Test-reduced teaching for stimulation of intrinsic motivation (TRUST): a randomized controlled intervention study.

BACKGROUND: The anatomy dissection course is a major part of the first two years of the traditional medical curriculum in Germany. The vast amount of content to be learned and the repeated examination is unanimously perceived by students and teachers as a major stress factor that contributes to the increase of psychosocial stress during the first two years of the course of study. Published interventions for specific stress reduction are scarce. METHODS: In a randomized, controlled design two intervention groups were compared with a control group (CG) over the whole dissection course (nine measuring points before, during and after first and second semester). The 'Stress Management intervention (IVSM)' targeted at the setting of personal standards, the 'Friendly Feedback intervention (IVFF)' at the context of frequent testing. Quantitative surveys were distributed at nine measuring points. The questionnaire comprised validated instruments and self-developed items regarding stress, positive and negative affect, anxiety, intrinsic and extrinsic motivation, self-efficacy, and perceived performance. RESULTS: Out of 195 students inscribed in the dissection course, 166 (85%) agreed to participate in the study. The experience of stress during the dissection course was significantly higher in the CG than in the IVFF. Anxiety and negative affect were lower in students of the IVFF while positive affect, intrinsic motivation, and self-efficacy were higher than in the CG. For anxiety and negative affect in the IVSM this was especially seen at the end of the second semester. The self-perceived increase in both knowledge and preparedness for the first big oral and written examination did not differ between the study groups. About three quarters of the participants would choose the intervention 'Friendly Feedback' if given the choice. CONCLUSIONS: Replacing formal tests with friendly feedback has proven to be an effective measure to reduce stress and negative affect and foster positive affect, self-efficacy, and intrinsic motivation, while it did not impair self-perceived academic performance.

Sleep Matters: CD4+ T Cell Memory Formation and the Central Nervous System.

The mechanisms of CD4+ T-cell memory formation in the immune system are debated. With the well-established concept of memory formation in the central nervous system (CNS), we propose that formation of CD4+ T-cell memory depends on the interaction of two different cell systems handling two types of stored information. First, information about antigen (event) and challenge (context) is taken up by antigen-presenting cells, as initial storage. Second, event and context information is transferred to CD4+ T cells. During activation, two categories of CD4+ T cell develop: effector CD4+ T cells, carrying event and context information, enabling them to efficiently focus their response to tissues under attack; and persisting CD4+ T cells, providing context-independent antigen-specific memories and long-term storage. This novel hypothesis is supported by the observation that mammalian sleep can improve both CNS and CD4+ T-cell memory.

Deep Sequencing Reveals Transient Segregation of T Cell Repertoires in Splenic T Cell Zones during an Immune Response.

Immunological differences between hosts, such as diverse TCR repertoires, are widely credited for reducing the risk of pathogen spread and adaptation in a population. Within-host immunological diversity might likewise be important for robust pathogen control, but to what extent naive TCR repertoires differ across different locations in the same host is unclear. T cell zones (TCZs) in secondary lymphoid organs provide secluded microenvironmental niches. By harboring distinct TCRs, such niches could enhance within-host immunological diversity. In contrast, rapid T cell migration is expected to dilute such diversity. In this study, we combined tissue microdissection and deep sequencing of the TCR ß-chain to examine the extent to which TCR repertoires differ between TCZs in murine spleens. In the absence of Ag, we found little evidence for differences between TCZs of the same spleen. Yet, 3 d after immunization with sheep RBCs, we observed a >10-fold rise in the number of clones that appeared to localize to individual zones. Remarkably, these differences largely disappeared at 4 d after immunization, when hallmarks of an ongoing immune response were still observed. These data suggest that in the absence of Ag, any repertoire differences observed between TCZs of the same host can largely be attributed to random clone distribution. Upon Ag challenge, TCR repertoires in TCZs first segregate and then homogenize within days. Such "transient mosaic" dynamics could be an important barrier for pathogen adaptation and spread during an immune response.

