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Plasmacytoid dendritic cells (pDCs) produce type I interferon (IFN-I) during HIV-1 infection in response to TLR7 stimulation. However, IFN-I-signaling has been shown to play opposite effects in HIV-1 and SIV infection. TLR7-driven type I interferon production in pDCs is higher in women than in men due to the cell-intrinsic actions of estrogen and X-chromosome complement. Indeed, TLR7 is encoded on the X-chromosome, and the TLR7 gene escapes the X-chromosome inactivation in immune cells of women which express significantly higher levels of TLR7 protein than male cells. Following HIV infection, women have a lower viremia during acute infection and exhibit stronger antiviral responses than men, which has been attributed to the increased capacity of female pDCs to produce IFN-α upon TLR7-stimulation. However, a deleterious functional impact of an excessive TLR7 response on acute viremia in women has been recently revealed by the analysis of the frequent rs179008 c.32A>T SNP of TLR7. This SNP was identified as a sex-specific protein abundance quantitative trait locus (pQTL) causing a difference in the TLR7 protein dosage and effector function in females only. T allele expression was associated with a lower TLR7 protein synthesis, blunted production of IFN-α by pDCs upon TLR7 stimulation, and an unexpectedly lower viral load during primary HIV-1 infection in women. In the present review, the author will revisit the role of TLR7-driven pDC innate function in the context of HIV-1 infection to discuss at what stage of primary HIV-1 infection the TLR7 rs179008 T allele is likely to be protective in women.

Voltage-gated calcium (CaWe generated mice deleted for CaWe demonstrated non-redundant and synergistic functions of CaThus, Ca

Women develop stronger immune responses than men, with positive effects on the resistance to viral or bacterial infections but magnifying also the susceptibility to autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, the dosage of the endosomal Toll-like receptor 7 (TLR7) is crucial. Murine models have shown that TLR7 overexpression suffices to induce spontaneous lupus-like disease. Conversely, suppressing TLR7 in lupus-prone mice abolishes SLE development. TLR7 is encoded by a gene on the X chromosome gene, denoted TLR7 in humans and Tlr7 in the mouse, and expressed in plasmacytoid dendritic cells (pDC), monocytes/macrophages, and B cells. The receptor recognizes single-stranded RNA, and its engagement promotes B cell maturation and the production of pro-inflammatory cytokines and antibodies. In female mammals, each cell randomly inactivates one of its two X chromosomes to equalize gene dosage with XY males. However, 15 to 23% of X-linked human genes escape X chromosome inactivation so that both alleles can be expressed simultaneously. It has been hypothesized that biallelic expression of X-linked genes could occur in female immune cells, hence fostering harmful autoreactive and inflammatory responses. We review here the current knowledge of the role of TLR7 in SLE, and recent evidence demonstrating that TLR7 escapes from X chromosome inactivation in pDCs, monocytes, and B lymphocytes from women and Klinefelter syndrome men. Female B cells where TLR7 is thus biallelically expressed display higher TLR7-driven functional responses, connecting the presence of two X chromosomes with the enhanced immunity of women and their increased susceptibility to TLR7-dependent autoimmune syndromes.

At the onset of adolescence, asthma becomes less prevalent in males than in females, suggesting a protective role of male sex hormones. Here, Laffont et al. show that androgens negatively control ILC2 development and ILC2-driven lung inflammation in male mice., Prevalence of asthma is higher in women than in men, but the mechanisms underlying this sex bias are unknown. Group 2 innate lymphoid cells (ILC2s) are key regulators of type 2 inflammatory responses. Here, we show that ILC2 development is greatly influenced by male sex hormones. Male mice have reduced numbers of ILC2 progenitors (ILC2Ps) and mature ILC2s in peripheral tissues compared with females. In consequence, males exhibit reduced susceptibility to allergic airway inflammation in response to environmental allergens and less severe IL-33–driven lung inflammation, correlating with an impaired expansion of lung ILC2s. Importantly, orchiectomy, but not ovariectomy, abolishes the sex differences in ILC2 development and restores IL-33–mediated lung inflammation. ILC2Ps express the androgen receptor (AR), and AR signaling inhibits their differentiation into mature ILC2s. Finally, we show that hematopoietic AR expression limits IL-33–driven lung inflammation through a cell-intrinsic inhibition of ILC2 expansion. Thus, androgens play a crucial protective role in type 2 airway inflammation by negatively regulating ILC2 homeostasis, thereby limiting their capacity to expand locally in response to IL-33.

Plasmacytoid dendritic cells (pDCs) are found in the CNS during neuroinflammation and have been reported to exert regulatory functions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of entry of pDCs into the CNS as well as their phenotype and innate functional properties, once recruited into the CNS, have not been thoroughly examined. Herein, we show that pDCs rapidly accumulate into the brain and spinal cord during the acute phase of EAE, and maintain the expression of numerous phenotypic markers typical of peripheral pDCs. Functionally, CNS-pDCs constitutively expressed IRF7 and were able to rapidly produce type I IFNs and IL-12p40 upon ex vivo TLR-9 stimulation. Using adoptive transfer experiments, we provide evidence that CNS-pDC are recruited from the blood and accumulate into the CNS during the acute phase of EAE. Accumulation of pDCs into the CNS was strongly inhibited in the absence of CD29, but not CD18, suggesting a major role for ß1 but not ß2 integrins. Indeed, blocking the CD49d α4-integrins during acute EAE drastically diminished CNS-pDC numbers. Together, our results demonstrate that circulating pDCs are actively recruited into the CNS during acute EAE through a mechanism largely dependent on CD49d/CD29-integrins.

17β-Estradiol (E2) suppresses the development of experimental autoimmune encephalomyelitis (EAE) through estrogen receptor (ER) α, yet the cellular targets remain elusive. We have used an adoptive transfer model of myelin oligodendrocyte glycoprotein-specific CD4

Allergic asthma is a chronic pulmonary inflammatory disease initiated by exposure to normally harmless allergens and marked by bronchial hyperreactivity. It affects more than 300 million people worldwide. Asthma often starts in childhood. Epidemiological studies show that there are sexual disparities in the prevalence and severity of asthma. Before the age of 10, the disease is more common in boys. This tendency reverses at puberty suggesting a regulating role of the sex hormones. In this synthesis, we summarize current knowledge on the role of sex hormones in allergic inflammation, with a particular focus on the impact of androgens on the development and function of recently introduced group 2 innate lymphoid cell subsets (ILC2) as critical actors in the initiation of allergic responses.♢.

