Cutaneous macroglobulinosis deposits in a patient with IgM paraproteinemia/incipient Waldenström macroglobulinemia.

A 43-year-old healthy patient developed disseminated flat skin-colored to red-brown papules over a few months. These papules were the result of cutaneous IgM deposits representing the first symptom of a hitherto undiagnosed IgM paraproteinemia. This extremely rare skin manifestation of IgM paraproteinemia e. g. possibly incipient Waldenström macroglobulinemia should be included in the histopathological differential of eosinophilic dermal deposits.

Vaccinia virus impairs directional migration and chemokine receptor switch of human dendritic cells.

A crucial event for the induction of an anti-viral immune response is the coordinated, phenotype-dependent migration of dendritic cells (DC) to sites of infection and secondary lymphoid organs. Here we show that the vaccinia virus (VV) strains Western Reserve (WR) and modified virus Ankara (MVA) inhibit directional migration of mature DC toward the lymphoid chemokines CCL19 and CXCL12 without affecting surface expression of the respective chemokine receptors or impairing undirected cellular locomotion. Instead, infection with VV results in a deficiency of extracellular signal-regulated kinase-1 and a disturbance of intracellular calcium mobilization, indicating a viral interference with signaling events downstream of the surface chemokine receptors. In immature DC, apart from inhibiting chemokine-induced migration of infected DC, infection with both VV strains increases expression of the inflammatory chemokine receptors CCR1 and CXCR1 on non-infected bystander DC, which depends on the activity of IFN-alpha. Although functional, these chemokine receptors are resistant to lipopolysaccharide-induced down-regulation. In addition, VV-infected and non-infected bystander DC fail to up-regulate the lymphoid chemokine receptor CCR7 upon activation, together pointing to a disability to undergo the chemokine receptor switch. This study shows that VV targets directional migration of professional antigen-presenting cells at multiple functional levels, revealing a potent viral strategy of immune escape.

Mature monocyte-derived dendritic cells respond more strongly to CCL19 than to CXCL12: consequences for directional migration.

The chemokine receptor CCR7 is crucial for migration of mature dendritic cells (DC) directed toward secondary lymphoid organs; however, there is little knowledge about the function of the homeostatic chemokine receptor CXCR4 in DC and its contribution to directional migration of DC during inflammation. By comparing the impact of chemokine receptor engagement on mature DC we found that the CCR7 ligand CCL19 holds a stronger chemotactic potency than the CXCR4 ligand CXCL12. Moreover, CCL19 elicited rapid, steep and long-lasting mobilization of intracellular calcium in individual cells and induced intense phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B, while the intracellular signals elicited by CXCL12 were in part distinct and significantly weaker. Analysis of chemokine receptor expression revealed that although CCR7 and CXCR4 were expressed by a similar percentage of DC, the mean fluorescence intensity of CCR7 was up to six times higher, suggesting a higher receptor density. Based on these correlations we propose that the type of chemokine signal in conjunction with the expression and functional activity of the respective chemokine receptor is also determining the migration rate and potency of a chemotactic response in mature DC. In conclusion, our data support the fundamental role of CCR7 for rapidly guiding DC toward secondary lymphoid organs at an extra- and intracellular molecular level and on the contrary render CXCR4 a weaker contributor to directional migration of DC during inflammation.

Functional analysis of tumor-specific Th cell responses detected in melanoma patients after dendritic cell-based immunotherapy.

Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3(243-258)), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.

Antigen loading of dendritic cells with whole tumor cell preparations.

Dendritic cells (DC) based vaccinations have been widely used for the induction of anti-tumoral immunity in clinical studies. Antigen loading of DC with whole tumor cell preparations is an attractive method whenever tumor cell material is available. In order to determine parameters for the loading procedure, we performed dose finding and timing experiments. We found that apoptotic and necrotic melanoma cells up to a ratio of one-to-one, equivalent to 1mg/ml protein per 1 x 10(6) DC, can be added to monocyte derived DC without effecting DC recovery extensively. Using the isolated protein content of tumor cells (lysate) as a parameter, up to 5 mg/ml protein per 1 x 10(6) DC can be added. To achieve significant protein uptake at least 1 mg/ml of protein have to be added for more than 24 h as tested with FITC-labelled ovalbumin. Maturation inducing cytokines can be added simultaneously with the tumor cell preparations to immature DC without affecting the uptake. Furthermore, we tested the feasibility of cryopreservation of loaded and matured DC to facilitate the generation of ready to use aliquots. DC were cryopreserved in a mix of human serum albumin, DMSO and 5% glucose. After thawing, surface expression of molecules indicating the mature status (CD83, costimulatory and MHC molecules), was found to be unaltered. Furthermore, cryopreserved DC kept the capability to stimulate allogenic T-cell proliferation in mixed leukocyte reactions at full level. Loaded and matured DC pulsed with influenza matrix peptide (IMP) retained the capacity to induce the generation of IMP-specific cytotoxic T-lymphocytes after cryopreservation as measured by ELISPOT and tetramer staining. The expression of the chemokine receptor CXCR-4 and CCR-7 remained unaltered during cryopreservation and the migratory responsiveness towards MIP-3beta was unaltered as measured in a migration assay. Thus we conclude that the large scale loading and maturation of DC with whole tumor cell preparations can be performed in a single session. These data will facilitate the clinical application of DC loaded with whole tumor cell preparations.

DNA viruses and genetic modification of dendritic cells.

An alternative approach to the use of tumor-peptide-loaded dendritic cells (DC) in immunotherapy would be the use of genetically modified DC using viral vectors expressing tumor-associated antigens (TAA). However, viruses have developed several immune escape mechanisms and, thus, one has to study the interaction between viruses and DC before these viruses can be used as an alternative strategy. Here we report that vaccinia virus (VV) as well as herpes simplex virus type 1 (HSV-1) are able to potently infect monocyte-derived DC, however, this infection leads to the inhibition of the DC-mediated T cell stimulation in vitro.