Antigen-pulsed epidermal Langerhans cells protect susceptible mice from infection with the intracellular parasite Leishmania major

Efficient vaccination against the parasite Leishmania major, the causative agent of human cutaneous leishmaniasis, requires the development of a resistance-promoting CD4+-mediated Th1 response. Epidermal Langerhans cells (LC) are critically involved in the induction of the primary immune response to Leishmania infection. They are able to ingest the parasites, to express MHC class II molecules with extraordinarily long half-life and to activate naive L. major-specific Th cells. Considering these unique properties, we studied the capacity of LC to mediate resistance to L. major in vivo. A single i.v. application of LC that had been pulsed with L. major antigen in vitro induced the protection in susceptible BALB/c mice against subsequent challenges with L. major parasites. Resistance could neither be induced by unpulsed LC, nor by L. major antigen alone or by L. major-pulsed macrophages. Development of resistance was paralleled by a reduced parasite burden and by a shift of the cytokine expression towards a Th1-like pattern. In contrast, control mice developed a Th2 response. In vitro exposure of LC to L. major antigen induced the expression of IL-12 (p40) mRNA. In conclusion, our data demonstrate that LC are able to serve as a natural adjuvant and to induce a protective immune response to L. major infection. This effect is based on the initiation of a Th1-like response that is likely to be mediated by IL-12.     

MHC class I-selected CD4-CD8-TCR-alpha beta+ T cells are a potential source of IL-4 during primary immune response

Differentiation of naive CD4+ lymphocytes into either Th1 or Th2 cells is influenced by the cytokine present during initial Ag priming. IL-4 is the critical element in the induction of Th2 response; however, its origin during a primary immune response is not well defined. In the present study, we characterized a novel potential source of IL-4, the class I-selected CD4-CD8-TCR-alpha beta+ T cells. In a first set of experiments, we demonstrated that CD4-CD8-TCR-alpha beta+ thymocytes produce a large amount of IL-4 after in vitro anti-CD3 stimulation. This phenomenon was not observed in class I-deficient mice, demonstrating that among these cells, the class I-selected subset was predominantly responsible for IL-4 production. Further studies focused on the in vivo IL-4-producing capacity of peripheral CD4-CD8-TCR-alpha beta+ T cells. To this end, a single injection of anti-CD3 mAb, which promptly induces IL-4 mRNA expression, was used. Peripheral CD4-CD8-TCR-alpha beta+ T cells express high levels of IL-4 mRNA in response to in vivo anti-CD3 challenge. Furthermore, analysis performed in mice lacking MHC class I or class II molecules demonstrates that both the class I-selected subset of CD4-CD8-TCR+ and CD4+ peripheral T lymphocytes are the major IL-4 producers after in vivo anti-CD3 stimulation. These findings suggest that class I-selected CD4-CD8-TCR-alpha beta+ and CD4+ T cell populations are important sources of IL-4 probably implicated in the development of specific Th2 immune responses.     

Identification of the immunodominant T cell epitope of p38, a major egg antigen, and characterization of the epitope-specific Th responsiveness during murine schistosomiasis mansoni

A recently cloned major Schistosoma mansoni egg Ag p38 induced and elicited strong Th1-type responsiveness in mice of H-2k haplotype. Now, we have identified the immunodominant T cell epitope of p38 and analyzed the dynamics of epitope-specific Th responsiveness during murine schistosomiasis mansoni. Overlapping recombinant and synthetic peptides that encompassed the full-length 354 amino acid of p38 were tested for proliferation and cytokine production in peptide- or p38-sensitized mice. The immunodominant T cell epitope of p38 that elicited pulmonary granuloma formation was localized within peptide P4 (amino acids 235-249). The P4-specific cytokine response of splenocytes that had been sensitized s.c. with p38, P4 or soluble egg Ags in IFA, or i.p. with 3000 eggs was predominantly as the Th1 type, with strong IL-2 and IFN-gamma, but trace amounts of IL-4 and IL-5 secretion. At 6.5 wk of infection, splenocytes and mesenteric lymph node cells responded to p38/P4 peptides with predominantly Th1-type responsiveness. This response did not switch to a Th2-type pattern from 8 wk onwards; rather, it underwent down-modulation. Moreover, the hepatic granuloma lymphocytes at 6.5 wk responded to p38/P4 predominantly with Th1-type cytokine production, indicating that they participate in early granuloma formation. From 8 wk onwards an immune deviation to the p38-specific response was observed that was manifested by rising IgG1, IgE, and IgG2a Ab production as opposed to declining Th1- and Th2-type cytokine secretion.     

