Dendritic cells but not B cells present antigenic complexes to class II-restricted T cells after administration of protein in adjuvant.

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.

Selective immunosuppression by administration of major histocompatibility complex (MHC) class II-binding peptides. I. Evidence for in vivo MHC blockade preventing T cell activation.

Draining lymph node cells (LNC) from mice immunized with hen egg white lysozyme (HEL) display at their surface antigen-MHC complexes able to stimulate, in the absence of any further antigen addition, HEL peptide-specific, class II-restricted T cell hybridomas. Chloroquine addition to these LNC cultures fails to inhibit antigen presentation, indicating that antigenic complexes of class II molecules and HEL peptides are formed in vivo. MHC class II restriction of antigen presentation by LNC from HEL-primed mice was verified by the use of anti-class II monoclonal antibodies. Coinjection of HEL and the I-Ak-binding peptide HEL 112-129 in mice of H-2k haplotype inhibits the ability of LNC to stimulate I-Ak-restricted, HEL 46-61-specific T cell hybridomas. Similar results are obtained in mice coinjected with the HEL peptides 46-61 and 112-129. Inhibition of T hybridoma activation can also be observed using as antigen-presenting cells irradiated, T cell-depleted LNC from mice coinjected with HEL 46-61 and HEL 112-129, ruling out the possible role of either specific or nonspecific suppressor T cells. Inhibition of T cell proliferation is associated with MHC-specific inhibition of antigen presentation and with occupancy by the competitor of class II binding sites, as measured by activation of peptide-specific T cell hybridomas. These results demonstrate that administration of MHC class II binding peptide competitors selectively inhibits antigen presentation to class II-restricted T cells, indicating competitive blockade of class II molecules in vivo.

Selective development of T helper (Th)2 cells induced by continuous administration of low dose soluble proteins to normal and beta(2)-microglobulin-deficient BALB/c mice.

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.

Selective immunosuppression by administration of major histocompatibility complex class II-binding peptides. II. Preventive inhibition of primary and secondary in vivo antibody responses.

The self-mouse lysozyme peptide corresponding to residues 46-62 (ML46-62) binds to the major histocompatibility complex (MHC) class II molecules I-A(k) and it selectively inhibits, when coinjected with antigen, priming of I-A(k)-restricted, antigen-specific T cells. We demonstrate that administration of ML46-62 also inhibits in vivo antibody responses induced by I-A(k)-restricted T helper cells. ML46-62 is able to prevent the primary anti-hen egg white lysozyme (HEL) antibody response induced by the entire HEL molecule in B10.A(4R) mice, expressing only I-A(k) molecules, but not in mice of H-2d haplotype. ML46-62 also strongly decreases, in B10.A(4R) mice, the antibody response to ribonuclease A, a protein antigen unrelated to the MHC blocker, indicating that MHC blockade is the mechanism leading to inhibition of antibody response. This is further supported by the concomitant decrease, in vivo, of complex formation between immunodominant HEL peptides and I-A(k) molecules, preventing I-A(k)-restricted T cell induction. Administration of ML46-62 after antigen priming does not affect ongoing antibody responses, as expected from MHC blockade. A single injection of ML46-62 at the time of protein antigen priming precludes not only the primary, but also the secondary antibody response to a subsequent challenge with soluble protein, even when the challenge is performed several months after priming. Coinjection of antigen and MHC antagonist inhibits production of all antibody isotypes equally well, suggesting that MHC class II blockade affects both Th1- and Th2-type T helper cells. Therefore, these results indicate that administration of MHC class II-binding peptides can efficiently and selectively prevent the induction of T cell-dependent primary and secondary in vivo antibody responses by blocking antigen presentation to class II-restricted T helper cells.

Interleukin 4-producing CD4 T cells arise from different precursors depending on the conditions of antigen exposure in vivo.