Correction to: Exam performance of different admission quotas in the first part of the state examination in medicine: a cross-sectional study.

An amendment to this paper has been published and can be accessed via the original article.

Exam performance of different admission quotas in the first part of the state examination in medicine: a cross-sectional study.

BACKGROUND: Most medical students in Germany are admitted via selection procedures, which are adjusted to the demands of the universities. At Lübeck medical school, scores from interviews that measure non-academic skills and pre-university GPAs are summed to arrive at an admission decision. This article seeks to illuminate the effectiveness of this selection procedure in comparison to other non-selected student groups. METHODS: Quota information and exam results from the first federal exam were linked for students admitted to Lübeck medical school between 2012 and 2015 (N = 655). Five different student groups (university-specific selection quota, pre-university GPA quota, waiting time quota, ex-ante quota and foreign students) were compared regarding exam attempts, written and oral grades, temporal continuity and examination success in the standard study period. RESULTS: While the pre-university GPA quota outperformed all other quotas regarding written and oral grades, it did not differ from the selection quota regarding exam attempts, temporal continuity and examination success in the standard study period. Students in the waiting time and ex-ante quotas performed inferior by comparison. The results of foreign students were the most problematic. CONCLUSION: Students selected by the university show high temporal continuity and examination success. These results, and possible advantages in physician eligibility, argue for the utilisation of non-academic skills for admission.

Evidence for a contributory role of a xenogeneic immune response in experimental epidermolysis bullosa acquisita.

Autoimmune diseases affect a large fraction of the population in Western countries. To elucidate the underlying causes, autoantibody transfer-induced mouse models have been established that greatly contributed to the understanding of the pathophysiology of these diseases. However, the role of a potentially co-occurring murine xenogeneic immune response to commonly utilized rabbit anti-mouse IgG remains poorly understood. Using the established rabbit anti-mouse type VII collagen (COL7) IgG-induced mouse model of the mucocutaneous blistering disorder epidermolysis bullosa acquisita (EBA), we found in this study a profound T- and B-cell response along with an altered cytokine expression profile in draining lymph nodes of mice injected with the xenogeneic IgG. This was associated with the formation of circulating and skin-bound mouse anti-rabbit IgG in wild-type but not CD154-deficient or B-cell-deficient JHT mice challenged with pathogenic rabbit IgG. Development of EBA skin lesions was attenuated in the two mouse strains lacking a B-cell response at later observation time points, but was not affected in mice treated with the T-cell trafficking blocker FTY720. Collectively, our results implicate a host's xenoreactive immune response to rabbit anti-mouse COL7 IgG, a confounding effect that may contribute to immune complex-driven inflammation and tissue damage in this antibody transfer-induced EBA mouse model, especially at later time points. In this regard, it may be recommended to finish the evaluation of results obtained by experiments employing antibody-transferred mouse models within the first 2 weeks after the pathogenic antibody injection.

Nanoparticles prepared from porcine cells support the healing of cutaneous inflammation in mice and wound re-epithelialization in human skin.

Previous reports have demonstrated that cell-derived nanoparticles (CDNPs) composed of bovine or porcine protein complexes exerted therapeutic effects against viral infections and cancer in mice and humans. Based on these observations, we asked whether CDNPs would improve inflammatory skin disorders. To address this, we utilized two distinct mouse models of cutaneous inflammation: the autoimmune skin-blistering disease epidermolysis bullosa acquisita (EBA) as an example of an autoantibody-induced cutaneous inflammation, and Leishmania major (L. major) infection as an example of a pathogen-induced cutaneous inflammation. In both models, we observed that CDNPs increased mRNA expression of the Th2 cytokine IL-4. Clinically, CDNPs decreased inflammation due to EBA and increased L. major-specific IgG1 levels without major effects on infected skin lesions. In addition, CDNPs supported the growth of keratinocytes in human skin cultures. In vitro studies revealed that CDNPs were taken up predominantly by macrophages, leading to a shift towards the expression of anti-inflammatory cytokine genes. Altogether, our data demonstrate that treatment with porcine CDNPs may be a new therapeutic option for the control of autoimmune-mediated inflammatory skin disorders.