CD4+ T cells polarize into effector Th subsets characterized by signature transcription factors and cytokines. Although T-bet drives Th1 responses and represses the alternative Th2, Th17, and Foxp3+ regulatory T cell fates, the role of the T-bet–related transcription factor eomesodermin (Eomes) in CD4+ T cells is less well understood. In this study, we analyze the expression and effects of Eomes in mouse CD4+ T lymphocytes. We find that Eomes is readily expressed in activated CD4+ Th1 T cells in vivo. Eomes+ CD4+ T cells accumulated in old mice, under lymphopenic conditions in a T cell transfer model of colitis, and upon oral Ag administration. However, despite its expression, genetic deletion of Eomes in CD4+ T cells did not impact on IFN-γ production nor increase Th2 or Th17 responses. In contrast, Eomes deficiency favored the accumulation of Foxp3+ cells in old mice, after in vivo differentiation of Eomes-deficient naive CD4+ T cells, and in response to oral Ag in a cell-intrinsic way. Enforced Eomes expression during in vitro regulatory T cell induction also reduced Foxp3 transcription. Likewise, bystander Eomes-deficient CD4+ T cells were more efficient at protecting from experimental autoimmune encephalitis compared with wild-type CD4+ T cells. This enhanced capacity of Eomes-deficient CD4+ T cells to inhibit EAE in trans was associated with an enhanced frequency of Foxp3+ cells. Our data identify a novel role for Eomes in CD4+ T cells and indicate that Eomes expression may act by limiting Foxp3 induction, which may contribute to the association of EOMES to susceptibility to multiple sclerosis.

T lymphocytes express not only cell membrane ORAI calcium release-activated calcium modulator 1 but also voltage-gated calcium channel (CaWe investigated the role of β and α2δ auxiliary subunits on CaWe used CaMouse and human TThese results stress the role of Ca

Human plasmacytoid dendritic cells (pDCs) play a major role in innate immunity through the production of type I IFNs after TLR engagement by pathogens. Sex-based differences in the innate function of human pDCs have been established, with pDCs from women exhibiting enhanced TLR7-mediated IFN-α production as compared with pDCs from males. In mice, we recently provided evidence for a role of estrogens as a positive regulator of pDC innate functions through cell-intrinsic estrogen receptor α signaling, but did not exclude a role for other X-linked factors, particularly in human pDCs. In this study, we investigated the respective contribution of X chromosome dosage and sex hormones using a humanized mouse model in which male or female NOD-SCID-β2m−/− were transplanted with human progenitor cells purified from either male or female cord blood cells. We showed that, in response to TLR7 ligands, the frequency of IFN-α– and TNF-α–producing pDCs from either sex was greater in female than in male host mice, suggesting a positive role for estrogens. Indeed, blockade of estrogen receptor signaling during pDC development in vitro inhibited TLR7-mediated IFN-α production by human pDCs, which expressed both ESR1 and ESR2 genes. Interestingly, we also found that X chromosome dosage contributed to this sex bias as female pDCs have an enhanced TLR7-mediated IFN-α response as compared with male ones, irrespective of the sex of the recipient mice. Together, these results indicate that female sex hormones, estrogens, and X chromosome complement independently contribute to the enhanced TLR7-mediated IFN-α response of pDCs in women.

The recognition of multiple ligands by a single TCR is an intrinsic feature of T cell biology, with important consequences for physiological and pathological processes. Polyspecific T cells targeting distinct self-antigens have been identified in healthy individuals as well as in the context of autoimmunity. We have previously shown that the 2D2 TCR recognizes the myelin oligodendrocyte glycoprotein epitope (MOG)35–55 as well as an epitope within the axonal protein neurofilament medium (NF-M15–35) in H-2b mice. In this study, we assess whether this cross-reactivity is a common feature of the MOG35–55-specific T cell response. To this end, we analyzed the CD4 T cell response of MOG35–55-immunized C57BL/6 mice for cross-reactivity with NF-M15–35. Using Ag recall responses, we established that an important proportion of MOG35–55-specific CD4 T cells also responded to NF-M15–35 in all mice tested. To study the clonality of this response, we analyzed 22 MOG35–55-specific T cell hybridomas expressing distinct TCR. Seven hybridomas were found to cross-react with NF-M15–35. Using an alanine scan of NF-M18–30 and an in silico predictive model, we dissected the molecular basis of cross-reactivity between MOG35–55 and NF-M15–35. We established that NF-M F24, R26, and V27 proved important TCR contacts. Strikingly, the identified TCR contacts are conserved within MOG38–50. Our data indicate that due to linear sequence homology, part of the MOG35–55-specific T cell repertoire of all C57BL/6 mice also recognizes NF-M15–35, with potential implications for CNS autoimmunity.

Brown Norway rats injected with aurothiopro-panolsulfonate sodium salt develop systemic autoimmunity. The aim of this study was to assess the influence of the sulfur-containing group in this experimental model of gold-induced autoimmunity. It was shown that the sulfur-containing group does not induce autoimmunity of itself, but potentiates the immunotoxic effects of gold.

Th2 cells orchestrate allergic asthma and the cytokines they produce (IL-4, IL-5, and IL-13) are deleterious in allergy. Therefore, it is important to identify key signaling molecules expressed by Th2 cells that are essential for their function. We have previously shown that dihydropyridines selectively modulate Th2 cell functions.Because dihydropyridines bind to and modulate voltage-dependent calcium (Ca(v)1) channel in excitable cells, we aimed at showing that Th2 cells selectively express functional Ca(v)1-related channels, the inhibition of which may prevent asthma.We looked for Ca(v)1 channel expression in Th2 and Th1 cells by real-time polymerase chain reaction and Western blotting. We sequenced the isoforms expressed by Th2 cells and tested whether Ca(v)1 antisense oligodeoxynucleotides (Ca(v)1AS) affected Ca(2+) signaling and cytokine production. Finally, we tested the effect of Ca(v)1AS in the passive asthma model by injection of ovalbumin-specific Th2 cells transfected with Ca(v)1AS into BALB/c mice challenged with intranasal ovalbumin and in the active model of asthma by intranasal delivery of Ca(v)1AS together with soluble ovalbumin in BALB/c mice previously immunized with ovalbumin in alum.We show that mouse Th2 but not Th1 cells expressed Ca(v)1.2 and Ca(v)1.3 channels. Th2 cells transfected with Ca(v)1AS had impaired Ca(2+) signaling and cytokine production, and lost their ability to induce airway inflammation on adoptive transfer. Furthermore, intranasal administration of Ca(v)1AS suppressed airway inflammation and hyperreactivity in an active model of asthma.These results indicate that Th2 cells selectively express Ca(v)1 channels that may be efficiently targeted in T lymphocytes to prevent experimental asthma.

Asthma is a chronic airway disease resulting from inappropriate Th2-cell biased activation. Interleukin (IL)-4, IL-5 and IL-13 produced by Th2 cells contribute to the inflammatory process. Attempts for inhibiting interleukin-4 or IL-5 gave disappointing results. The simultaneous inhibition of several Th2-cytokines could be a more promising issue. Several arguments support the concept that Th2-cells express selective markers that could be targeted in asthma. Our group showed that Th2-cells selectively up-regulated dihydropyridine-sensitive Ca(2+) (DHP-Ca) channels essential for Ca(2+) signaling and type-2 cytokine production. Indeed, DHP-Ca antagonist effectively prevented or even reverted airway inflammation, airway remodeling and airway hyperresponsiveness in experimental models of asthma. Although it remains to be formally demonstrated that human lung infiltrating T-lymphocytes in asthmatic patients express DHP- Ca(2+) channels, we hypothesize that targeting DHP-Ca channels in T-lymphocytes could represent an efficient strategy in the treatment of asthma.This review article also discussed patents relevant to the field.