The precursor origin of T helper (Th) cell subsets in vivo has been difficult to study and remains poorly investigated. We have previously shown that chronic administration of soluble protein antigen induces selective development of antigen-specific CD4 Th2 cells in genetically predisposed mouse strains. To analyze the origin of effector T cells in this model, we designed a competitive polymerase chain reaction-based approach to track public BV-J rearrangement expressed by CD4 T cells specific for hen egg white lysozyme (HEL) in BALB/c mice. We show that public T cell clones are predominantly associated with type 1 or 2 effector Th cells recovered after primary immunization in complete or incomplete Freund's adjuvant, respectively. Conversely, continuous administration of soluble antigen, which induces strong memory Th2 response, is associated with a dose-dependent reduction of public clone size by a mechanism resembling clonal anergy. Thus, soluble HEL-induced Th2 cells do not express the public complementarity determining region 3 motifs characteristic of immunogenic challenge in the presence of adjuvant. These results demonstrate that there are multiple pathways of induction of Th2 responses depending on the condition of antigen exposure in vivo, i.e., clonal immune deviation versus recruitment of a different pool of precursor cells.

Exogenous peptides compete for the presentation of endogenous antigens to major histocompatibility complex class II-restricted T cells.

Antigen-presenting cells (APC) transfected with a construct encoding the hen egg-white lysozyme (HEL) amino acid sequence 1-80 constitutively present HEL peptides complexed to major histocompatibility complex (MHC) class II molecules to specific T cell hybridomas, indicating that endogenous cellular antigens can be efficiently presented to class II-restricted T cells. Here we show that exogenous peptide competitors added to HEL-transfected APC can inhibit the presentation of endogenous HEL peptides to class II-restricted T cells. The inhibition is specific for the class II molecule binding the competitor peptide, and it affects to the same extent presentation of exogenous or endogenous HEL peptides. These results, demonstrating that an exogenous competitor can inhibit class II-restricted T cell activation induced by endogenous as well as exogenous antigen, suggest lack of strict compartmentalization between endogenous and exogenous pathways of antigen presentation. Since autoreactive T cells may recognize endogenous, as well as exogenous antigens, the results have implications for the treatment of autoimmune diseases by MHC blockade.

Role of inflammatory cytokines in the effect of estradiol on atheroma.

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.

Lethal host-versus-graft disease and hypereosinophilia in the absence of MHC I-T-cell interactions.

Neonatal injection of semiallogeneic spleen cells in BALB/c mice induces a self-limited state of chimerism that promotes the differentiation of donor-specific CD4 T cells toward the Th2 phenotype. Here we show that injection of spleen cells from beta2-microglobulin-deficient (BALB/c x C57BL/6) F1 mice into BALB/c newborns with a disrupted beta2-microglobulin (beta2m) gene results in a lethal lymphoproliferative disorder associated with uncontrolled Th2 response, long-term persistence of donor B cells, and sustained blood eosinophilia. Autoimmune manifestations are also enhanced and characterized by a severe autoantibody-mediated glomerulonephritis. Histological examination of the spleen shows a hyperplasia of periarteriolar lymphoid sheaths, with accumulation of eosinophils and basophils, and variable degree of fibrosis. Perivascular lymphoid infiltrates with eosinophils are also found in the lung and are correlated with disease severity. Such abnormalities are almost absent using beta2m-sufficient mice. These data demonstrate that induction of lymphoid chimerism in the absence of MHC class I-T-cell interactions results in a lethal form of host-versus-graft disease that represents a unique model of Th2-dependent chronic inflammatory disease associated with an hypereosinophilic syndrome in mice.

Understanding the controversy about hormonal replacement therapy: insights from estrogen effects on experimental and clinical atherosclerosis.

Whereas hormonal replacement/menopause therapy (HRT) in post-menopausal women increases the coronary artery risk, epidemiological studies (protection in pre-menopaused women) suggest and experimental studies (prevention of the development of fatty streaks in animals) demonstrate a major atheroprotective action of estradiol (E2). The understanding of the deleterious and beneficial effects of estrogens is thus required. The immuno-inflammatory system plays a key role in the development of fatty streak deposit as well as in the atherosclerotic plaque rupture. Whereas E2 favors an anti-inflammatory effect in vitro (cultured cells), it rather elicits pro-inflammation in vivo, at the level of several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. Endothelium is another important target for E2, since it stimulates endothelial NO and prostacyclin production, thus promoting beneficial effects of vasorelaxation and platelet aggregation inhibition. Prostacyclin, but not NO, appears to be involved in the atheroprotective effect of E2. Estradiol accelerates also endothelial regrowth, thus favoring vascular healing. Finally, most of these effects of E2 are mediated by estrogen receptor alpha, and are independent of estrogen receptor beta. In summary, a better understanding of the mechanisms of estrogen action is required not only on the normal and atheromatous arteries, but also on innate and adaptive immune responses. This should help cardiovascular disease prevention optimization after menopause. These mouse models should help to screen existing and future Selective Estrogen Receptor Modulators (SERMs).