The role of microfibrillar-associated protein 4 (MFAP4) in the formation and function of splenic compartments during embryonic and adult life.

Microfibrillar-associated protein 4 (MFAP4) is an extracellular protein belonging to the fibrinogen-related protein superfamily and is recognized as an integrin ligand with suggested functions in pulmonary and vascular tissue homeostasis. MFAP4 expression in the spleen is increased during infections; however, the significance of MFAP4 for the function of the spleen is unknown. Immunohistochemistry, morphometry and real-time RT-PCR were used to analyze wild-type and MFAP4-deficient spleens. In addition, they were compared with splenic tissue, which was newly formed 8 weeks after avascular implantation into adult mice in order to obtain information about the role of MFAP4 in the formation of splenic tissue during ontogeny and adult life. The present study shows that MFAP4 is co-localized with laminin in the B- and T-cell zones of the spleen, in addition to capsular and trabecular expression. MFAP4 is most likely produced by fibroblastic reticulum cells and follicular dendritic cells of the spleen but can also be imported via the blood from other tissues. The development of splenic tissue is not disturbed in MFAP4-deficient mice. However, in splenic tissue regenerating under MFAP4-deficient conditions, the number of FDCs is significantly decreased but is corrected by MFAP4 imported from other tissues. No differences were observed for lymphocyte numbers or splenic structure. The data indicate that MFAP4 promotes FDC development in regenerating splenic tissue and warrant further investigations regarding the MFAP4 dependency of splenic B-cell maturation.

Skin microbiota-associated inflammation precedes autoantibody induced tissue damage in experimental epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune blistering skin disease characterized by autoantibodies against type VII collagen (COL7). Immunization of SJL/J mice with recombinant murine COL7 results in break of tolerance and skin blisters. Strikingly, despite circulating autoantibodies, the same genetic background and identical environmental conditions, 20% of mice remain healthy. To elucidate the regulation of the transition from the presence of autoantibodies to overt autoimmune disease, we characterized the innate and adaptive immune response of mice that remain healthy after immunization and compared it to mice that developed skin disease. Both clinically healthy and diseased SJL/J mice showed circulating autoantibodies and deposition of complement-fixing IgG2c autoantibodies and C3 at the dermal-epidermal junction. However, only in diseased animals significant neutrophil infiltration and increase in FcgRIV expression were observed in the skin. In contrast, the expression of T cell signature cytokines in the T cell zone of the draining lymph node was comparable between clinically healthy and diseased animals after immunization. Surprisingly, health was associated with a decreased expression of CD11c, TNFA and KC (CXCL1) in the skin prior to immunization and could be predicted with a negative predictive value of >80%. Furthermore, mice that did not develop clinical disease showed a significantly higher richness and distinctly clustered diversity of their skin microbiota before immunization. Our data indicate that the decision whether blisters develop in the presence of autoantibodies is governed in the skin rather than in the lymph node, and that a greater richness of cutaneous bacterial species appears to be protective.

Activated CD4+ T cells enter the splenic T-cell zone and induce autoantibody-producing germinal centers through bystander activation.

CD4(+) T (helper) cells migrate in huge numbers through lymphoid organs. However, little is known about traffic routes and kinetics of CD4(+) T-cell subsets within different organ compartments. Such information is important because there are indications that CD4(+) T cells may influence the function of microenvironments depending on their developmental stage. Therefore, we investigated the migration of resting (naïve), activated, and recently activated (memory) CD4(+) T cells through the different compartments of the spleen. Resting and recently activated CD4(+) T cells were separated from thoracic duct lymph and activated CD4(+) T cells were generated in vitro by cross-linking the T-cell receptor and CD28. The present study shows that all three CD4(+) T-cell subsets selectively accumulate in the T-cell zone of the spleen. However, only activated T cells induce the formation of germinal centers (GCs) and autoantibodies in rats and mice. Our results suggest that in a two-step process they first activate B cells independent of the T-cell receptor repertoire and CD40 ligand (CD154) expression. The activated B cells then form GCs whereby CD154-dependent T-cell help is needed. Thus, activated T cells may contribute to the development of autoimmune diseases by activating autoreactive B cells in an Ag-independent manner.

