Christiane Steeg

Sialylated ligands on pathogenic Trypanosoma cruzi interact with Siglec-E (sialic acid-binding Ig-like lectin-E)

Trypanosoma cruzi causes a suppression of the immune system leading to persistence in host cells. The trans‐sialidase expressed by T. cruzi is a major virulence factor and transfers sialic acid from host glycoconjugates to mucin‐like molecules on the parasite. Here we demonstrate that these sialylated structures play a role in the immunosuppression. We used two T. cruzi strains, whose TS activity correlated with their pathogenicity. The Tulahuen strain, characterized by a high TS activity efficiently infected mice, whereas the Tehuantepec strain showing a reduced TS activity could not establish a patent parasitemia. In vitro analysis revealed that these two strains invaded phagocytic and non‐phagocytic host cells at a comparable rate, but they exhibited different potentials to modulate dendritic cell function. In contrast to Tehuantepec, the Tulahuen strain suppressed the production of the proinflammatory cytokine IL‐12 and subsequent T‐cell activation. This inhibitory effect was absent upon desialylation of the parasite. Therefore, we analysed whether sialylated structures of T. cruzi interact with the inhibitory sialic acid‐binding protein Siglec‐E on DC. Indeed, Siglec‐E interacted with the pathogenic Tulahuen strain, but showed a diminished binding to the Tehuantepec strain. Ligation of Siglec‐E on DC using antibodies confirmed this inhibitory effect on DC function.

CD83 is a regulator of murine B cell function in vivo

The transmembrane glycoprotein CD83 has been described as a specific maturation marker for dendritic cells and several lines of evidence suggest that CD83 regulates thymic T cell maturation as well as peripheral T cell activation. Here we show for the first time that CD83 is involved also in the regulation of B cell function. CD83 is up‐regulated on activated B cells in vivo, specifically in the draining lymph nodes of Leishmania major‐infected mice. The ubiquitous transgenic (Tg) expression of CD83 interferes with Leishmania‐specific T cell‐dependent and with T cell‐independent antibody production. This defect is restricted to the B cell population since the antigen‐specific T cell response of CD83Tg mice to L. major infection is unchanged. The defective immunoglobulin (Ig) response is due to Tg expression of CD83 on the B cells because wild‐type B cells display normal antigen‐specific responses in CD83Tg hosts and CD83Tg B cells do not respond to immunization in a mixed wild‐type/CD83Tg bone marrow chimera. Finally, the treatment of non‐Tg C57BL/6 mice with anti‐CD83 mAb induces a dramatic increase in the antigen‐specific IgG response to immunization, thus demonstrating a regulatory role for naturally induced CD83 on wild‐type B cells.

Limited Role of CD4+Foxp3+ Regulatory T Cells in the Control of Experimental Cerebral Malaria

Cerebral malaria (CM) associated with Plasmodium berghei ANKA (PbA) infection is an accepted model of human CM. CM during PbA infection critically depends on sequestration of T cells into the brain. Several studies aimed to address the role of regulatory T cells (Treg) in modulating this pathogenic T cell response. However, these studies are principally hampered due to the fact that until recently no reagents were available to deplete Foxp3+ Treg specifically. To study the function of Treg in the genesis of CM, we used depletion of Treg mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin receptor-enhanced GFP fusion protein under the control of the foxp3 gene locus. These mice allow for a selective depletion of Foxp3+ Treg by diphtheria toxin injection, and also their specific detection and purification during an ongoing infection. Using depletion of Treg mice, we found only a small increase in the absolute numbers of Foxp3+ Treg during PbA infection and, consequently, the ratio of Treg to T effector cells (Teff) decreased due to the rapid expansion of Teff. Although the latter sequester in the brains of infected mice, almost no Treg were found in the brains of infected mice. Furthermore, we demonstrate that depletion of Treg has no influence on sequestration of Teff and on the clinical outcome, and only minor influence on T cell activation. Using ex vivo analysis of purified Treg from either naive mice or PbA-infected mice, we found that both exhibit similar inhibitory capacity on Teff.

Enhanced Activation of CD83-Positive T Cells*

CD83 is a marker molecule for mature dendritic cells (DCs) but is also substantially expressed on activated T cells in humans and mice. Its function is unknown, but CD83 knockout mice show an impaired thymic maturation of CD4‐positive cells and soluble CD83 inhibits partially antigen‐specific responses in vitro pointing to a role of CD83 in the immune system. Here we show that CD83‐positive T cells produce strongly increased amounts of interferon‐γ and interleukin‐2. In contrast, constitutive expression of CD83 on DCs alters neither the activation of DCs following addition of lipopolysaccharide nor the ability to present antigenic peptides. Thus, the expression of CD83 on T cells has direct functional consequences for tuning the activation threshold.

Interleukin-12 but not interleukin-18 is required for immunity to Trypanosoma cruzi in mice.

Protective immunity to the parasite Trypanosoma cruzi in mice depends on a pro-inflammatory T cell response involving the production of interferon-gamma (IFN-gamma). In conjunction with interleukin-12 (IL-12), IL-18 promotes the synthesis of IFN-gamma and a T helper type 1 immune response. We investigated the requirements of IL-12 and IL-18 in murine T. cruzi infection by use of C57BL/6 mice genetically deficient in either cytokine. IL-12p40(-/-) mice succumbed to infection at doses of 100 parasites, whereas IL-18(-/-) and wild-type mice resisted infectious doses up to 1000 parasites to the same extent. Levels of parasitemia were comparable between the latter groups, as were tissue parasite burdens according to quantitative real-time PCR. In contrast, IL-12p40(-/-) mice displayed vastly increased levels of parasites both in blood and in tissue. IFN-gamma concentrations in the serum of infected mice and in supernatants of splenocytes stimulated in vitro were decreased in IL-18(-/-) mice, whereas in IL-12p40(-/-) mice, IFN-gamma was undetectable in the serum and drastically reduced in cell supernatants. Levels of IL-12 production were generally comparable between wild-type and IL-18(-/-) mice, as were levels of IL-4, IL-2 and nitric oxide. Thus, the requirement for endogenous pro-inflammatory cytokines for a protective murine immune response against T. cruzi is satisfied by the expression of IL-12, while IL-18 is dispensable.

