Enhanced FoxP3 expression and Treg cell function in pregnant and estrogen-treated mice.

Estrogen (E2) upregulates the FoxP3 gene that marks regulatory CD4+CD25+ T cells (Treg cells). However, E2 also inhibits the ability of antigen presenting cells (APC) to activate T cells. It is possible that these opposing functions might affect the degree of overt suppression during pregnancy and autoimmunity. To evaluate E2 effects on Treg cell function, we quantified FoxP3 levels and Treg suppression in CD4+CD25+ T cells from pregnant and E2-treated mice, and overt Treg suppression in E2- vs. placebo-pretreated mice with autoimmune encephalomyelitis. The data clearly demonstrate that enhanced expression of FoxP3, which occurs in pregnant mice and in mice treated exogenously with E2 pellets, results in a concomitant increase in functional suppression within the CD4+CD25(bright) Treg fraction of splenocytes. The similarities in FoxP3 expression and Treg cell function in E2-treated and pregnant mice implicate E2 as a major contributor for increasing Treg function during pregnancy. Surprisingly, suppression was not enhanced when Treg cells from E2-treated mice were activated with APC and CD4+CD25- responder T cells from the same E2-treated mice, a result consistent with impaired APC activation of Treg cells. In contrast, Treg suppression was strikingly enhanced in combined cell cultures from E2-pretreated mice that were protected from EAE induced with neuroantigen in complete Freund's adjuvant. These results suggest that E2 treatment may have opposing effects on Treg cells vs. APC that both contribute to overt suppression, but such effects are overcome and focused towards enhanced suppression in inflammatory environments produced during pregnancy and EAE.

17Beta-estradiol treatment profoundly down-regulates gene expression in spinal cord tissue in mice protected from experimental autoimmune encephalomyelitis.

It is now well documented that experimental autoimmune encephalomyelitis (EAE) can be effectively prevented by estrogen therapy. Previously, we identified a limited set of genes that were altered in spleens of mice protected from EAE by 17beta-estradiol (E2) treatment. As a continuation of these studies, we present here transcriptional changes in genes expressed in spinal cord tissue. The Affymetrix microarray system was used to screen more than 12,000 genes from E2-treated double transgenic (BV8S2 and AV4) female mice protected from EAE vs. control mice with severe EAE. We found that estrogen therapy had a profound inhibitory effect on the expressions of many immune-related genes in spinal cords. Estrogen significantly affected the transcription of 315 genes, 302 of which were down-regulated and only 13 that were up-regulated by > or = 2.4 fold. A number of genes encoding the histocompatibility complex, cytokines/receptors, chemokines, adhesion molecules, and signal transduction proteins were strongly down-regulated (> 20 fold) in estrogen-treated mice to levels similar to those of the spinal cord tissue from unmanipulated mice. The identification of genes with altered expression patterns in the spinal cords of estrogen-treated mice provides unique insight into the process that ultimately results in protection against EAE.

MHC class II derived recombinant T cell receptor ligands protect DBA/1LacJ mice from collagen-induced arthritis.

We previously demonstrated the therapeutic effects of MHC class II derived recombinant T cell receptor ligands (RTL), single-chain two domain complexes of the alpha1 and beta1 domains of MHC class II molecules genetically linked with an immunodominant peptide, in experimental autoimmune encephalomyelitis. In the current study, we produced a monomeric murine I-Aq-derived RTL construct covalently linked with bovine collagen type II peptide (bCII257-270) suitable for use in DBA/1LacJ mice that develop collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis, after immunization with bCII protein in CFA. In this study, we demonstrate that the I-Aq-derived RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term modulation of T cells specific for arthritogenic Ags. Our results showed that the I-Aq/bCII257-270 molecule could systemically reduce proinflammatory IL-17 and IFN-gamma production and significantly increase anti-inflammatory IL-10, IL-13, and FoxP3 gene expression in splenocytes. Moreover, I-Aq/bCII257-270 molecule could also selectively inhibit IL-1beta, IL-6, and IL-23 expression in local joint tissue. This is the first report demonstrating effective prevention of joint inflammation and clinical signs of CIA with an I-Aq-derived RTL, thus supporting the possible clinical use of this approach for treating rheumatoid arthritis in humans.

Treg suppressive activity involves estrogen-dependent expression of programmed death-1 (PD-1).