Understanding the oestrogen action in experimental and clinical atherosclerosis.

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.

Selective immunosuppression.

Experimental models of autoimmune diseases have demonstrated that such disease can be prevented or treated by selectively interfering with activation of any of these cell types: antigen-presenting cells, autoreactive T cells and regulatory T cells. Luciano Adorini and colleagues discuss these approaches to selective immunosuppression and examine how similar strategies may become applicable to the treatment of human autoimmune diseases.

Selective immunosuppression of class II-restricted T cells by MHC class II-binding peptides.

Administration of major histocompatibility complex (MHC) class II-binding synthetic peptides can induce selective immunosuppression via different mechanisms of action. In this review four different ways to induce selective immunosuppression by peptide administration will be examined: MHC blockade, T cell receptor (TCR) antagonism, induction of peripheral tolerance, and activation of regulatory cells. These approaches to selective immunosuppression target three types of cells: antigen-presenting cells, antigen-specific T cells, and regulatory T cells. Understanding these forms of immunosuppression will provide new insights in basic immunology and may offer strategies for selective immunointervention in autoimmune diseases, allograft rejection, and allergy.

Manipulation of the Th1/Th2 cell balance: an approach to treat human autoimmune diseases?

Differentiated T cells produce a restricted set of lymphokines, allowing their subdivision into two major subsets: Th1 and Th2 cells. This has lead to a new paradigm for immunoregulation based on the Th1/Th2 dichotomy. A strict compartmentalization of T cells into Th1 and Th2 is clearly an oversimplification: regulatory and effector mechanisms in the immune system encompass much more than Th1 and Th2 cells. This oversimplification is nevertheless useful to carry out experiments designed to test the paradigm. Based on results obtained in different experimental models of autoimmune diseases, the subdivision of T cells into Th1 and Th2 subsets has been extended to suggest that Th1 cells contribute to the pathogenesis of several organ-specific autoimmune diseases, whereas Th2 cells may inhibit disease development. Although more slowly and maybe less clearly, a similar dichotomy is starting to emerge in human autoimmune diseases. It will soon be possible to formally test immunointervention based on Th1/Th2 cell manipulation in clinical situations: the tools and a conceptual frame are already available. In this review we will examine two key factors affecting the Th1/Th2 balance: antigen and the role of cytokines influencing the development of Th1 and Th2 cells. The rational manipulation of these two variables may ultimately lead to an effective control of Th1 and Th2 cells potentially able to alter the natural course of human autoimmune diseases.

Flow cytometric analysis of intracellular interferon-gamma synthesis in rat CD4 T cells.

To date the techniques used to analyse cytokine expression by rat T cells do not give information about the simultaneous production of different cytokines from individual cells. Recently, a method for analysing the intracellular production of cytokines at the single cell level using flow cytometry has been developed. It is well established that the most critical requirement for successful intracellular cytokine staining is the availability of appropriate antibodies. In rat, it is possible to stain for intracellular IL-4 and IL-10 (Th2 cytokines) using the commercially available antibodies but not for Th1 cytokines. In the present work, we show that DB1, a mouse anti-rat IFN-gamma monoclonal antibody, could be used for intracytoplasmic staining of IFN-gamma producing rat CD4 T cells. The specificity of the staining was confirmed using a molar excess of unlabelled antibodies or recombinant cytokine. Finally, intracellular staining for IFN-gamma correlates with cytokine production in culture supernatant as evaluated by ELISA analysis.

A spontaneous hybridoma producing autoanti-idiotypic antibodies that recognize a V kappa-associated idiotope in mercury-induced autoimmunity.

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.