In contrast, CD62L and CCR7 expression in CD45RA− Treg cells was less stable and decreased from 58 ± 8% CD62L+CCR7+ cells after isolation to 32 ± 20% after culture for 3 weeks. Similar differences were observed with respect to cytokine production, as determined by intracellular staining: Whereas less than 5% (n=8) of expanded CD45RA+ Treg cells expressed IL-2 and/or IFN-γ, almost 40% of expanded CD45RA− Treg cells produced one or both of these pro-inflammatory cytokines upon stimulation with PMA/ionomycin. Interestingly, expanded CD45RA− Treg cells also contained a defined subpopulation of IL-10-producing cells (6.9 ± 5.6%; n=8), which was absent in expanded CD45RA+ Treg cells. Both subpopulations showed suppressive activity after polyclonal restimulation, however, suppression of Tresp cell proliferation by expanded CD45RA+ Treg cells was much more profound with only 13.6 ± 4% proliferating cells as compared to 35.4 ± 7.7% in the presence of expanded CD45RA− Treg cells and 79.6 ± 18.7% in the absence of Treg cells, as measured in a CFSE-dilution assay at a 1:4 ratio of Treg and Tresp cells (n=7). Most importantly, when determined on a single cell level by intracellular staining, 93.6 ± 1 % (n=3) of expanded CD45RA+ Treg cells still expressed FOXP3 after 3 weeks in culture, whereas only 10.8 ± 6.8% of expanded CD45RA− Treg cells remained FOXP3+. No re-expression could be induced in FOXP3− expanded CD45RA− Treg cells by short-term restimulation via CD3/CD28, whereas expanded FOXP3+ CD45RA+ Treg cells transiently upregulated FOXP3, with peak expression levels as early as 24h after stimulation and return to baseline levels by 48 to 72h. Based on these unexpected findings that only naive CD45RA+, but not memory-type CD45RA− CD4+CD25high T cells give rise to homogeneous Treg cell lines, we suggest that isolation and expansion of CD45RA+ CD4+CD25high T cells is the best strategy for adoptive Treg cell therapies.

Interleukin-2-Dependent Mechanisms of Tolerance and Immunity In Vivo

IL-2 is a critical T cell growth factor in vitro, but predominantly mediates tolerance in vivo. IL-2 is mainly produced by CD4(+) Th cells, but the role of Th cell-derived IL-2 in vivo is controversial. We demonstrate that during immunity to a tumor/self-Ag, the predominant role of Th cell-derived IL-2 was to maintain IL-2Ralpha (CD25) on CD4(+) T regulatory cells (T(reg)), which resulted in their maintenance of the T(reg) cell lineage factor, Forkhead/winged helix transcription factor (Foxp3), and tolerance. However, in the absence of T(reg) cells, Th cell-derived IL-2 maintained effector T cells and caused autoimmunity. IL-2R signaling was indispensable for T(reg) cell homeostasis and efficient suppressor function in vivo, but, surprisingly, was not required for their generation, because IL-2(-/-) and CD25(-/-) mice both contained Foxp3(+) T cells in the periphery. IL-2R signaling was also important for CD8(+) T cell immunity, because CD25(-/-) tumor-reactive CD8(+) T cells failed to affect established tumors. Conversely, IL-2R signaling was not required for Th cell function. Lastly, administration of anti-IL-2 plus exogenous IL-15 to tumor-bearing mice enhanced the adoptive immunotherapy of cancer. Therefore, Th cell-derived IL-2 paradoxically controls both tolerance and immunity to a tumor/self-Ag in vivo.

Estrogen-mediated immunomodulation involves reduced activation of effector T cells, potentiation of treg cells, and enhanced expression of the PD-1 costimulatory pathway

Estrogen (E2)-induced immunomodulation involves dual effects on antigen-presenting cells (APC) and CD4(+)CD25(+) regulatory T cells (Treg) but not a direct effect on effector T cells. In this report, we further investigated the effects of E2 on APC and Treg function. We found that E2 treatment in vivo strongly reduced recovery of APC from the peritoneal cavity and inhibited induction of the inflammatory cytokines interleukin (IL)-12 and interferon-gamma but enhanced secretion of IL-10. Moreover, E2-conditioned bone marrow-derived dendritic cells (BM-DC) could both enhance Treg activity and directly inhibit responder T cells in the absence of Treg cells. We examined whether this E2-induced inhibitory activity of BM-DC might involve costimulation through the recently described PD-1 pathway. Both E2 and pregnancy markedly enhanced PD-1 expression in several types of APC, including macrophages, B cells, and especially dendritic cells (DC). Similarly to E2-induced enhancement of FoxP3 expression and experimental autoimmune encephalomyelitis protection, E2-induced enhancement of PD-1(+) cells was also mediated through estrogen receptor alpha (Esr1) in DC and macrophages but not in B cells. Based on antibody inhibition studies, PD-1 interaction with its ligands, PDL-1 and especially PDL-2, could mediate either positive or negative regulatory signaling in both mature and immature E2-conditioned DC, depending, respectively, on a relatively high (10:1) or low (1:1) ratio of T cells:BM-DC. These novel findings indicate that E2-induced immunomodulation is mediated in part through potentiation in BM-DC of the PD-1 costimulatory pathway.