Estrogen [17-beta-estradiol (E2)] is a potent driver of the FoxP3+ regulatory T cell (Treg) compartment. Recently, Tregs were further characterized by intracellular expression of the negative co-stimulatory molecule, programmed death-1 (PD-1). To clarify the role of PD-1 versus FoxP3 in E2-enhanced Treg suppression, we evaluated both markers and functional suppression in wild-type, estrogen receptor knockout (ERKO) mice and PD-1 KO mice. We demonstrate that intracellular PD-1 expression is also E2 sensitive, since E2 treatment increased intracellular PD-1 levels in CD4+FoxP3+ cells, and PD-1 expression and Treg suppression were reduced in ERKO mice. Surprisingly, PD-1 KO mice retained normal levels of FoxP3 expression, but Tregs from these mice lacked functional suppression. However, E2 pre-treatment of PD-1 KO mice partially restored functional Treg suppression without enhancing FoxP3 expression. Thus, functional Treg suppression in immunized mice without E2 pre-treatment was more closely linked to PD-1 expression than to FoxP3 expression. However, although enhanced PD-1 expression was E2 dependent, functional suppression was still enhanced by E2 pre-treatment in the absence of PD-1. These data clearly demonstrate that E2 can affect multiple regulatory elements that influence Treg suppression, including both PD-1-dependent and PD-1-independent pathways.

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.

Middle-age male mice have increased severity of experimental autoimmune encephalomyelitis and are unresponsive to testosterone therapy.

Treatment with sex hormones is known to protect against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, little is known about how age affects the course of EAE or response to hormone treatment. This study demonstrates striking differences between middle-age vs young C57BL/6 male mice in the clinical course of EAE and response to both testosterone (T4) and estrogen (E2) hormone therapy. Unlike young males that developed an acute phase of EAE followed by a partial remission, middle-age males suffered severe chronic and unremitting EAE that was likely influenced by alterations in the distribution and function of splenic immunocytes and a significant reduction in suppressive activity of CD4+CD25+ regulatory T cells in the spleen and spinal cord. Middle-age males had reduced numbers of splenic CD4+ T cells that were generally hypoproliferative, but enhanced numbers of splenic macrophages and MHC class II-expressing cells, and increased secretion of the proinflammatory factors IFN-gamma and MCP-1. Surprisingly, middle-age males were unresponsive to the EAE-protective effects of T4 and had only a transient benefit from E2 treatment; young males were almost completely protected by both hormone treatments. T4 treatment of young males inhibited proliferation of myelin oligodendrocyte glycoprotein 35-55-specific T cells and secretion of TNF-alpha and IFN-gamma. The effects of T4 in vivo and in vitro were reversed by the androgen receptor antagonist, flutamide, indicating that the regulatory effects of T4 were mediated through the androgen receptor. These data are the first to define age-dependent differences in EAE expression and response to hormone therapy.

CD30 expression identifies the predominant proliferating T lymphocyte population in human alloimmune responses.

CD30 is an inducible member of the TNFR superfamily that is expressed on activated T and B cells and some lymphoid malignancies. We have previously shown that human CD30(+) T cells elicited with allogeneic APC are a major source of IFN-gamma and IL-5 production. In the present study we have used alloantigen, as well as anti-CD3 plus anti-CD28 mAb stimulation, to further characterize human CD30(+) T cells with respect to function and the expression of other activation-dependent cell surface molecules, including the related TNFR family members OX-40 and 4-1BB (CD137). Our results indicate that human CD30(+) T cells are a subset of activated T cells that also express CD25 and CD45RO. Moreover, we observed that allogeneic APC consistently induced a greater proportion of CD30(+) cells within the activated T cell population than did stimulation with plate-bound anti-CD3 plus anti-CD28 mAb or stimulation with soluble anti-CD3 plus anti-CD28 and autologous APC. The enhanced induction of CD30 expression by alloantigen was not common to other inducible TNFR family members because anti-CD3 plus anti-CD28 mAbs were far more effective in inducing expression of 4-1BB and OX-40. Furthermore, CD30 expression marked the predominant proliferating T cell population induced by alloantigen as determined by CFSE staining and flow cytometry. These results indicate that CD30, but not 4-1BB or OX-40, is preferentially induced by alloantigen, suggesting that CD30 may be important in human alloimmune responses.

Opposing roles for TGF-beta1 and TGF-beta3 isoforms in experimental autoimmune encephalomyelitis.

Hormones can exert significant protective effects on autoimmune diseases by activating immunoregulatory mechanisms. One of the possible mechanisms of hormonal protection might be through the anti-inflammatory effects of the TGF-beta molecule. The present study investigated the changes in expression of two TGF-beta isoforms, TGF-beta1 and TGF-beta3, in C57BL/6 and TCR transgenic (T/R+) B10.PL mice that manifested or were protected against clinical signs of experimental autoimmune encephalomyelitis (EAE) with 17beta-estradiol (E2) treatment. We here demonstrate an inverse relationship between expression of TGF-beta1 that is enhanced in mice with EAE, and TGF-beta3 that is enhanced in E2-protected mice. The differential expression of TGF-beta isoforms was observed in spinal cord tissue but not spleen. Additionally TGF-beta1 expression was evident both in whole spinal cord tissue and mononuclear cells isolated from inflamed tissue, in contrast to TGF-beta3 that was only detected in spinal cord tissue but not in mononuclear cells. Further studies revealed that CD3 and especially MAC-1 positive cells were the main source of TGF-beta1 in the mononuclear CNS population. Of crucial importance, the TGF-beta3 isoform displayed anti-proliferative properties towards encephalitogenic cells in vitro. We propose that the TGF-beta1 and TGF-beta3 isoforms play opposing roles in the expression of EAE.