Impact of air pollution on the mortality and emergencies of chronic obstructive pulmonary disease and asthma in Barcelona

The murine model of infection with Leishmania major has allowed the demonstration of a causal relationship between, on the one hand, genetically determined resistance to infection and the development of a Th1 CD4+ cell response, and on the other hand, genetically determined susceptibility and Th2 cell maturation. Using this murine model of infection, the role of cytokines in directing the functional differentiation pathway of CD4+ T cell precursors, has been demonstrated in vivo. Thus, IL-12 and IFN-gamma have been shown to favour Th1 cell development and IL-4 is crucial for the differentiation of Th2 responses. Maturation of a Th2 response in susceptible BALB/c mice following infection with L. major is triggered by the IL-4 produced during the first two days after parasite inoculation. This IL-4 rapidly renders parasite specific CD4+ T cells precursors unresponsive to IL-12. A restricted population of CD4+ T cells expressing the V beta 4V alpha 8 TCR heterodimer and recognizing a single epitope on the LACK (Leishmania Activated C-Kinase) antigen of L. major is responsible for this rapid production of IL-4, instructing subsequent differentiation towards the Th2 phenotype of CD4+ T cells specific for several parasite antigens.     

Expansion of IL-3-responsive IL-4-producing non-B non-T cells correlates with anemia and IL-3 production in mice infected with blood-stage Plasmodium chabaudi malaria

A prominent switch of CD4+ T cells from Th1 to Th2 type response occurs in mice infected with the non-lethal malaria parasite Plasmodium chabaudi chabaudi AS around the time of peak parasitemia. This is reflected by a decrease in IFN-gamma- and an increase in IL-4-producing cells. The peak occurs approximately 9-10 days after infection and is accompanied by anemia. The mechanism behind the switch in Th cell response is poorly understood. We here report on the production of IL-4 from a non-T cell source during P. chabaudi infection in BALB/c mice. Flow cytometric analysis of spleen and peripheral blood leukocytes (PBL) showed a dramatic increase in the percentage of non-B non-T (NBNT) cells 9-23 days after P. chabaudi infection with peak values by day 15 (approximately 30 % of splenocytes and approximately 55 % of PBL being NBNT cells). The expansion of NBNT cells correlated closely with the appearance of a cell type secreting IL-4 and IL-6 following stimulation with IL-3 and/or cross-linking of FcgammaR. Compared to cells from uninfected animals, NBNT cells from P. chabaudi-infected mice were shown to be hyper-responsive to IL-3. The levels of the hematopoietic cytokine IL-3 were elevated in supernatants from unstimulated spleen cell cultures as well as in serum at the same time points at which NBNT cell-derived IL-4 and IL-6 were detected from spleen cultures and PBL. Thus, IL-3-responsive IL-4-producing NBNT cells may provide cytokines supporting the switch from Th1 to a Th2 response which is important for the final clearance of the parasite in P. chabaudi malaria.     

