Andreas O. Eggert

Unique appearance of proliferating antigen-presenting cells expressing DC-SIGN (CD209) in the decidua of early human pregnancy.

Intact human pregnancy can be regarded as an immunological paradox in that the maternal immune system accepts the allogeneic embryo without general immunosuppression. Because dendritic cell (DC) subsets could be involved in peripheral tolerance, the uterine mucosa (decidua) was investigated for DC populations. Here we describe the detailed immunohistochemical and functional characterization of HLA-DR-positive antigen-presenting cells (APCs) in early pregnancy decidua. In contrast to classical macrophages and CD83(+) DCs, which were found in comparable numbers in decidua and nonpregnant endometrium, only decidua harbored a significant population of HLA-DR(+)/DC-SIGN(+) APCs further phenotyped as CD14(+)/CD4(+)/CD68(+/-)/CD83(-)/CD25(-). These cells exhibited a remarkable proliferation rate (9.2 to 9.8% of all CD209(+) cells) by double staining with Ki67 and proliferating cell nuclear antigen. Unique within the DC-family, the majority of DC-SIGN(+) decidual APCs were observed in situ to have intimate contact with CD56(+)/CD16(-)/ICAM-3(+) decidual natural killer cells, another pregnancy-restricted cell population. In vitro, freshly isolated CD14(+)/DC-SIGN(+) decidual cells efficiently took up antigen, but could not stimulate naive allogeneic T cells at all. Treatment with an inflammatory cytokine cocktail resulted in down-regulation of antigen uptake capacity and evolving capacity to effectively stimulate resting T cells. Fluorescence-activated cell sorting analysis confirmed the maturation of CD14(+)/DC-SIGN(+) decidual cells into CD25(+)/CD83(+) mature DCs. In summary, this is the first identification of a uterine immature DC population expressing DC-SIGN, that appears only in pregnancy-associated tissue, has a high proliferation rate, and a conspicuous association with a natural killer subset.

Generation and functional characterization of mouse monocyte-derived dendritic cells.

Dendritic cells (DC) are potent antigen‐presenting cells with the unique capacity to initiate primary immune responses. As a result, DC are currently used in clinical studies to induce immunity against infectious disease and malignant cells. However, multiple DC subsets exist and it has been suggested that the type of DC may affect the immune response induced. The vast majority of DC used in experimental mouse tumor models is derived from bone marrow progenitors. In contrast, most in vitro as well as in vivo human studies involve the use of DC generated from adherent peripheral blood‐derived monocytes in the presence of GM‐CSF and IL‐4. In the current report, we describe for the first time the generation and characterization of mouse monocyte‐derived DC (MODC). The results indicate that mouse MODC display similar morphology, phenotype and immunostimulatory activity as compared to bone marrow‐derived DC. Both DC subsets were able to efficiently take up and subsequently cross‐present protein antigen to cytotoxic T cells. Moreover, we demonstrate that vaccination with peptide‐loaded MODC mediates induction of tumor‐reactive immunity in vivo. The isolation and characterization of mouse MODC will provide a valuable research tool to investigate fundamental aspects of DC biology and which DC subsets are most suitable to induce anti‐tumor immunity.

Analysis of dendritic cell trafficking using EGFP-transgenic mice.

Dendritic cells (DCs) are professional antigen presenting cells, well equipped to initiate an immune response. For effective induction of an immune response, DC should migrate from the periphery to the lymph node to present the antigen to T lymphocytes. Currently, tumor-antigen loaded DCs are used in clinical vaccination trials in cancer patients. To investigate the migratory capacity of DC in vivo, a variety of fluorescent and radioactive labels have been used. Here we introduce a novel tool to study DC migration in vivo: DCs generated from enhanced green fluorescent protein (EGFP)-transgenic mice. DC from EGFP-transgenic mice display typical DC behavior and can be matured without affecting their autofluorescence in vitro. In addition, the continuously produced cytoplasmic EGFP in living cells functions as a viability marker, since EGFP released from dying cells does not stain DC from C57Bl/6 mice upon coculture. In vivo migration studies using EGFP-DC and indium-111-labeled DC were performed to determine the efficiency of i.d. versus s.c. administered DC to reach the draining lymph node. The analysis demonstrates that i.d. injection increases the amount of EGFP-DC/indium-111-labeled DC in the lymph node compared to s.c. injection. Subsequent quantitative, phenotypical and ultrastuctural analysis demonstrate that DC generated from EGFP-transgenic mice are well suited to study the migratory behavior, distribution and phenotype of DC in vivo.