Increased IFN-α production contributes to the pathogenesis of infectious and autoimmune diseases. Plasmacytoid dendritic cells (pDCs) from females produce more IFN-α upon TLR7 stimulation than pDCs from males, yet the mechanisms underlying this difference remain unclear. In this article, we show that basal levels of IFN regulatory factor (IRF) 5 in pDCs were significantly higher in females compared with males and positively correlated with the percentage of IFN-α–secreting pDCs. Delivery of recombinant IRF5 protein into human primary pDCs increased TLR7-mediated IFN-α secretion. In mice, genetic ablation of the estrogen receptor 1 (Esr1) gene in the hematopoietic compartment or DC lineage reduced Irf5 mRNA expression in pDCs and IFN-α production. IRF5 mRNA levels furthermore correlated with ESR1 mRNA levels in human pDCs, consistent with IRF5 regulation at the transcriptional level by ESR1. Taken together, these data demonstrate a critical mechanism by which sex differences in basal pDC IRF5 expression lead to higher IFN-α production upon TLR7 stimulation in females and provide novel targets for the modulation of immune responses and inflammation.

A growing body of evidence from basic and clinical studies supports the therapeutic potential of estrogens in multiple sclerosis (MS), originating from the well-established reduction in relapse rates observed among women with MS during pregnancy. The biological effects of estrogens are mediated by estrogen receptors (ERα and ERβ). Estrogens or selective ER-agonists have been shown to exert potent neuroprotective or anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. A central question in EAE is to identify the cellular targets that express a functional ER isotype, and the mechanisms underlying the neuroprotective and anti-inflammatory effects of estrogens. Using pharmacological approaches targeting ER-specific functions, and genetic tools such as conditional knockout mice in which ERα or ERβ are selectively deleted in specific cell populations, a clearer picture is now emerging of the various cellular targets and downstream molecules responsible for estrogen-mediated protection against central nervous system autoimmunity.

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.

Estrogen treatment has been shown to exert a protective effect on experimental autoimmune encephalomyelitis (EAE), and is under clinical trial for multiple sclerosis. Although it is commonly assumed that estrogens exert their effect by modulating immune functions, we show in this study that 17β-estradiol (E2) treatment can inhibit mouse EAE without affecting autoantigen-specific T cell responsiveness and type 1 cytokine production. Using mutant mice in which estrogen receptor α (ERα) has been unambiguously inactivated, we found that ERα was responsible for the E2-mediated inhibition of EAE. We next generated irradiation bone marrow chimeras in which ERα expression was selectively impaired in inflammatory T lymphocytes or was limited to the radiosensitive hemopoietic compartment. Our data show that the protective effect of E2 on clinical EAE and CNS inflammation was not dependent on ERα signaling in inflammatory T cells. Likewise, EAE development was not prevented by E2 treatment in chimeric mice that selectively expressed ERα in the systemic immune compartment. In conclusion, our data demonstrate that the beneficial effect of E2 on this autoimmune disease does not involve ERα signaling in blood-derived inflammatory cells, and indicate that ERα expressed in other tissues, such as CNS-resident microglia or endothelial cells, mediates this effect.

Beta(2)-microglobulin (beta(2)m)-derived peptides are minor transplantation Ags in mice as beta(2)m-positive skin grafts (beta(2)m(+/+)) are rejected by genetically beta(2)m-deficient recipient mice (beta(2)m(-/-)). We studied the effector pathways responsible for the rejection induced by beta(2)-microglobulin-derived minor transplantation Ags. The rejection of beta(2)m(+/+) skin grafts by naive beta(2)m(-/-) mice was dependent on both CD4 and CD8 T cells as shown by administration of depleting mAbs. Experiments performed with beta(2)m(-/-)CD8(-/-) double knockout mice grafted with a beta(2)m(+/+) MHC class I-deficient skin showed that sensitized CD4 T cells directed at beta(2)m peptides-MHC class II complexes are sufficient to trigger rapid rejection. Rejection of beta(2)m(+/+) grafts was associated with the production of IL-5 in vitro, the expression of IL-4 and IL-5 mRNAs in the grafted tissue, and the presence within rejected grafts of a considerable eosinophil infiltrate. Blocking IL-4 and IL-5 in vivo and depleting eosinophils with an anti-CCR3 mAb prevented graft eosinophil infiltration and prolonged beta(2)m(+/+) skin graft survival. Lymphocytes from rejecting beta(2)m(-/-) mice also displayed an increased production of IFN-gamma after culture with beta(2)m(+/+) minor alloantigens. In vivo neutralization of IFN-gamma inhibited skin graft rejection. Finally, beta(2)m(+/+) skin grafts harvested from B6(lpr/lpr) donor mice, which lack a functional Fas molecule, survived longer than wild-type beta(2)m(+/+) skin grafts, showing that Fas-Fas ligand interactions are involved in the rejection process. We conclude that IL-4- and IL-5-dependent eosinophilic rejection, IFN-gamma-dependent mechanisms, and Fas-Fas ligand interactions are effector pathways in the acute rejection of minor transplantation Ags.

Although much progress has been made in characterization of the signaling pathways that control Th cell commitment, little is known about the early events that govern differentiation of IL-4-producing T lymphocytes in vivo. We have previously shown that chronic administration of low dose, soluble hen egg white lysozyme (HEL) induced the selective development of Ag-specific Th2 in genetically predisposed BALB/c mice. Here, we show that these memory/effector Th2 cells express a unique TCR Vβ repertoire, different from the TCR Vβ profile of primary effector cells from HEL-adjuvant-primed mice. This Th2-associated repertoire contains a highly frequent public clonotype characterized by preferred TCR AV and BV gene segment usage along with conserved sequences in the third hypervariable regions of both TCR chains. This Th2 clonotype, which is not recruited in primary effector T cells from HEL-adjuvant-immunized mice, recognized an IAd-restricted HEL determinant, preferentially processed by dendritic cells, but not by B cells. Thus, IL-4-producing CD4 T cells that expand following chronic Ag sensitization emerge from a distinct pool of precursors, supporting the hypothesis that ligand-TCR interactions play a crucial role in the regulation of Ag-specific Th2 cell development in vivo.

The mechanisms that influence the polarization of CD4 T cells specific for allogeneic MHC class II molecules in vivo are still poorly understood. We have examined the pathway of alloreactive CD4 T cell differentiation in a situation in which only CD4 T cells could be activated in vivo. In this report we show that priming of adult mice with allogeneic APC, in the absence of MHC class I-T cell interactions, induces a strong expansion of type 2 cytokine-producing allohelper T cells. These alloantigen-specific CD4 T cells directly recognize native allogeneic MHC class II molecules on APC and secrete, in addition to the prototypic Th2 cytokines IL-4, IL-5, and IL-10, large amounts of TGF-β. The default Th2-phenotype acquisition is not genetically controlled and occurred both in BALB/c and C57BL/6 mice. CD8 T cells are the principal cell type that controls CD4 T cell differentiation in vivo. Furthermore, we demonstrate that strong Th2 priming can be induced not only with allogeneic splenocytes but also with a low number of bone marrow-derived dendritic cells. Finally, using a passive transfer system, we provide direct evidence that CD8 T cell expansion in situ promotes alloreactive Th1 cell development principally by preventing their default development to the Th2 pathway in a mechanism that is largely IFN-γ independent. Therefore, this work demonstrates that type 2 cytokine production represents a dominant pathway of alloreactive CD4 T cell differentiation in adult mice, a phenomenon that was initially thought to occur only during the neonatal period.