Generation, persistence, and modulation of Th0 effector cells: role of autocrine IL-4 and IFN-gamma

Many studies have classified CD4 responses into either Th1-like or Th2-like, based on cytokine secretion profiles, but little significance has been placed on Th0 cells. This has largely resulted from studies that suggested that Th0 populations primarily comprise individual Th1 and Th2 cells. Here, we show that priming of Ag-specific naive CD4 cells with moderate dose IL-4 generates a Th0 population that is evident after 3 days in vitro and becomes prevalent after successive encounters with Ag over a 9-day period. By intracellular cytokine staining, the majority (>60%) of effector cells generated in this way produce either IL-4, IFN-gamma and IL-2, or IL-4 and IFN-gamma without IL-2. Endogenous IFN-gamma secreted over the initial 3 days of culture was critical for generating Th0 cells, since neutralization allowed IL-4 to induce differentiation into Th2-like cells. Successive encounters with Ag were required for generating Th0 cells, and their stability and persistence were governed by the balance of endogenous IL-4 and IFN-gamma secreted during the later stages of differentiation. Studies blocking Fas-induced cell death showed that this process played no role in Th0 cell generation, and differential death of committed Th1 or Th2 cells was not required for Th0 persistence. These data suggest that Th0 cells can be as prevalent as Th1- or Th2-like cells after naive CD4 activation, that the relative levels of autocrine IL-4 and IFN-gamma are important to the lack of commitment, and that not all cells are predestined to the Th1 or Th2 phenotypes early in the response.     

Is epsilon-aminocaproic acid as effective as aprotinin in reducing bleeding with cardiac surgery?: a meta-analysis

Infection of BALB/c mice with Trypanosoma cruzi resulted in up-regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4+ T cells, activation-induced CD4+ T cell death (AICD), and in Fas: FasL-mediated cytotoxicity. When CD4+ T cells from infected mice were co-cultured with T. cruzi-infected macrophages, onset of AICD exacerbated parasite replication. CD4+ T cells from T. cruzi-infected FasL-deficient BALB gld/gld mice had no detectable AICD in vitro and their activation with anti-TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld/gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild-type mice. Secretion of Th2 cytokines IL-10 and IL-4 by CD4+ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti-IL-4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild-type mice. These results indicate that, besides controlling CD4+ T cell AICD and parasite replication in vitro, an intact Fas: FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2-type immune response to the parasite.     

CD40-mediated stimulation contributes to lymphocyte proliferation, antibody production, eosinophilia, and mastocytosis during an in vivo type 2 response, but is not required for T cell IL-4 production

CD40/CD40 ligand interactions are required for the development of T cell-dependent Ab responses in vivo. The role of these cell surface molecules in contributing to T cell cytokine production and the development of effector populations other than B cells and T cells is, however, less well defined. We have examined the in vivo effects of blocking CD40/CD40 ligand interactions on the type 2 mucosal immune response that follows oral inoculation of mice with the nematode parasite, Heligmosomoides polygyrus. Administration of anti-gp39 (CD40L) mAb (MR1) blocked H. polygyrus-induced elevations in serum IgG1 levels and inhibited elevations in blood eosinophils and mucosal mast cells at day 14 after inoculation. Anti-gp39 mAb markedly inhibited B cell blastogenesis 8 days after H. polygyrus inoculation but did not inhibit elevations in B cell class II MHC expression. Maximal elevations in B7-2 expression required signaling through both CD40 and the IL-4R. Elevations in T cell cytokine gene expression and elevations in the number of IL-4-secreting cells were unaffected by treatment with anti-gp39 mAb, although IL-4 production was inhibited by anti-IL-4R mAb. These results suggest that CD40/CD40L interactions are not required to activate T cells to produce cytokines but are required for the activation and proliferation of other effector cells associated with the type 2 response.     

The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R beta2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R beta2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R beta2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R beta2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA beta2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-gamma, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R beta2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.     

