The biological role of Langerin+ dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin+ DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of Langerin+ DCs in vivo. For the first time, infection experiments with DT-treated Lang-DTR mice revealed that proliferation of L. major-specific CD8+ T cells is significantly reduced during the early phase of the immune response following depletion of Langerin+ DCs. Consequently, the total number of activated CD8+ T cells within the draining lymph node and at the site of infection is diminished. Furthermore, we show that the impaired CD8+ T cell response is due to the absence of Langerin+ dDCs and not Langerhans cells. Nevertheless, the CD4+ T cell response is not altered and the infection is cleared as effectively in DT-treated Lang-DTR mice as in control mice. This clearly demonstrates that Langerin+ DCs are, in general, dispensable for an efficient adaptive immune response against L. major parasites. Thus, we propose a novel concept that, in the experimental model of leishmaniasis, priming of CD4+ T cells is mediated by Langerin− dDCs, whereas Langerin+ dDCs are involved in early priming of CD8+ T cells.

A possible function of eukaryotic heat shock protein 60 (Hsp60) as endogenous danger signal has been controversially discussed in the past. Hsp60 was shown to induce the secretion of proinflammatory cytokines in professional antigen-presenting cells and to enhance the activation of T cells in primary stimulation. However, in vitro activation of macrophages by Hsp60 was attributed to contaminating endotoxin in the recombinant Hsp60 protein preparations. Here, we employ low endotoxin recombinant human Hsp60 and murine Hsp60 expressed by eukaryotic cell lines to dissect the Hsp60 protein-mediated effects from biologic effects that are mediated by prokaryotic contaminants in the Hsp60 protein preparation. The induction of tumor necrosis factor-␣ secretion in mouse macrophages is lost after endotoxin removal and is not mediated by Hsp60 expressed in eukaryotic systems. In contrast, the Hsp60-mediated enhancement of antigen-specific T cell activation does not correlate with endotoxin contamination. Moreover, Hsp60 that is expressed on the surface of different eukaryotic cell lines increases the activation of T cells in primary stimulation. Taken together, we provide evidence that endogenous Hsp60, which is thought to be released from dying infected cells in vivo, has a biological function that is not due to contaminating pathogen-associated molecules.

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Heat shock protein (Hsp) 60 is thought to function as endogenous danger signal by activating professional antigen-presenting cells (APC) through toll-like receptor (TLR) 4 and CD14, a mechanism that is also used by bacterial LPS. We recently showed that Hsp60 binds LPS and enhances LPS-induced immune stimulation. On the other hand, we also observed immune stimulation by Hsp60 independent of LPS which was partially mediated by Hsp60-induced IFN alpha. Here, we study the mechanisms involved in immune stimulation mediated by endotoxin-free Hsp60. We show that T cell co-stimulation induced by LPS-free Hsp60 was independent of TLR4 and the TLR-associated myeloide differentiation factor 88-signaling pathway. LPS-free Hsp60 did not induce IL-6, IL-12 or tumor necrosis factor alpha production in APC nor were these cytokines needed for Hsp60-mediated T cell co-stimulation in the absence of LPS. In contrast to endotoxin-free Hsp60, T cell co-stimulation induced by LPS or Hsp60/LPS complexes strictly depended on IL-12 and functional TLR-4. Furthermore, we show that LPS-free Hsp60 enhances IFN alpha expression in APC and that this cytokine represents one important mediator in immune stimulation by Hsp60 in the absence of LPS. Taken together, we provide evidence that endotoxin-free Hsp60 and LPS or Hsp60/LPS complexes employ different signaling mechanisms to transduce co-stimulatory signals.

The murine transmembrane glycoprotein CD83 is an important regulator for both thymic T cell maturation and peripheral T cell response. CD83 deficiency leads to a block in the thymic maturation of CD4-positive T cells, and interference with peripheral CD83/CD83 ligand interaction by addition of soluble CD83 suppresses immune responses in vivo and in vitro. Here we report the generation of a mouse transgenic for a fusion protein consisting of the extracellular domain of murine CD83 fused to the constant part of human IgG1 heavy chain. Thymic selection of CD4-positive T cells was unchanged in CD83Ig transgenic and in CD83Ig/OT-2 double-transgenic mice. However, thymic and peripheral CD4-positive T cells derived from CD83Ig/OT-2 transgenic mice displayed a reduced cytokine response to antigenic stimulation in vitro, whereas CD83Ig/OT-1-derived CD8-positive T cells showed normal cytokine secretion. The T cell defect was relevant in vivo, since a sub-lethal infection with Trypanosoma cruzi led to an increased parasitemia and reduced survival rate of CD83Ig transgenic mice compared to wild-type C57BL/6 mice. In contrast, in vivo application of recombinant CD83Ig did not result in an increase in parasitemia. Taken together our data suggest that thymic selection in the presence of CD83Ig leads to an intrinsic T cell defect of CD4-positive T cells resembling the phenotype described for CD4-positive T cells derived from CD83-deficient mouse strains.