The inability of certain antigen processing mutant cell lines to present intact proteins to T cells and to form SDS-stable MHC class II dimers has been shown to result from defective expression of HLA-encoded DMA and DMB genes. We have utilized some of these mutants to determine species compatibility of antigen presentation components. Mouse MHC class II I-A d cDNA was transfected into the human B cell lymphoblastoid cell lines 8.1.6, 7.9.6 (a mutant cell line derived from 8.1.6) and an independent deletion mutant T2 (called 8.1.6d, 7.9.6d and T2.d respectively). These cells were then examined for various functions in antigen presentation. Interestingly, none of the cells transfected with I-A d presented peptides derived from intact proteins to specific T cell hybridomas. However, presentation of synthetic peptides by these cells was normal. The ability to form SDS-stable dimers was dramatically reduced in the transfectants. In addition, I-A d molecules at the cell surface appeared loaded predominantly with the invariant chain peptides, CLIP. These properties of the I-A d transfectants are identical to those described for HLA class II molecules expressed in HLA-DM mutants. Perhaps the most interesting finding was the inability of I-A d in 8.1.6 to present protein antigens. Since 8.1.6 cells present antigens to HLA-DR, DP, DQ-restricted T cells and also have intact HLA-DM and invariant chain (Ii) functions, these results argue that some component of human antigen processing machinery is incompatible with I-A d molecules.

We have compared the capacity of dendritic cells (DC) and B cells to present peptide-class II complexes following administration of protein in adjuvant or in soluble form. Three different antigen-presenting cell (APC) populations were separated from draining lymph node cells from mice immunized s.c. with hen egg-white lysozyme (HEL) in adjuvant or with adjuvant only followed by soluble HEL: DC (N418+, class II+, B220-, low buoyant density), large B cells (B220+, low buoyant density) and small B cells (B220+, high buoyant density). HEL peptide-class II complexes displayed by these APC were evaluated by their capacity to activate HEL-specific T hybridoma cells. Following immunization with HEL in adjuvant, DC are the only lymph node APC population expressing detectable HEL peptide-class II complexes. Conversely, after i.v. administration of soluble HEL in mice previously injected with adjuvant only, lymph node B cells are much more efficient than DC in presenting peptide-class II complexes to T cells. Therefore, different modes of protein antigen administration lead to selective expression of antigenic complexes by different APC populations. These data correlate with the observation that, unlike B cells, DC recruited in lymph nodes of mice injected with adjuvant only present in vitro processed protein antigen much less efficiently than synthetic peptides, probably as a consequence of their maturation in vivo.

17β-Estradiol (E2) has been shown to regulate GM-CSF– or Flt3 ligand–driven dendritic cell (DC) development through estrogen receptor (ER) α signaling in myeloid progenitors. ERα regulates transcription of target genes through two distinct activation functions (AFs), AF-1 and AF-2, whose respective involvement varies in a cell type– or tissue-specific manner. In this study, we investigated the role of ERα AFs in the development and effector functions of inflammatory DCs, steady-state conventional DCs, and plasmacytoid DCs (pDC), using mouse lacking either AF-1 or AF-2. In agreement with previous works, we showed that E2 fostered the differentiation and effector functions of inflammatory DCs through ERα-dependent upregulation of IFN regulatory factor (IRF)-4 in GM-CSF–stimulated myeloid progenitors. Interestingly, whereas AF-1 was required for early IRF-4 upregulation in DC precursors, it was dispensable to enhance IRF-4 expression in differentiated DCs to a level compatible with the development of the more functional Ly6C− CD11b+ DC subset. Presence of E2 had no effect on progenitors from either knock-in mice with 7-aa deletion in helix 12 of ERα, lacking AF-2, or ERα−/− mice. By contrast, in Flt3 ligand–driven DC differentiation, activation of AF-1 domain was required to promote the development of more functionally competent conventional DCs and pDCs. Moreover, lack of ERα AF-1 blunted the TLR7-mediated IFN-α response of female pDCs in vivo. Thus, our study demonstrates that ERα uses AF-1 differently in steady-state and inflammatory DC lineages to regulate their innate functions, suggesting that selective ER modulators could be used to target specific DC subsets.

International audience; Plasmacytoid dendritic cells (pDCs) produce large amounts of type I interferons (IFN-α/β) in response to viral or endogenous nucleic acids through activation of their endosomal Toll-like receptors (TLR-7 and TLR-9). Enhanced TLR-7-mediated IFN-α production by pDCs in women, compared with men, has been reported, but whether sex hormones, such as estrogens, are involved in this sex-based difference is unknown. Here we show, in humanized mice, that the TLR-7-mediated response of human pDCs is increased in female host mice relative to male. In a clinical trial, we establish that treatment of postmenopausal women with 17β-estradiol markedly enhances TLR-7- and TLR-9-dependent production of IFN-α by pDCs stimulated by synthetic ligands or by nucleic acid-containing immune complexes. In mice, we found exogenous and endogenous estrogens to promote the TLR-mediated cytokine secretion by pDCs through hematopoietic expression of estrogen receptor (ER) α. Genetic ablation of ERα gene in the DC lineage abrogated the enhancing effect of 17β-estradiol on their TLR-mediated production of IFN-α, showing that estrogens directly target pDCs in vivo. Our results uncover a previously unappreciated role for estrogens in regulating the innate functions of pDCs, which may account for sex-based differences in autoimmune and infectious diseases.

Estrogens influence the incidence and the course of numerous immune or inflammatory diseases in humans and in experimental models. For instance, estrogens prevent the accumulation of granulocytes in acute inflammatory murine models, but the respective actions on neutrophil and eosinophil trafficking remain to be clarified. We demonstrate here that in a model of TGC-induced sterile peritonitis in ovx mice, chronic E2 administration electively and strongly inhibited peritoneal eosinophil accumulation. E2 decreased BM eosinophil number, contributing to a marked prevention of the TGC-induced eosinophil blood mobilization. These effects on eosinophil mobilization and peritoneal accumulation were abolished in ER-α−/− mice, demonstrating the crucial role of this nuclear receptor. Grafting ER-α−/− mice with ER-α+/+ BM cells restored the suppressive effect of E2 on peritoneal eosinophilia, although the action on eosinophil blood mobilization was still abrogated. We therefore explored additional mechanisms and found that E2 reduced the peritoneal concentrations of key eosinophil prosurvival factors (IL-5, IL-9, and IL-25) and enhanced eosinophil apoptosis during the inflammatory process. Furthermore, this proapoptotic effect of E2 was abrogated in IL-5-overexpressing Tg mice. To conclude, we demonstrate for the first time that ER-α activation by exogenous E2 administration strongly inhibits eosinophil accumulation during acute inflammation in a nonreproductive target site for estrogen through combined actions on eosinophil mobilization and apoptosis. This specific, suppressive effect of chronic E2 replacement therapy on eosinophils has to be integrated to further understand the evolution of eosinophil-associated diseases in menopausal women.