Induction of IL-12 p40 messenger RNA expression and IL-12 production of macrophages via CD40-CD40 ligand interaction

The mechanism of IL-12 production has been studied by stimulating macrophages or B cell lines with LPS, Staphylococcus aureus, or phorbol diester. However, since IL-12 plays an important role in the activation of T cells interacting with APC, it is important to study the mechanism of IL-12 production induced by T helper cell-APC interaction. We and others have demonstrated that IL-12 is produced in cultures where Th1 cells are stimulated with Ag or APC. In the present experiments, we studied a role of CD40-CD40 ligand (CD40L) interaction in IL-12 production and obtained the following results: 1) incubation of normal Th1 clone with APC in the presence of Ag induced IL-12 p40 and p35 mRNA accumulation and IL-12 production, and the addition of anti-CD40L blocked the p40 mRNA accumulation and IL-12 production but not p35 mRNA accumulation; 2) when Th1 clone from a CD40L-deficient mouse was used in the incubation, p35 mRNA accumulation was induced, but neither p40 mRNA accumulation nor IL-12 production was induced; 3) CD40L+ Th1 clone, or insect cell membrane expressing mouse CD40L, induced p40 mRNA accumulation and IL-12 production but not p35 mRNA accumulation. These results indicate that the CD40-CD40L interaction plays a critical role in IL-12 p40 mRNA accumulation and bioactive IL-12 production and that p35 mRNA accumulation was regulated via a different mechanism than CD40-CD40L interaction. Most of the cells producing IL-12 were Mac-1+ macrophages.     

Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells

Dendritic cells are the most relevant antigen-presenting cells (APC) for presentation of antigens administered in adjuvant to CD4+ T cells. Upon interaction with antigen-specific T cells, dendritic cells (DC) expressing appropriate peptide-MHC class II complexes secrete IL-12, a cytokine that drives Th1 cell development. To analyze the T cell-mediated regulation of IL-12 secretion by DC, we have examined their capacity to secrete IL-12 in response to stimulation by antigen-specific Th1 and Th2 DO11.10 TCR-transgenic cells. These cells do not differ either in TCR clonotype or CD40 ligand (CD40L) expression. Interaction with antigen-specific Th1, but not Th2 cells, induces IL-12 p40 and p75 secretion by DC. The induction of IL-12 production by Th1 cells does not depend on their IFN-gamma secretion, but requires direct cell-cell contact mediated by peptide/MHC class II-TCR and CD40-CD40L interactions. Th2 cells not only fail to induce IL-12 secretion, but they inhibit its induction by Th1 cells. Unlike stimulation by Th1, inhibition of IL-12 production by Th2 cells is mediated by soluble molecules, as demonstrated by transwell cultures. Among Th2-derived cytokines, IL-10, but not IL-4 inhibit Th1-driven IL-12 secretion. IL-10 produced by Th2 cells appears to be solely responsible for the inhibition of Th1 -induced IL-12 secretion, but it does not account for the failure of Th2 cells to induce IL-12 production by DC. Collectively, these results demonstrate that Th1 cells up-regulate IL-12 production by DC via IFN-gamma-independent cognate interaction, whereas this is inhibited by Th2-derived IL-10. The inhibition of Th1 -induced IL-12 production by Th2 cells with the same antigen specificity represents a novel mechanism driving the polarization of CD4+ T cell responses.     

Differential effects of IL-12 receptor blockade with IL-12 p40 homodimer on the induction of CD4+ and CD8+ IFN-gamma-producing cells