Specific peptide‐mediated immunity against established melanoma tumors with dendritic cells requires IL‐2 and fetal calf serum‐free cell culture

Melanoma, despite its aggressive growth characteristics, is an antigenic tumor expressing several characterized neo‐ and differentiation antigens. Dendritic cells (DC) when pulsed with definedpeptides have been shown to effectively induce melanoma‐specific T cell responses in humans and mice. These protect animals from challenge with melanoma, but so far have failed to induce significant tumor regressions. To study the efficacy of DC‐based anti‐tumor vaccinations, we set up a therapeutic model using C57BL/6J mice with established pulmonary and subcutaneous metastases induced by the B16‐melanoma cell line B78‐D14. Mice were vaccinated twice with 20,000 antigen‐presenting cells, either bone marrow‐derived DC or epidermal Langerhans cells (LC), which were loaded with the tyrosinase‐related protein 2 (TRP2) peptide. Generally, DC cultured with fetal calf serum (FCS) induced a dominant unspecific response. This was not seen using LC cultured without serum; however, vaccination with TRP2‐loaded FCS‐free LC alone failed to influence the growth of established B16 tumors. A reproducible reduction of tumor size and weight was only obtained if LC vaccinations with TRP2 were followedby a 5‐day treatment of mice with 200,000 IU IL‐2 intraperitoneally twice/daily. Omitting the TRP2 peptide abolished the efficacy of this combined treatment, demonstrating the crucial role of priming a melanoma‐specific T cell response. Microcytotoxic assays performed with spleen‐derived T cells and melanoma as well as congenic fibroblast lines as targets confirmed the TRP2‐dependent specificity of LC‐induced immune responses. Thus, despite the fact that tumor‐specific T cells were primed, an additional IL‐2‐dependent stimulus was needed to translate this immune response into a therapeutic effect against established tumors.

Immunological tumor destruction in a murine melanoma model by targeted LTα independent of secondary lymphoid tissue

BackgroundWe previously demonstrated that targeting lymphotoxin α (LTα) to the tumor evokes its immunological destruction in a syngeneic B16 melanoma model. Since treatment was associated with the induction of peritumoral tertiary lymphoid tissue, we speculated that the induced immune response was initiated at the tumor site.Methods and resultsIn order to directly test this notion, we analyzed the efficacy of tumor targeted LTα in LTα knock-out (LTα−/−) mice which lack peripheral lymph nodes. To this end, we demonstrate that tumor-targeted LTα mediates the induction of specific T-cell responses even in the absence of secondary lymphoid organs. In addition, this effect is accompanied by the initiation of tertiary lymphoid tissue at the tumor site in which B and T lymphocytes are compartmentalized in defined areas and which harbor expanded numbers of tumor specific T cells as demonstrated by in situ TRP-2/Kb tetramer staining. Mechanistically, targeted LTα therapy seems to induce changes at the tumor site which allows a coordinated interaction of immune competent cells triggering the induction of tertiary lymphoid tissue. ConclusionThus, our data demonstrate that targeted LTα promotes an accelerated immune response by enabling the priming of T cells at the tumor site.

Stromal cells as the major source for matrix metalloproteinase-2 in cutaneous melanoma

Matrix metalloproteinases (MMPs) are essential for tumor progression, invasion and metastases formation. Expression of these proteinases is not only restricted to the tumor cells themselves, but also is found in normal stromal cells. Moreover, immunohistochemistry suggests stromal cells as the major source. To scrutinize this hypothesis we established a slowly growing, syngeneic tumor model using the B16-melanoma cell line B78D14. In vitro analysis demonstrated that B78D14 cells secreted MMP-2, MT1-MMP, and to a lesser degree MMP-9; in addition they expressed both MT1-MMP and EMMPRIN on their surface. In subcutaneous (s.c.) tumors of these cells MMP-2 expression was predominantly present at the tumor-stroma border indicating stromal cells as primary source for this protease in vivo. Indeed, double staining experiments and in situ zymography confirmed that tumor adjacent stromal cells at the invasive front expressed MMP-2 and only at this site activated MMP-2 was detectable. Notably, in an experimental pulmonary metastases model neither tumor nor stromal cells expressed MMP-2, suggesting that the capacity of stromal cells is largely dependent on the surrounding microenvironment.

Bcl-2 expression in rituximab refractory cutaneous B-cell lymphoma.