International audience; Sex hormones influence immune responses and the development of autoimmune diseases including MS and its animal model, EAE. Although it has been previously reported that ovariectomy could worsen EAE, the mechanisms implicated in the protective action of endogenous ovarian hormones have not been addressed. In this report, we now show that endogenous estrogens limit EAE development and CNS inflammation in adult female mice through estrogen receptor α expression in the host non-hematopoietic tissues. We provide evidence that the enhancing effect of gonadectomy on EAE development was due to quantitative rather than qualitative changes in effector Th1 or Th17 cell recruitment into the CNS. Consistent with this observation, adoptive transfer of myelin oligodendrocyte glycoprotein-specific encephalitogenic CD4(+) T lymphocytes induced more severe EAE in ovariectomized mice as compared to normal female mice. Finally, we show that gonadectomy accelerated the early recruitment of inflammatory cells into the CNS upon adoptive transfer of encephalitogenic CD4(+) T cells. Altogether, these data show that endogenous estrogens, through estrogen receptor α, exert a protective effect on EAE by limiting the recruitment of blood-derived inflammatory cells into the CNS.

17β-estradiol (E2) has been shown to promote the expression of inflammatory mediators by LPS-activated tissue resident macrophages through estrogen receptor α (ERα) signaling. However, it remained to be determined whether E2 similarly influences macrophages effector functions under inflammatory conditions in vivo, and whether this action of E2 resulted from a direct effect on macrophages. We show in this study that chronic E2 administration to ovariectomized mice significantly increased both cytokine (IL-1β, IL-6, and TNF-α) and inducible NO synthase mRNA abundance in thioglycolate (TGC)-elicited macrophages. The proinflammatory action of E2 was also evidenced at the level of released IL-1β and IL-6 by ex vivo LPS-activated macrophages. E2 concomitantly inhibited PI3K activity as well as Akt phosphorylation in TGC-elicited macrophages, suggesting that E2 promoted TLR-dependent macrophage activation by alleviating this suppressive signaling pathway. Indeed, this effect was abolished in the presence of the inhibitor wortmannin, demonstrating a key functional link between inhibition of PI3K activity and the E2 action on macrophage functions. Endogenous estrogens levels circulating in ovary-intact mice were sufficient to promote the above described actions. Finally, thanks to a CreLox strategy, targeted disruption of ERα gene in macrophages totally abolished the effect of E2 on the expression of inflammatory mediators by both resident and TGC-elicited peritoneal macrophages. In conclusion, we demonstrate that estrogens, through the activation of ERα in macrophages in vivo, enhance their ability to produce inflammatory mediators and cytokines upon subsequent TLR activation.

Background— The prevention of early atheroma by estrogens has been clearly demonstrated in all animal models and appears to be mediated through a direct action on the arterial wall rather than through an effect on the lipoprotein profile. The goal of the present study was to evaluate which cellular target is crucial in this beneficial action of estradiol. Methods and Results— We first confirmed the key role of estrogen receptor-α (ERα) in the atheroprotective effect of estradiol, because this action was completely abolished in mice deficient in both the low-density lipoprotein receptor (LDLr) and ERα. Second, using chimeric mice with an ERα deficiency in the hematopoietic lineage, we showed the persistence of the protective action of estradiol, which suggests the involvement of extrahematopoietic ERα. Third, we showed that loxP-flanked ERα mice (ERα flox/flox ) bred with Tie2-Cre + mice on an LDLr −/− background had complete inactivation of ERα in most hematopoietic and all endothelial cells. Remarkably, in this mouse model, the atheroprotective effect of estradiol was completely abolished. Fourth, the atheroprotective effect of estradiol remained abolished in Tie2-Cre + ERα flox/flox LDLr −/− mice transplanted with either Tie2-Cre + ERα flox/flox or ERα −/− bone marrow, whereas it was present in analogous chimeric Tie2-Cre − ERα flox/flox LDLr −/− receivers expressing endothelial ERα. Conclusions— We demonstrate directly and for the first time that endothelial ERα represents a key target of the atheroprotective effect of estradiol, whereas hematopoietic ERα is dispensable. Selective estrogen receptor modulators that mimic the endothelial action of estradiol should now be considered in atheroprotection.

Objectives— E2 accelerates reendothelialization through estrogen receptor α (ERα), and we now aimed at defining the precise local and systemic cellular actors of this process. Methods and Results— The respective roles of endothelial and hematopoietic targets of E2 were investigated in a mouse carotid injury model, using confocal microscopy, to follow endothelium repair. Grafting ERα −/− mice with ERα +/+ bone marrow (BM) was not sufficient to restore the accelerative effect of E2 on reendothelialization, demonstrating the necessary role of extrahematopoietic ERα. Using an endothelial-specific inactivation of ERα (Cre-Lox system), we showed that endothelial ERα plays a pivotal role in this E2 action. Conversely, in ERα +/+ grafted with ERα −/− BM, the E2 regenerative effect was abolished, demonstrating that ERα-expressing hematopoietic cells are also needed. As eNOS expression in BM was required for this action, both endothelial progenitor cells and platelets could be the hematopoietic targets that participate to this beneficial E2 effect. Conclusions— We demonstrate that endothelial ERα plays a pivotal role in E2-mediated reendothelialization. However, endothelial targeting alone is not sufficient because the concomitant stimulation of a subpopulation of BM ERα is necessary. This cooperation should be taken into account in strategies aimed at optimizing in-stent reendothelialization.

11 pages; International audience; Natural killer (NK)-cell alloreactivity is exploited in bone marrow transplantation to improve clinical outcome. Likewise, in solid organ transplantation, it has been recently shown that recipient NK cells may limit alloreactive T-cell responses through their capacity to prevent the persistence of graft-derived allogeneic dendritic cells (DCs). In a model of CD4(+) T cell-mediated allogeneic skin graft rejection, we show that the absence of host NK-cell alloreactivity was characterized by enhanced expansion of alloreactive effector T lymphocytes, including Th2 cells, and massive eosinophilic infiltrates in the rejected tissues. In CD8(+) T cell-deficient C57BL/6 (H-2(b)) recipients injected with allogeneic BALB/c (H-2(d)) DCs, we demonstrated that NK cells expressing the H-2D(d)-specific Ly49D activating receptor were implicated in the regulation of alloreactive CD4(+) T-cell responses. Moreover, we showed that Ly49D(+) CD127(-) NK cells were recruited within DC draining lymph nodes and rapidly eliminated allogeneic H-2(d) DCs through the perforin pathway. In normal mice, we further demonstrated that NK cells by quickly eliminating allogeneic DCs strongly inhibited alloreactive CD8(+) T-cell responses. Thus, NK cells act as early regulators of alloreactive T-cell priming in allotransplantation through their capacity to kill allogeneic DCs in draining lymph nodes.