The role of IL-12 role in regulating Th1/Th2 balance is attributed in part to the ability of this cytokine to induce IFNgamma production by NK and Th1 cells, which in turn promotes Th1 and inhibits Th2 development. In the present study, the requirement for IL-12 in the development of alloantigen-reactive Th1 was assessed by adding neutralizing anti-IL-12 Abs or the IL-12 receptor antagonist p40 homodimer to primary MLC. The resulting cell populations were assessed for Th1 development by measuring IFN-gamma production upon restimulation with alloantigens. While the addition of anti-IL-12 Abs to primary MLC did not influence subsequent cytokine production, addition of p40 homodimer markedly enhanced, rather than decreased, Th1 development. To determine which T cell population produced enhanced levels of IFN-gamma in response to p40 homodimer, CD4+ or CD8+ T cells were depleted from the MLC. While p40 homodimer was inhibitory to selected CD4+ Th1 development, it enhanced IFN-gamma production by CD8+ T cells. To test the in vivo relevance of these findings, mouse heterotopic cardiac allograft recipients were treated with either p40 homodimer, anti-CD8 mAb, or with both p40 homodimer and anti-CD8 mAb. Treatment of allograft recipients with p40 homodimer had no effect on the in vivo sensitization of IFN-gamma-producing cells and resulted in accelerated allograft rejection relative to unmodified recipients. However, p40 homodimer markedly prolonged allograft survival in mice depleted of CD8+ T cells. Hence, p40 homodimer stimulates CD8+ Th1 development in vitro but inhibits CD4+ T cell function both in vitro and in vivo.     

Blockade of CD40-CD40 ligand pathway induces tolerance in murine contact hypersensitivity

Interactions between CD40 on antigen-presenting cells and its ligand (CD40L) on T cells has been implicated in T cell-mediated immune responses. Previously, we have shown that contact hypersensitivity (CHS), a cell-mediated cutaneous immune response in reaction to haptens, could be subclassified based on whether the hapten primed for Th1 or Th2 cytokines in cells isolated from draining lymph nodes. We also found that tolerance to a Th2-priming hapten could be induced only by simultane blockade of the CD40-CD40L and B7-CD28 at the time of sensitization. Here we demonstrate that blockade of CD40-CD40L signaling alone induces long-lasting unresponsiveness to the Th1 hapten 2,4-dinitrofluorobenzene (DNFB), and inhibits antigen-specific T cell proliferation in vitro. We find that CD40-CD40L signaling is required in the sensitization but not elicitation phase of DNFB-induced CHS, as treatment of mice with anti-CD40L monoclonal antibody (mAb) does not affect the response to hapten challenge in previously sensitized and untreated animals. Examination of cytokine production shows that anti-CD40L mAb decreases interferon-gamma production by draining lymph node cells from DNFB-sensitized mice, and reciprocally increases interleukin (IL)-4 production. Consistent with this Th1 to Th2 immune deviation, anti-CD40L mAb prevents the induction of IL-12 mRNA in regional lymph nodes, an event which is normally seen within 12 h following hapten sensitization. In contrast, suppression of CHS by CTLA4Ig decreased the production of all cytokines by draining lymph node cells. Together, these data show that blockade of the CD40-CD40L pathway by itself is sufficient to induce tolerance to DNFB-induced CHS, and that this is associated with blockade of IL-12 induction and Th1 to Th2 immune deviation.     

Maturation of human neonatal CD4+ and CD8+ T lymphocytes into Th1/Th2 effectors

The increased susceptibility of neonates to infections has been ascribed to the immaturity of their immune system. More particularly, T cell-dependent responses were shown to be biased towards a Th2 phenotype. Our studies on the in vitro maturation of umbilical cord blood T cells suggest that the Th2 bias of neonatal response cannot be simply ascribed to intrinsic properties of neonatal T cells. Phenotypically, neonatal CD4+ T cells are more immature than their adult CD45RO-/RA+ naive counterparts and they contain a subset (10-20%) of CD45RO-/RA+ CD31- cells which is very low in adults and displays some unique functional features. The activation and maturation of neonatal CD4+ T cells is particularly dependent upon the strength of CD28-mediated cosignal which dictates not only the cytokine profile released upon primary activation but also the response to IL-12. Activation of adult as well as neonatal CD4+ T cells in the context of low CD28 costimulation yields to the production of low levels of only one cytokine, i.e. IL-2. In contrast, strong CD28 costimulation supports the production of high levels of type 1 (IL-2, IFN gamma and TNF beta) and low levels of type 2 (IL-4 and IL-13) cytokines by neonatal T cells. The low levels of naive T cell-derived IL-4 are sufficient to support their development into high IL-4/IL-5 producers by an autocrine pathway. The ability of IL-12 to prime neonatal CD4+ T cells for increased production of IL-4 (in addition to IFN gamma) is observed only when CD28 cosignal is minimal. Under optimal activation conditions (i.e. with anti-CD3/B7.1 or allogenic dendritic cells) the response and the maturation of neonatal and adult naive T cells are similar. Thus the Th2 bias of neonatal immune response cannot be simply ascribed to obvious intrinsic T cell defect but rather to particular conditions of Ag presentation at priming. Unlike CD4+ T cells, neonatal CD8+ T cells strictly require exogenous IL-4 to develop into IL-4/IL-5 producers. Most importantly, anti-CD3/B7-activated neonatal CD8 T cells coexpress CD4 as well as CCR5 and CXCR4 and are susceptible to HIV-1 infection in vitro.     

