Uwe Ritter

Priming of CD8+ and CD4+ T Cells in Experimental Leishmaniasis Is Initiated by Different Dendritic Cell Subtypes

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.

Epidermal Langerhans Cells Are Dispensable for Humoral and Cell-Mediated Immunity Elicited by Gene Gun Immunization

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8+ T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4+ Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4+ and CD8+ T cells, IFN-γ secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity. Together, our data show that gene gun immunization is capable of inducing humoral and cell-mediated immune reactions independently of LC.

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.

Leishmania major parasite stage-dependent host cell invasion and immune evasion

Leishmania pathogenesis is primarily studied using the disease‐inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease‐relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage‐specific DsRed protein expression. We found parasite stage‐specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote‐specific complement receptor 3‐mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1‐mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage‐specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease‐propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigotedriven immune response in leishmaniasis.—Wenzel, U. A., Bank, E., Florian, C., Förster, S., Zimara, N., Steinacker, J., Klinger, M., Reiling, N., Ritter, U., van Zandbergen, G. Leishmania major parasite stage‐dependent host cell invasion and immune evasion. FASEB J. 26, 29–39 (2012). www.fasebj.org

CEACAM1+ myeloid cells control angiogenesis in inflammation

Local inflammation during cutaneous leishmaniasis is accompanied by accumulation of CD11b(+) cells at the site of the infection. A functional role for these monocytic cells in local angiogenesis in leishmaniasis has not been described so far. Here, we show that CD11b(+) cells express high levels of the myeloid differentiation antigen carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). In experimental cutaneous leishmaniasis in C57BL/6 wild-type (B6.WT) and B6.Ceacam1(-/-) mice, we found that only B6.Ceacam1(-/-) mice develop edemas and exhibit impairment of both hemangiogenesis and lymphangiogenesis. Because CEACAM1 expression correlates with functional angiogenesis, we further analyzed the role of the CD11b(+) population. In B6.Ceacam1(-/-) mice, we found systemic reduction of Ly-6C(high)/CD11b(high) monocyte precursors. To investigate whether CEACAM1(+) myeloid cells are causally related to efficient angiogenesis, we used reverse bone marrow transplants (BMTs) to restore CEACAM1(+) or CEACAM1(-) bone marrow in B6.Ceacam1(-/-) or B6.WT recipients, respectively. We found that angiogenesis was restored by CEACAM1(+) BMT only. In addition, we observed reduced morphogenic potential of inflammatory cells in Matrigel implants in CEACAM1(-) backgrounds or after systemic depletion of CD11b(high) macrophages. Taken together, we show for the first time that CEACAM1(+) myeloid cells are crucial for angiogenesis in inflammation.

Myeloid suppressor cells require membrane TNFR2 expression for suppressive activity

TNF and TNF receptor type 2 (TNFR2) have been shown to be important for generation of myeloid‐derived suppressor cells (MDSC). In order to analyze whether and how TNFR2 passes the effect of TNF on, myeloid cells from TNFR2‐deficient mice were compared to respective cells from wild‐type mice. Primary TNFR2‐deficient myeloid cells showed reduced production of NO and IL‐6 which was attributable to CD11b+ CD11c− Ly6C+ Ly6G− immature monocytic MDSC. TNFR2‐deficient MDSC isolated from bone marrow were less suppressive for T cell proliferation compared to WT‐derived MDSC. These differences on myeloid cells between the two mouse lines were still observed after co‐culture of bone marrow cells from the two mouse lines together during myeloid cell differentiation, which demonstrated that the impaired functional capacity of TNFR2‐deficient cells was independent of soluble factors but required membrane expression of TNFR2. Similarly, adoptive transfer of TNFR2‐deficient bone marrow cells into wild‐type hosts did not rescue the TNFR2‐specific phenotype of bone marrow‐derived myeloid cells. Therefore, membrane TNFR2 expression determines generation and function of monocytic MDSC.