Random migration and signal integration promote rapid and robust T cell recruitment.

To fight infections, rare T cells must quickly home to appropriate lymph nodes (LNs), and reliably localize the antigen (Ag) within them. The first challenge calls for rapid trafficking between LNs, whereas the second may require extensive search within each LN. Here we combine simulations and experimental data to investigate which features of random T cell migration within and between LNs allow meeting these two conflicting demands. Our model indicates that integrating signals from multiple random encounters with Ag-presenting cells permits reliable detection of even low-dose Ag, and predicts a kinetic feature of cognate T cell arrest in LNs that we confirm using intravital two-photon data. Furthermore, we obtain the most reliable retention if T cells transit through LNs stochastically, which may explain the long and widely distributed LN dwell times observed in vivo. Finally, we demonstrate that random migration, both between and within LNs, allows recruiting the majority of cognate precursors within a few days for various realistic infection scenarios. Thus, the combination of two-scale stochastic migration and signal integration is an efficient and robust strategy for T cell immune surveillance.

B cells, dendritic cells, and macrophages are required to induce an autoreactive CD4 helper T cell response in experimental epidermolysis bullosa acquisita.

In autoimmune bullous dermatoses (AIBD), autoantibodies induce blisters on skin or mucous membranes, or both. Mechanisms of continued autoantibody production and blistering have been well characterized using AIBD animal models. Mechanisms leading to the initial autoantibody production, however, have not been investigated in detail. Epidermolysis bullosa acquisita (EBA) is an AIBD associated with autoantibodies to type VII collagen (COL7). The majority of EBA patients' sera recognize the noncollagenous domain 1, including the von Willebrand factor A-like domain 2 (vWFA2). In experimental EBA induced by immunization with GST-COL7, disease manifestation depended on the genetic background, a Th1 polarization, and the GST-tag. In this model, nude mice neither produced autoantibodies nor blisters. It has remained uncertain which APC and T cell subsets are required for EBA induction. We established a novel EBA model by immunization with vWFA2 fused to intein (lacking the GST-tag). All tested mouse strains developed autoantibodies, but blisters were exclusively observed in mice carrying H2s. In immunized mice, CD4 T cells specific for vWFA2 were detected, and their induction required presence of B cells, dendritic cells, and macrophages. Anti-vWFA2 autoantibodies located at the lamina densa bound to the dermal side of salt-split skin and induced blisters when transferred into healthy mice. Absence of CD8 T cells at time of immunization had no effect, whereas depletion of CD4 T cells during the same time period delayed autoantibody production and blisters. Collectively, we demonstrate the pathogenic relevance of Abs targeting the vWFA2 domain of COL7 and show the requirement of APC-induced CD4 T cells to induce experimental EBA.

IL-7-producing stromal cells are critical for lymph node remodeling.

Nonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7-Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7-producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin-mediated depletion of IL-7-producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7-producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.

Specific immunoadsorption of pathogenic autoantibodies in pemphigus requires the entire ectodomains of desmogleins.

Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are life-threatening autoimmune blistering skin diseases. They are characterized by circulating autoantibodies which bind to the ectodomains of desmoglein (Dsg) 1 and Dsg3. These antibodies induce acantholysis in skin and mucous membranes. In severe cases of pemphigus, immunoadsorption is applied to remove total IgG from patient plasma using protein A or other ligands. To develop a specific adsorber for anti-Dsg antibodies, epitope mapping studies of Dsg1 and Dsg3 ectodomains were conducted. Dsg variants were expressed on the surface of HEK-293 cells and analysed for reactivity with pemphigus and control sera by indirect immunofluorescence technique. For Dsg1, a construct consisting of domain 1 directly fused to domain 5, seemed to be suitable for specific immunoadsorption of anti-Dsg1 antibodies. The recognized epitopes were mainly conformation-dependent. However, adsorption of pemphigus foliaceus IgG using this protein coupled to a Sepharose matrix did not completely remove pathogenicity from the sera, as proven by a keratinocyte dissociation assay. In contrast, full-length Dsg1 and Dsg3 ectodomains were able to specifically adsorb anti-Dsg antibodies and to efficiently eliminate pathogenicity. Therefore, the complete and correctly folded ectodomains of both desmogleins are required for therapeutic immunoadsorption.

Defining the quantitative limits of intravital two-photon lymphocyte tracking.

Two-photon microscopy has substantially advanced our understanding of cellular dynamics in the immune system. Cell migration can now be imaged in real time in the living animal. Strikingly, the migration of naive lymphocytes in secondary lymphoid tissue appears predominantly random. It is unclear, however, whether directed migration may escape detection in this random background. Using a combination of mathematical modeling and experimental data, we investigate the extent to which modern two-photon imaging can rule out biologically relevant directed migration. For naive T cells migrating in uninfected lymph nodes (LNs) at average 3D speeds of around 18 µm/min, we rule out uniform directed migration of more than 1.7 µm/min at the 95% confidence level, confirming that T cell migration is indeed mostly random on a timescale of minutes. To investigate whether this finding still holds for longer timescales, we use a 3D simulation of the naive T cell LN transit. A pure random walk predicts a transit time of around 16 h, which is in good agreement with experimental results. A directional bias of only 0.5 µm/min-less than 3% of the cell speed-would already accelerate the transit twofold. These results jointly strengthen the random walk analogy for naive T cell migration in LNs, but they also emphasize that very small deviations from random migration can still be important. Our methods are applicable to cells of any type and can be used to reanalyze existing datasets.

Recombinant IL-6 treatment protects mice from organ specific autoimmune disease by IL-6 classical signalling-dependent IL-1ra induction.

Cytokines are key regulators of physiological inflammatory responses, while aberrant cytokine expression contributes to pathogenesis of autoimmune diseases. We noted increased IL-6 levels in human and murine epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune bullous dermatoses (AIBD) induced by autoantibodies to type VII collagen (COL7). In contrast to rheumatoid arthritis, blockade of IL-6 led to strikingly enhanced experimental EBA, while treatment with recombinant IL-6 was protective. This was due to classical IL-6 signalling and independent of IL-6 trans-signalling, as treatment of mice with sgp130Fc had no impact on EBA manifestation. Induction of EBA in mice led to increased IL-1ra levels in skin and serum, while blockade of IL-6 completely inhibited IL-1ra expression induced by autoantibodies to COL7. In line, treatment of mice with EBA with recombinant IL-6 induced IL-1ra concentrations exceeding those of untreated animals with EBA, and IL-1ra (anakinra) administration significantly impaired experimental EBA induction. We here identified a novel anti-inflammatory pathway in an organ-specific autoimmune disease. Modulation of this IL-1ra pathway by classical IL-6 signalling demonstrates anti-inflammatory and protective activities of IL-6 in vivo.

Heat-shock protein 90 inhibition in autoimmunity to type VII collagen: evidence that nonmalignant plasma cells are not therapeutic targets.