Estrogen treatment induces a novel population of regulatory cells, which suppresses experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a debilitating neurological disease characterized by a progressive loss of motor and sensory function, eventually leading to paralysis and death. The primary cause of neurological impairment is demyelination of the central nervous system (CNS) caused by an inflammatory autoimmune response. Previous studies have shown that the severity of MS is reduced during pregnancy, suggesting that the increased level of sex hormones may reduce the autoimmune response. Recently, we have shown that estrogen treatment confers protection from experimental autoimmune encephalomyelitis (EAE), which is an animal model for MS. However, the cellular basis of estrogen's action remains unknown. In the current study, we demonstrate that estrogen treatment led to the induction of a novel subpopulation of regulatory cells in spleen and CNS, which also occurs naturally in pregnant mice. These previously uncharacterized cells display a low level expression of CD45 (CD45(dim)) and no detectable expression of many cell surface markers related to TCR signaling, including CD3 and TCR. However, these cells retained expression of VLA-4, an extracellular protein involved in cellular migration. Several lines of evidence suggest that these novel cells, defined as CD45(dim)VLA-4(+) cells, may play a role in the protective effects of estrogen in EAE. Injection of purified CD45(dim)VLA-4(+) cells conferred protection from spontaneous EAE (Sp-EAE). In contrast, injection of CD45(high)VLA-4(+) cells exacerbated the disease course. CD45(dim)VLA-4(+) cells also suppressed antigen-specific proliferation of primed lymphocytes in coculture. A better understanding of how CD45(dim)VLA-4(+) cells suppress the harmful immune response of EAE may help in explaining the induction of immune tolerance during pregnancy and lead to novel therapeutic approaches to combat MS and other autoimmune diseases.

Transfer of severe experimental autoimmune encephalomyelitis by IL-12- and IL-18-potentiated T cells is estrogen sensitive.

The aim of this study was to evaluate the roles of IL-18 and IL-12 in potentiating the encephalitogenic activity of T cell lines specific for myelin oligodendrocyte glycoprotein (MOG(35-55)). MOG-specific T cells stimulated with anti-CD3 and anti-CD28 in the presence of IL-12 or IL-18 alone transferred only mild experimental autoimmune encephalomyelitis (EAE) into a low percentage of recipients. However, T cells cocultured with both cytokines transferred aggressive clinical and histological EAE into all recipients. Coculture of T cells with IL-12 enhanced the secretion of IFN-gamma, but not TNF-alpha, whereas coculture with IL-18 enhanced the secretion of TNF-alpha, but not INF-gamma. However, coculture with both IL-18 and IL-12 induced high levels of both TNF-alpha and IFN-gamma. Additionally, IL-12 selectively enhanced mRNA expression of CCR5, whereas IL-18 selectively enhanced the expression of CCR4 and CCR7, and CCR4 and CCR5 were coexpressed on the surface of T cells cocultured with IL-12 and IL-18. Finally, estrogen treatment, previously found to inhibit both TNF-alpha and IFN-gamma production, completely abrogated all signs of passive EAE. These data demonstrate that optimal potentiation of encephalitogenic activity can be achieved by conditioning MOG-specific T cells with the combination of IL-12 and IL-18, which, respectively, induce the secretion of IFN-gamma/CCR5 and TNF-alpha/CCR4/CCR7, and that estrogen treatment, which is known to inhibit both proinflammatory cytokines, can completely ablate this aggressive form of passive EAE.

CNS gene expression pattern associated with spontaneous experimental autoimmune encephalomyelitis.

Transgenic mice with T-cell receptor (TCR) specific for myelin basic protein (MBP)-Ac1-11 peptide and homozygous for the RAG-1 mutation (T/R- mice) spontaneously develop acute progressive experimental autoimmune encephalomyelitis (Sp-EAE) mediated by CD4+ T cells. Microarray analysis of spinal cord tissue obtained from symptomatic versus non-symptomatic T/R- mice revealed strongly upregulated transcripts for genes involved in antigen presentation and processing, signal transduction, transcription regulation, metabolism, development, cell cycle, and many other processes involved in the induction of clinical and pathological signs of Sp-EAE. Several highly expressed genes were related directly to inflammation, including cytokines/receptors, chemokines/receptors, acute phase, complement molecules, and others. Many CNS-specific genes were also upregulated in sick mice. Abundance of message for the Tg TCR BV8S2 gene as well as several monocyte/macrophage-associated genes would suggest that both components play a crucial role in the pathogenesis of Sp-EAE. The profile of transcriptional changes found during the development of Sp-EAE provides the first description of the encephalitogenic process in the absence of purposeful immunization with myelin peptides and immune-enhancing adjuvants. This unique approach is the first to implicate molecules and pathways that contribute naturally to onset of paralysis and demyelination, and thus may provide unique insights and novel treatment strategies for human diseases such as multiple sclerosis.