Dendritic cells (DC) are critical actors in the initiation of primary immune responses and regulation of self-tolerance. The steroid sex hormone 17beta-estradiol (E(2)) has been shown to promote the differentiation of DCs from bone marrow (BM) precursors in vitro. However, the estrogen receptor (ER) involved in this effect has not yet been characterized. Using recently generated ERalpha- or ERbeta-deficient mice, we investigated the role of ER isotypes in DC differentiation and acquisition of effector functions. We report that estrogen-dependent activation of ERalpha, but not ERbeta, is required for normal DC development from BM precursors cultured with GM-CSF. We show that reduced numbers of DCs were generated in the absence of ERalpha activation and provide evidence for a cell-autonomous function of ERalpha signaling in DC differentiation. ERalpha-deficient DCs were phenotypically and functionally distinct from wild-type DCs generated in the presence of estrogens. In response to microbial components, ERalpha-deficient DCs failed to up-regulate MHC class II and CD86 molecules, which could account for their reduced capacity to prime naive CD4(+) T lymphocytes. Although they retained the ability to express CD40 and to produce proinflammatory cytokines (e.g., IL-12, IL-6) upon TLR engagement, ERalpha-deficient DCs were defective in their ability to secrete such cytokines in response to CD40-CD40L interactions. Taken together, these results provide the first genetic evidence that ERalpha is the main receptor regulating estrogen-dependent DC differentiation in vitro and acquisition of their effector functions.

International audience; 1. Although hormonal therapy (HT) may increase the risk of coronary heart disease (CHD) and stroke in postmenopausal women, epidemiological studies (protection in premenopausal women) suggest and experimental studies (prevention of fatty streak development in animals) demonstrate a major atheroprotective action of estradiol (E2). The understanding of the deleterious and beneficial effects of oestrogens is thus required. 2. The immuno-inflammatory system plays a key role in the development of fatty streak deposit as well as in the rupture of the atherosclerotic plaque. Although E2 favours an anti-inflammatory effect in vitro (cultured cells), it rather elicits a pro-inflammatory response in vivo involving several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. The functional role of several cytokines was explored in hypercholesterolemic mice. The atheroprotective effect of E2 was fully maintained in mice deficient in interferon-g or interleukin-12, as well as IL-10. In contrast, the protective effect of estradiol was abolished and even reversed in hypercholesterolemic mice given a neutralizing anti-transforming growth factor-b (TGF-b) antibody. Endothelium is another important target for E2, since it not only potentiates endothelial nitric oxide and prostacyclin production, but also controls trafficking of the populations of the immuno-inflammatory system. 3. To conclude, the respective actions of oestrogens on the cell populations involved in the pathophysiology of atherothrombosis may be influenced, among others, by the timing of HT initiation, the status of the vessel wall and, as recently demonstrated the status of the TGF-b pathway.

Anti-idiotypic (Id) antibodies have been suggested to play a role in the self regulation process observed in Brown-Norway rats developing mercury-induced autoimmunity. However, the presence of such antibodies has not yet been directly demonstrated. For that purpose, spleen cells from a mercury-injected rat were fused and the resulting hybridomas tested for their anti-Id activity against monoclonal anti-glomerular basement membrane (GBM) antibodies produced in this model. A monoclonal antibody (mAb) was obtained that specifically reacted in an enzyme-linked immunosorbent assay with an anti-GBM mAb and to a much lesser extent with another one produced in the same fusion. In Western blot experiments this autoanti-Id mAb reacted under reducing conditions with the kappa L chains but not with the H chains of the two anti-GBM mAb. It did not react with the kappa L chains of eight other rat mAb. This mAb is therefore an autoanti-Id mAb that recognizes a V kappa-associated Id expressed on two anti-GBM mAb. These results demonstrate that anti-GBM antibodies and their corresponding autoanti-Id antibodies are simultaneously produced during this disease. Whether or not these autoanti-Id antibodies have a regulatory and/or a pathogenic role in this disease remains to be established.

Mercury-induced autoimmune glomerulonephritis in the Brown-Norway (BN) rat is characterized by the successive appearance of linear and granular glomerular IgG deposits. Anti-laminin autoantibodies represent the major part of the anti-glomerular basement membrane (GBM) antibodies produced in this model. Fusions were performed in this model and four anti-GBM monoclonal antibodies (mAb) were obtained. Three of them were laminin specific. Using rabbit anti-idiotype antibodies, cross-reactive idiotypes (CRId) were characterized on anti-laminin antibodies. They were expressed on the three anti-laminin mAb, on kidney-eluted and circulating anti-laminin antibodies. CRId-bearing immunoglobulins were detected transiently in the circulation and paralleled the anti-laminin antibody activity. By immunofluorescence studies on kidney cryostat sections two different CRId were defined. One was localized close to the antigen-combining site since it was not revealed on kidney-bound antibodies, in contrast with the second CRId. This latter CRId was also found deposited in a typical linear pattern in the early phase of the disease and in a granular pattern in the late phase, demonstrating that these CRId are components of immune deposits. Taken together, these results suggest that in this model of T-dependent polyclonal B cell activation, restricted sets of V genes encode for at least a part of the anti-GBM autoantibodies.

RATIONALE: Ca(2+) signaling controls the production of T helper (Th) type 2 cytokines known to be deleterious in asthma. Recently, we showed that Ca(2+) signaling was dihydropyridine (DHP)-sensitive in Th2 lymphocytes and that the DHP derivate, nicardipine, used in the treatment of cardiovascular pathologies, prevents Th2-dependent B cell polyclonal activation. OBJECTIVES: We tested the effect of nicardipine in experimental allergic asthma. METHODS: BALB/c mice immunized with ovalbumin (OVA) in alum and challenged with intranasal OVA were treated with nicardipine once the Th2 response, or even airway inflammation, was induced. We also tested the effect of nicardipine in asthma induced by transferring OVA-specific Th2 cells in BALB/c mice exposed to intranasal OVA. We checked the impact of nicardipine on T-cell responses and airway inflammation. MEASUREMENTS AND MAIN RESULTS: Nicardipine inhibited in vitro Ca(2+) response in Th2 cells. In vivo, it impeded the development of Th2-mediated airway inflammation and reduced the capacity of lymphocytes from lung-draining lymph nodes to secrete Th2, but not Th1, cytokines. Nicardipine did not affect antigen presentation to CD4(+) T lymphocytes, nor the initial localization of Th2 cells into the lungs of mice exposed to intranasal OVA; however, it reduced the production of type 2 cytokines and the amplification of the Th2 response in mice with asthma. Conversely, nicardipine had no effect on Th1-mediated airway inflammation. CONCLUSIONS: Nicardipine improves experimental asthma by impairing Th2-dependent inflammation. This study could provide a rationale for developing drugs selectively targeting DHP receptors of Th2 lymphocytes, potentially beneficial in the treatment of asthma.

Whereas hormone replacement/menopause therapy (HRT) in postmenopausal women increases the coronary artery risk, epidemiological studies (protection in premenopaused women) suggest and experimental studies (prevention of the development of fatty streaks in animals) demonstrate a major atheroprotective action of oestradiol (E2). The understanding of the deleterious and beneficial effects of oestrogens is thus required. The immuno-inflammatory system plays a key role in the development of fatty streak deposit as well as in the rupture of the atherosclerotic plaque. Whereas E2 favours an anti-inflammatory effect in vitro (cultured cells), it rather elicits in vivo a proinflammation at the level of several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. Endothelium is another important target for E2, as it potentiates endothelial NO and prostacyclin production, thus promoting the beneficial effects as vasorelaxation and inhibition of platelet aggregation. Prostacyclin, but not NO, appears to be involved in the atheroprotective effect of E2. E2 also accelerates endothelial regrowth, thus favouring vascular healing. Finally, most of these effects of E2 are mediated by oestrogen receptor alpha, and are independent of oestrogen receptor beta. In summary, a better understanding of the mechanisms of oestrogen action not only on the normal and atheromatous arteries, but also on innate and adaptive immune responses is required and should help to optimize the prevention of cardiovascular disease after menopause. These mouse models should help to screen existing and future selective oestrogen receptor modulators.