Long-term CD4 Th1 and Th2 memory following acute lymphocytic choriomeningitis virus infection

CD4 T cells play a central role in viral immunity. They provide help for B cells and CD8 T cells and can act as effectors themselves. Despite their importance, relatively little is known about the magnitude and duration of virus-specific CD4 T-cell responses. In particular, it is not known whether both CD4 Th1 memory and CD4 Th2 memory can be induced by viral infections. To address these issues, we quantitated virus-specific CD4 Th1 (interleukin 2 [IL-2] and gamma-interferon) and Th2 (IL-4) responses in mice acutely infected with lymphocytic choriomeningitis virus (LCMV). Using two sensitive assays (enzyme-linked immunospot assay and intracellular stain) to measure cytokine production at the single-cell level, we found that both CD4 Th1 and Th2 responses were induced during primary LCMV infection. At the peak (day 8) of the response, the frequency of LCMV-specific CD4 Th1 cells was 1/35 to 1/160 CD4 T cells, and the frequency of Th2 cells was 1/400. After viral clearance, the numbers of virus-specific CD4 T cells dropped to 1/260 to 1/3,700 and then were maintained at this level indefinitely. Upon rechallenge with LCMV, both CD4 Th1 and Th2 memory cells made an anamnestic response in vivo. These results show that unlike some microbial infections in which only Th1 or Th2 responses are seen, an acute viral infection can induce a mixed CD4 T-cell response with long-term memory.     

T cell-derived IL-4 and dendritic cell-derived IL-12 regulate the lymphokine-producing phenotype of alloantigen-primed naive human CD4 T cells

Previous studies on human Th subset development were restricted to the analysis of naive T cells activated with anti-CD3 mAb in the absence of physiologic APC. In this study, we have analyzed the role of cytokines and physiologic APC on T cell maturation in an Ag-specific system, in which naive neonatal CD4 T cells were primed with allogeneic dendritic cells (DC). We found that the cytokine profile of primed cells was dependent upon 1) the ratio between T cells and allogeneic DC and 2) the endogenous production of IL-4 and IL-12. Neutralization of IL-4 during primary MLR increased IFN-gamma production at priming and shifted the phenotype of primed cells from Th0 to Th1. These effects were IL-12 dependent, in that they were suppressed by anti-IL-12 Abs. The production of IL-12 in primary MLR was further evidenced by the presence of IL-12 p40 in the culture supernatant fluids. IL-12 production was suppressed by exogenous IL-4 and increased by anti-IL-4 blocking mAbs, indicating that endogenous IL-4 down-regulated IL-12 production by DC. Finally, IL-12 was produced as a result of T cell/DC interaction involving the CD40/CD40 ligand and CD28/B7 costimulation pathways, as revealed by the inhibitory effect of anti-CD40 ligand mAb and CTLA-4Ig. These observations suggest that in neutral conditions, Ag presentation by DC results in the coordinate production of naive T cell-derived IL-4 and DC-derived IL-12 that in concert shape the cytokine profile of Th cells.