A new view on cutaneous dendritic cell subsets in experimental leishmaniasis

Because of their anatomical distribution epidermal Langerhans cells (LCs) are discussed to be crucial for antigen uptake and subsequent presentation to naïve T cells in skin-draining lymph nodes. The use of LC-specific markers like Langerin or knock-in mice expressing green fluorescent protein under the control of the Langerin promotor now facilitates the dissection of LCs from other dendritic cell (DC) subsets. Surprisingly, current data indicate that LCs are not generally involved in the induction of cellular immune responses. Moreover, the widely accepted paradigm postulating that LCs in principle act as T cell activators is contested by recent publications. Consequently, the biological role of LCs, in particular in cutaneous immune responses, needs to be revisited. The experimental model of leishmaniasis represents a suitable model to study the origin of an antigen-specific T cell response in mice. With this model the transport and presentation of skin derived Leishmania (L.) major antigens can be monitored in vivo. Furthermore, the quality of T cell-DC interactions can be determined. Considering recent progress in LC research we propose a novel concept of LCs in T cell meditated immunity against L. major parasites.

Leishmania major Infection in Humanized Mice Induces Systemic Infection and Provokes a Nonprotective Human Immune Response

Background Leishmania (L.) species are the causative agent of leishmaniasis. Due to the lack of efficient vaccine candidates, drug therapies are the only option to deal with cutaneous leishmaniasis. Unfortunately, chemotherapeutic interventions show high toxicity in addition to an increased risk of dissemination of drug-resistant parasites. An appropriate laboratory animal based model is still missing which allows testing of new drug strategies in the context of human immune cells in vivo. Methodology/Principal Findings Humanized mice were infected subcutaneously with stationary phase promastigote L. major into the footpad. The human immune response against the pathogen and the parasite host interactions were analyzed. In addition we proved the versatility of this new model to conduct drug research studies by the inclusion of orally given Miltefosine. We show that inflammatory human macrophages get infected with Leishmania parasites at the site of infection. Furthermore, a Leishmania-specific human-derived T cell response is initiated. However, the human immune system is not able to prevent systemic infection. Thus, we treated the mice with Miltefosine to reduce the parasitic load. Notably, this chemotherapy resulted in a reduction of the parasite load in distinct organs. Comparable to some Miltefosine treated patients, humanized mice developed severe side effects, which are not detectable in the classical murine model of experimental leishmaniasis. Conclusions/Significance This study describes for the first time L. major infection in humanized mice, characterizes the disease development, the induction of human adaptive and innate immune response including cytokine production and the efficiency of Miltefosine treatment in these animals. In summary, humanized mice might be beneficial for future preclinical chemotherapeutic studies in systemic (visceral) leishmaniasis allowing the investigation of human immune response, side effects of the drug due to cytokine production of activated humane immune cells and the efficiency of the treatment to eliminate also not replicating (“hiding”) parasites.

Laser Ablation–Inductively Coupled Plasma Mass Spectrometry: An Emerging Technology for Detecting Rare Cells in Tissue Sections

Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation–inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 109 particles/ml for 1 h incorporated an average of 3.39 × 108 Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 105 counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific 154Sm-tagged anti–HLA-DR or 174Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology.

Myeloid‐derived suppressor cell functionality and interaction with Leishmania major parasites differ in C57BL/6 and BALB/c mice

Myeloid‐derived suppressor cells (MDSCs) represent a heterogeneous population of CD11b+ cells. According to the surface molecules Ly6G and Ly6C (where Ly6G and Ly6C are lymphocyte antigen 6, locus G and C, respectively), MDSCs are further divided into monocytic (Mo‐MDSCs, CD11b+/Ly6Chigh/Ly6G−) and polymorphonucleated suppressor cells (PMN‐MDSCs, CD11b+/Ly6Cint/Ly6G+). Most published manuscripts focus on the suppressive role of MDSCs in cancer, whereas their impact on adaptive immunity against obligatory intracellular parasites is not well understood. Furthermore, it is not clear how the genetic background of mice influences MDSC functionality. Therefore, we implemented an experimental model of leishmaniasis, and analyzed MDSC maturation and the impact of MDSCs on the parasite‐specific T‐cell responses in resistant C57BL/6 and susceptible BALB/c mice. This experimental setup demonstrated the impaired ability of BALB/c mice to produce Mo‐MDSCs when compared with C57BL/6 mice. This phenotype is detectable after subcutaneous infection with parasites and is specifically represented by a reduced accumulation of Mo‐MDSCs at the site of infection in BALB/c mice. Moreover, infected C57BL/6‐derived MDSCs were able to suppress Leishmania‐specific CD4+ T‐cell proliferation, whereas BALB/c‐derived MDSCs harboring parasites lost this suppressive function. In conclusion, we demonstrate that (i) genetic background defines MDSC differentiation; and (ii) Leishmania major parasites are able to modulate the suppressive effect of MDSCs in a strain‐dependent manner.