Myasthenia gravis (MG) is an organ-specific autoimmune disease caused in most cases by autoantibodies against the nicotinic acetylcholine receptor (AChR). It is now well documented that many autoimmune diseases, including MG, are more prevalent in women than in men, and that fluctuations in disease severity occur during pregnancy. These observations raise the question of the potential role of sex hormones, such as estrogens, as mediators of sex differences in autoimmunity. In the present study, we have analyzed the effect of 17β-estradiol (E2) on the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), an animal model of MG. We show that treatment with E2 before Ag priming is necessary and sufficient to promote AChR-specific Th1 cell expansion in vivo. This time-limited exposure to E2 enhances the production of anti-AChR IgG2ab (specific for b allotype; e.g., B6) and IgG2b, but not IgG1, and significantly increases the severity of EAMG in mice. Interestingly, the E2-mediated augmentation in AChR-specific Th1 response correlates with an enhanced production of IL-12 by splenic APCs through the recruitment of CD8α+ dendritic cells. These data provide the first evidence that estrogen enhances EAMG, and sheds some light on the role of sex hormones in immune responses and susceptibility to autoimmune disease in women.

Th1 cells that produce IFN-γ are essential in the elimination of intracellular pathogens, and Th2 cells that synthetize IL-4 control the eradication of helminths. However, highly polarized Th1 or Th2 responses may be harmful and even lethal. Thus, the development of strategies to selectively down-modulate Th1 or Th2 responses is of therapeutic importance. Herein, we demonstrate that dihydropyridine receptors (DHPR) are expressed on Th2 and not on Th1 murine cells. By using selective agonists and antagonists of DHPR, we show that DHPR are involved in TCR-dependent calcium response in Th2 cells as well as in IL-4, IL-5, and IL-10 synthesis. Nicardipine, an inhibitor of DHPR, is beneficial in experimental models of Th2-dependent pathologies in rats. It strongly inhibits the Th2-mediated autoimmune glomerulonephritis induced by injecting Brown Norway (BN) rats with heavy metals. This drug also prevents the chronic graft vs host reaction induced by injecting CD4+ T cells from BN rats into (LEW × BN)F1 hybrids. By contrast, treatment with nicardipine has no effect on the Th1-dependent experimental autoimmune encephalomyelitis triggered in LEW rats immunized with myelin. These data indicate that 1) DHPR are a selective marker of Th2 cells, 2) these calcium channels contribute to calcium signaling in Th2 cells, and 3) blockers of these channels are beneficial in the treatment of Th2-mediated pathologies.

It is widely accepted that females have superior immune responses than males, but the ways by which sex hormones may enhance T cell responses are still poorly understood. In the present study, we analyzed the effect of estrogens on CD4 T cell activation and differentiation after immunization with exogenous antigens. We show that administration of low doses of 17beta-estradiol (E2) to castrated female mice results in a striking increase of antigen-specific CD4 T cell responses and in the selective development of IFN-gamma-producing cells. Quantitative assessment of the frequency of T cells bearing a public TCR beta chain CDR3 motif demonstrated that the clonal size of primary antigen-specific CD4 T cells was dramatically increased in immune lymph nodes from E2-treated mice. By using mice with disrupted estrogen receptor (ER) alpha or beta genes, we show that ERalpha, but not ERbeta, was necessary for the enhanced E2-driven Th1 cell responsiveness. Furthermore, ERalpha expression in hematopoietic cells was essential, since E2 effects on Th1 responses were only observed in mice reconstituted with bone marrow cells from ERalpha+/+, but not ERalpha-deficient mice. These results demonstrate that estrogen administration promotes strong antigen-specific Th1 cell responses in a mechanism that requires functional expression of ERalpha in hematopoietic cells.

The interactions between CD28 and its ligand CD86 are critical for the regulation of T cell responses. However, it is not clear whether CD4+ T cells expressing low and high avidity TCR are equally dependent on CD28 costimulation for their activation and expansion. To address this issue, we have used multimers of I-Ad molecules linked to a peptide derived from the Leishmania major homolog for the receptor of activated C kinase (LACK) antigen to compare the fate of LACK-specific CD4+ T cells in Leishmania-infected BALB/c mice which have been treated or not with anti-CD86 mAb. Although the administration of anti-CD86 mAb did not completely prevent the expansion of LACK-specific T cells, their frequency and number were markedly reduced. In mice treated with anti-CD86 mAb as well as in control animals, L. major induced the clonal expansion of LACK-specific T cells which expressed a canonical low avidity Valpha8/Vbeta4 TCR. Taken together, our results suggest that the molecular interactions between CD28 on T cells and CD86 on APC serve to amplify and modulate T cell responses without promoting breadth in the TCR repertoire.

The T cell receptor (TCR) alpha and beta chains are encoded by a series of stochastic rearrangements between variable (V), diversity (D) for TCR beta chain only, and joining (J) gene segments, creating hypervariable complementarity-determining region 3 (CDR3) regions that contact the peptide/MHC complex and confer specificity. In the present paper, we applied the recently developed real-time quantitative RT-PCR technique to the detection of rearranged TCR beta chain mRNA transcripts. We designed BV- and BJ-specific primers together with TaqMan probes specific for the CDR3 regions of the clones of interest. As an external reference, we used plasmids containing the entire TCR beta chains, making it possible to normalize the number of specific rearranged BV-J mRNA copies among the total number of TCR beta chains. Here, we present data validating this fluorogenic PCR-based method for the quantification of several TCR clonotypes characteristic of the CD4 T cell response to hen egg white lysozyme (HEL) in mice of the H-2d haplotype. This accurate and sensitive procedure permits the precise determination of T cell clone frequencies ranging from 10(-2) to less than 10(-5) in normal biological samples; it may provide an alternative approach when frequencies are too low to be assessed by flow cytometry.

Interleukin-12 is a key regulatory cytokine produced by antigen-presenting cells (APC) which drives the development of interferon-gamma (IFN-gamma)-producing cells and promotes cell-mediated immunity. Following subcutaneous immunization with protein antigen in adjuvant, dendritic cells (DC) but not small nor large B cells in immune lymph nodes express antigenic complexes and secrete substantial amounts of bioactive IL-12 p75 upon antigen-specific interaction with T cells. We have analyzed secretion of IL-12 p40 and p75 by cell populations enriched in DC, macrophages or B cells in response to nonspecific stimulation or to interaction with antigen-specific CD4+ cells. These APC populations do not produce IL-12 constitutively but, upon stimulation with heat-fixed Staphylococcus aureus and IFN-gamma, IL-12 p40 and p75 are secreted by DC and macrophages, whereas B cells fail to produce IL-12. B cells also fail to secrete IL-12 in response to stimulation with LPS and IFN-gamma. Co-culture with CD4+ T hybridoma cells and antigen induces IL-12 secretion by DC. Up-regulation of IL-12 secretion by interaction with antigen-specific CD4+ T cells is abrogated by anti-class II monoclonal antibodies (mAb), by soluble CD40 molecules and by anti-CD40 ligand mAb, demonstrating a positive feedback between T cells and DC mediated by TCR-peptide/class II and by CD40-CD40 ligand interactions. Expression of class II and CD40 molecules is comparable in B cells and DC, and both APC types activate CD4+ T cells. Yet, even upon interaction with antigen-specific T cells, B cells fail to secrete IL-12. The capacity of B cells to present antigen but not to secrete IL-12 may explain their propensity to selectively drive T helper type 2 cell development.