Expression of CD83 in the murine immune system

CD83 is used as a marker for mature dendritic cells (DC) in man. We have developed a new monoclonal antibody (mAb), Michel-17, that specifically recognizes mouse CD83. We show that murine CD83 is expressed mainly on mature DC and on activated T cells. Histological analysis of serial spleen sections revealed a CD83 expression pattern resembling that of MIDC-8, a known murine DC marker molecule. In contrast to other costimulatory receptors, cross-linking of CD83 with the mAb Michel-17 on DC or T cells does not induce any activation signals. Our data describe for the first time the expression pattern of murine CD83, which is comparable to that of human CD83.The unique mAb Michel-17 will help to elucidate the biological functions of the CD83 molecule in more detail.

Interaction of natural killer cells with Trypanosoma cruzi-infected fibroblasts.

The protozoan parasite Trypanosoma cruzi circulates in the blood as trypomastigotes and invades a variety of cells to multiply intracellularly as amastigotes. The acute phase triggers an immune response that restricts the proliferation of the parasite. However, parasites are able to persist in different tissues causing the pathology of Chagas’ disease. Natural killer (NK) cells play an important role in innate resistance to a variety of pathogens. In the present study we demonstrate that NK cells trigger trypanocidal mechanisms in infected L929 cells that are critically dependent on inducible nitric oxide (NO) synthase (iNOS) induction which is, to a major degree, triggered by interferon (IFN)‐γ provided by NK cells. This work provides a more detailed analysis of how NK cells as a part of the innate immune system participate in the control of parasites that reside intracellularly in fibroblast‐like L929 cells.

B and T Lymphocyte Attenuator Restricts the Protective Immune Response against Experimental Malaria

The immune response against the blood stage of malaria has to be tightly regulated to allow for vigorous antiplasmodial activity while restraining potentially lethal immunopathologic damage to the host like cerebral malaria. Coinhibitory cell surface receptors are important modulators of immune activation. B and T lymphocyte attenuator (BTLA) (CD272) is a coinhibitory receptor expressed by most leukocytes, with the highest expression levels on T and B cells, and is involved in the maintenance of peripheral tolerance by dampening the activation of lymphocytes. The function of BTLA is described in several models of inflammatory disorders and autoimmunity, but its function in infectious diseases is less well characterized. Also, little is known about the influence of BTLA on non-T cells. In this study, we analyzed the function of BTLA during blood-stage malaria infection with the nonlethal Plasmodium yoelii strain 17NL. We show that BTLA knockout mice exhibit strongly reduced parasitemia and clear the infection earlier compared with wild-type mice. This increased resistance was seen before the onset of adaptive immune mechanisms and even in the absence of T and B cells but was more pronounced at later time points when activation of T and B cells was observed. We demonstrate that BTLA regulates production of proinflammatory cytokines in a T cell-intrinsic way and B cell intrinsically regulates the production of P. yoelii 17NL-specific Abs. These results indicate that the coinhibitory receptor BTLA plays a critical role during experimental malaria and attenuates the innate as well as the subsequent adaptive immune response.

Depletion of Regulatory T Cells Augments a Vaccine-Induced T Effector Cell Response against the Liver-Stage of Malaria but Fails to Increase Memory

Regulatory T cells (Treg) have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4+CD25+ Treg were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3+CD25− Treg. To obtain more insights in the specific function of Treg during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT) receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG). As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP) of Plasmodium berghei (Pb) was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC). Using this system we demonstrate here that the number of CSP-specific T cells increases when Treg are depleted during prime but also during boost immunization. Importantly, despite this increase of T effector cells no difference in the number of antigen-specific memory cells was observed.

Genes from Chagas Susceptibility Loci That Are Differentially Expressed in T. cruzi-Resistant Mice Are Candidates Accounting for Impaired Immunity

Variation between inbred mice of susceptibility to experimental Trypanosoma cruzi infection has frequently been described, but the immunogenetic background is poorly understood. The outcross of the susceptible parental mouse strains C57BL/6 (B6) and DBA/2 (D2), B6D2F1 (F1) mice, is highly resistant to this parasite. In the present study we show by quantitative PCR that the increase of tissue parasitism during the early phase of infection is comparable up to day 11 between susceptible B6 and resistant F1 mice. A reduction of splenic parasite burdens occurs thereafter in both strains but is comparatively retarded in susceptible mice. Splenic microarchitecture is progressively disrupted with loss of follicles and B lymphocytes in B6 mice, but not in F1 mice. By genotyping of additional backcross offspring we corroborate our earlier findings that susceptibility maps to three loci on Chromosomes 5, 13 and 17. Analysis of gene expression of spleen cells from infected B6 and F1 mice with microarrays identifies about 0.3% of transcripts that are differentially expressed. Assuming that differential susceptibility is mediated by altered gene expression, we propose that the following differentially expressed transcripts from these loci are strong candidates for the observed phenotypic variation: H2-Eα, H2-D1, Ng23, Msh5 and Tubb5 from Chromosome 17; and Cxcl11, Bmp2k and Spp1 from Chromosome 5. Our results indicate that innate mechanisms are not of primary relevance to resistance of F1 mice to T. cruzi infection, and that differential susceptibility to experimental infection with this protozoan pathogen is not paralleled by extensive variation of the transcriptome.