Rituximab has been established as an effective and safe therapy for cutaneous B-cell lymphoma (CBCL). Different survival pathways, that is the Raf/MEK/Erk- or the p38MAPK cascade, have been suggested as downstream mediators of rituximab and may be involved in treatment failure. Biopsies from four patients, suffering from different subtypes of CBCL, which were obtained at various time points of relapse during or after therapy with 375 mg rituximab per m2 of body surface area, were analysed for the expression of CD20, CD3, Ki-67, Raf-kinase inhibitory protein (RKIP) and bcl-2 by immunohistochemistry. No CD20-loss variants, that is the suggested main tumour escape mechanism to rituximab therapy, were observed in any specimen of relapsing CBCL. Notably, the expression of proapoptotic RKIP remained increased in these tumour samples. This was concomitated by a constant to slightly reduced proliferation status as demonstrated by Ki-67 staining. However, relapsing CBCL exhibited a strong upregulation of the antiapoptotic molecule bcl-2 in comparison to pretherapeutic levels. The immunohistochemical analyses of this case series of rituximab refractory CBCL suggest that upregulation of bcl-2 may play a major role in therapy resistance.

Shift from Systemic to Site-Specific Memory by Tumor-Targeted IL-2

IL-2 has been approved for treatment of patients with cancer. Moreover, it has been used as a component of vaccines against cancer. In this regard, we have recently demonstrated that dendritic cell-based peptide vaccination in mice required IL-2 to mount an effective immune response against established melanoma metastases. In this study, we confirm this observation by use of tumor-targeted IL-2. However, the development of a protective systemic memory was substantially impaired by this measure, i.e., mice, which successfully rejected s.c. tumors of B16 melanoma after vaccination with dendritic cells pulsed with tyrosinase-related protein 2-derived peptides plus a boost with targeted IL-2, failed to reject a rechallenge with experimental pulmonary metastases. Detailed analysis revealed a change in the distribution of the tumor-reactive T cell population: although targeted IL-2 expanded the local effector population, tyrosinase-related protein 2-reactive T cells were almost completely depleted from lymphatic tissues.

Identification and characterization of survivin‐derived H‐2Kb‐restricted CTL epitopes

Survivin is overexpressed in several malignancies and in tumor‐associated endothelium making it an attractive target for therapeutic cytotoxic T‐cell responses. Thus, it would be important to test this notion in preclinical models. Consequently, we screened the murine survivin sequence for potential binding Kb‐restricted octamer peptide epitopes. Two epitopes, which bind strongly to Kb, were selected to test their immunogenicity in vivo. Spleen cells from mice vaccinated by intradermal injection of mature DC pulsed with these peptides displayed reactivity to the respective epitopes. The natural processing and presentation of these epitopes by tumor cells was evident by the killing of murine melanoma cells by vaccination‐induced T cells. Subcutaneous challenge with syngeneic melanoma demonstrated the protective immunity of this vaccination. Notably, analysis of the vessel density in subcutaneous tumors revealed that survivin‐specific vaccination significantly reduced the number of intratumoral vessels. In summary, we demonstrated the immunogenicity of two Kb‐restricted peptide epitopes derived from the murine survivin protein; moreover, survivin‐specific vaccination not only resulted in a reduction of tumor cells but also the tumor supplying blood vessels. The presented preclinical model for survivin‐directed vaccination may serve as a valuable tool to improve already running clinical trials in a syngeneic tumor model.

CD28‐mediated costimulation impacts on the differentiation of DC vaccination‐induced T cell responses

Costimulatory signals such as the ones elicited by CD28/B7 receptor ligation are essential for efficient T cell activation but their role in anti‐tumour immune responses remains controversial. In the present study we compared the efficacy of DC vaccination‐induced melanoma specific T cell responses to control the development of subcutaneous tumours and pulmonary metastases in CD28‐deficient mice. Lack of CD28‐mediated costimulatory signals accelerated tumour development in both model systems and also the load of pulmonary metastases was strongly increased by the end of the observation period. To scrutinize whether lack of CD28 signalling influences priming, homing or effector function of Trp‐2180−188/Kb‐reactive T cells we investigated the characteristics of circulating and tumour infiltrating T cells. No difference in the frequency of Trp‐2180−188/Kb‐reactive CD8+ T cells could be demonstrated among the cellular infiltrate of subcutaneous tumours after DC vaccination between both genotypes. However, the number of IFN‐γ‐producing Trp‐2‐reactive cells was substantially lower in CD28‐deficient mice and also their cytotoxicity was reduced. This suggests that CD28‐mediated costimulatory signals are essential for differentiation of functional tumour‐specific CD8+ T‐effector cells despite having no impact on the homing of primed CD8+ T cells.