We have analyzed the relative contribution of dendritic cells (DC) and B cells in the presentation of peptide-class II complexes in an inflammatory situation in vivo. Draining lymph node cells from mice immunized subcutaneously with hen egg-white lysozyme (HEL) in adjuvant display HEL peptide-major histocompatibility complex class II complexes able to stimulate, in the absence of any further antigen addition, specific T hybridoma cells. The antigen-presenting capacity of three different antigen-presenting cell (APC) populations recruited in lymph nodes, DC (N418+, class II+, B220-, low buoyant density), large B cells (B220+, low buoyant density), and small B cells (B220+, high buoyant density), was analyzed. After immunization with HEL in adjuvant, DC are the only lymph node APC population expressing detectable HEL peptide-class II complexes. These results indicate that lymph node DC and not B cells are the APC initiating the immune response in vivo after administration of antigen in adjuvant.

Continuous administration of soluble proteins, delivered over a 10-d period by a mini-osmotic pump implanted subcutaneously, induces a long-lasting inhibition of antigen-specific T cell proliferation in lymph node cells from BALB/c mice subsequently primed with antigen in adjuvant. The decreased T cell proliferative response is associated with a down-regulation of the T helper cell (Th)1 cytokines interleukin (IL)-2 and interferon (IFN)-gamma and with a strong increase in the secretion of the Th2 cytokines IL-4 and IL-5 by antigen specific CD4+ T cells. This is accompanied by predominant inhibition of antigen-specific antibody production of IgG2a and IgG2b, rather than IgG1 isotype. Interestingly, inhibition of Th1 and priming of Th2 cells is also induced in beta(2) microglobulin-deficient BALB/c mice, indicating that neither CD8+ nor CD4+ NK1.1+ T cells, respectively, are required. The polarization in Th2 cells is stably maintained by T cell lines, all composed of CD4+/CD8- cells expressing T cell receptor for antigen (TCR) alpha/beta chains, derived from BALB/c mice treated with continuous antigen administration, indicating that they originate from Th2 cells fully differentiated in vivo. This polarization is induced in BALB/c mice by continuous administration of any protein antigen tested, including soluble extracts from pathogenic microorganisms. Priming of Th2 cells is dose dependent and it is optimal for low rather than high doses of protein. Blocking endogenous IL-4 in vivo inhibits expansion of antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen, indicating the involvement of two independent mechanisms. Consistent with this, Th2 cell development, but not inhibition of Th1 cells, depends on non-major histocompatibility complex genetic predisposition, since the Th2 response is amplified in BALB/c as compared to DBA/2, C3H, or C57BL/6 mice whereas tested. These findings support the hypothesis that continuous release of low amounts of protein antigens from pathogenic microorganisms may polarize the immune response toward a Th2 phenotype in susceptible mouse strains.

MHC class II-binding peptides administered in vivo can be effective and selective inhibitors of T cell activation. To selectively interfere with the activation of autoreactive T cells immunotherapy by peptides can be essentially directed to three cellular targets: APC, autoreactive T cells and regulatory T cells. Experimental models of autoimmune diseases have demonstrated that disease can be prevented or treated by peptides acting on each cellular target. The APC can be functionally inactivated by blocking the binding site of MHC class II molecules, thus preventing antigen presentation to T cells. We have demonstrated that injection of a class II-binding peptide can induce MHC class II blockade in vivo [22], resulting in inhibition of class II-restricted T cell activation and of T cell-dependent antibody responses. This is already a validated approach to prevent experimental autoimmune diseases [3, 32] and it may become applicable to the treatment of human autoimmune diseases. The autoreactive T cells could be inactivated by peptide-specific tolerance induction [14, 24], but it is necessary to define all potential autoantigenic epitopes to ensure complete and effective tolerance. Specific inhibition of autoreactive T cells could also be induced by antigen analogues acting as TCR antagonists [15], but this again requires, at a minimum, detailed knowledge of the autoantigen. Regulatory T cells represent a potentially interesting target for selective immunointervention by peptides. The restricted TCR V gene expression by autoreactive pathogenic T cells in some animal models of autoimmune diseases, for example the shared TCR V gene usage characteristic of EAE [1], raises the possibility of controlling self reactivity at the network level, as originally proposed by Cohen and colleagues [9]. Administration of peptides corresponding to TCR sequences utilized by autoreactive T cells has indeed been reported to down-regulate EAE, presumably via induction of anti-idiotypic T cells with suppressive activity [26, 43]. However, this approach does not always result in suppression of encephalitogenic effector T cells, and it may actually lead to enhanced chronic EAE [16]. It is also possible to induce regulatory T cells with suppressive activity by administration of peptides derived from the candidate autoantigen [24, 39] and this specific peptide therapy has been used to prevent the development of autoimmune diabetes in NOD mice [17]. However, if effective tolerance induction requires knowledge of all autoantigenic epitopes, induction of suppressor T cells by antigen is further complicated by our lack of definite molecular explanations for this phenomenon. Therefore, several immunoregulatory mechanisms induced by administration of class II-binding peptides may be responsible, in vivo, for interference with the activation of antigen-specific T cells. Unfortunately, with the possible exception of MHC blockade, the mechanisms involved are far from being clearly understood. What is needed is a more detailed analysis of these mechanisms in experimental autoimmune models to discern the precise mode of action of immunoregulatory peptides. This knowledge may then be applied to the treatment of human autoimmune diseases, keeping in mind that the clinical situation will invariably be even more complex.

Cross-reactive idiotypes (CRId) borne on autoanti-glomerular basement membrane antibodies of Brown-Norway (BN) rats with mercury-induced glomerulonephritis have been described in the preceding study (Guery, J.-C. et al., Eur. J. Immunol. 1990. 20:93). BN rats treated with sodium aurothiopropanol sulfonate or D-penicillamine, as well as (LEW X BN)F1 hybrids transferred with BN rat spleen cells, developed quite similar autoimmune abnormalities. In the present study, it is shown that immunoglobulins bearing such "public" idiotypes are also produced and deposited in the kidney in these three models. The CRId here described may, therefore, be considered as a marker of sets of recurrently expressed V region genes during the course of these autoimmune disorders. Anti-self class II T cells are present in the three models of toxin-induced autoimmunity and anti-allo class II T cells are responsible for the chronic graft-vs.-host reaction. The same B cell clones are probably triggered during these processes as a consequence of a polyclonal B cell activation mediated by anti-class II T cells.