Blocking heat-shock protein 90 (Hsp90) induces death of malignant plasma cells by activation of the unfolded protein response, a signaling pathway activated by accumulation of misfolded proteins within the endoplasmic reticulum. We hypothesized that nontransformed plasma cells are also hypersensitive to Hsp90 inhibition because of their high amount of protein biosynthesis. To investigate this hypothesis, 2 different Hsp90 inhibitors, the geldanamycin derivative 17-DMAG and the nontoxic peptide derivative TCBL-145, were applied to mice with experimental epidermolysis bullosa acquisita, an autoimmune bullous disease characterized by autoantibodies against type VII collagen of the dermal-epidermal junction. Both inhibitors ameliorated clinical disease of type VII collagen-immunized mice, suppressed auto-antibody production, and reduced dermal neutrophilic infiltrate. Interestingly, total plasma cell numbers, type VII collagen-specific plasma cells, and germinal center B cells were unaffected by anti-Hsp90 treatment in vivo. However, T-cell proliferation was potently inhibited, as evidenced by the reduced response of isolated lymph node cells from immunized mice to in vitro restimulation with anti-CD3/CD28 antibody or autoantigen in the presence of Hsp90 inhibitors. Our results suggest that Hsp90 blockade has no impact on normal or autoreactive plasma cells in vivo and indentify T cells as targets of anti-Hsp90 treatment in autoimmunity to type VII collagen.

Genetic identification and functional validation of FcγRIV as key molecule in autoantibody-induced tissue injury.

Autoantibody-mediated diseases are clinically heterogeneous and often fail conventional therapeutic strategies. Gene expression profiling has helped to identify new molecular pathways in these diseases, although their potential as treatment targets largely remains to be functionally validated. Based on weighted gene co-expression network analysis, we determined the transcriptional network in experimental epidermolysis bullosa acquisita (EBA), a paradigm of an antibody-mediated organ-specific autoimmune disease characterized by autoantibodies directed against type VII collagen. We identified 33 distinct and differentially expressed modules, including Fcγ receptor (FcγR) IV and components of the neutrophil-associated enzyme system in autoantibody transfer-induced EBA. Validation experiments, including functional analysis, demonstrated that FcγRIV expression on neutrophils crucially contributes to autoantibody-induced tissue injury in the transfer model of EBA. Mice lacking the common γ-chain of activating FcγRs, deficient in FcγRIV or treated with FcγRIV function blocking antibody, but not mice deficient in FcγRI, FcγRIIB, FcγRIII or both FcγRI and FcγRIII, were effectively protected from EBA. Skin disease was restored in γ-chain-deficient mice locally reconstituted with neutrophils from wild-type, but not from γ-chain-deficient, mice. Our findings both genetically and functionally identify a novel disease-related molecule, FcγRIV, in an autoantibody-mediated disorder, which may be of importance for the development of novel targeted therapies.

Repetitive immunization breaks tolerance to type XVII collagen and leads to bullous pemphigoid in mice.

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease of the elderly associated with considerable morbidity and mortality. As unspecific immunosuppressants are still the mainstay of BP therapy, several animal models, based on the passive transfer of autoantibodies or immune cells, have been developed to obtain a better understanding of the pathogenesis of BP and evaluate novel therapeutic interventions. We describe in this study an experimental model inducing BP by immunization of immunocompetent mice with a recombinant form of the immunodominant 15th noncollagenous domain of murine BP180 (type XVII collagen). The homologous noncollagenous 16A domain of human BP180 has previously been identified as an immunodominant region in human BP. Immunization of female SJL/J mice with the murine peptide led to clinical disease within 14 wk in 56% of mice. In contrast, none of the other strains developed blisters despite the presence of autoantibodies. The clinical disease manifested for at least 8 wk without further manipulation. This novel immunization-induced model reflects key immunopathological characteristics of human BP, including binding of complement-fixing autoantibodies along the dermal-epidermal junction, elevated total IgE serum levels, and infiltration of skin lesions with eosinophilic granulocytes. The use of immunocompetent mice and the induction of sustained clinical disease not requiring additional interventions make this immunization-induced mouse model most suitable to further explore the pathogenesis of BP and novel therapeutic interventions for this and other autoantibody-mediated diseases.

Complement-fixing anti-type VII collagen antibodies are induced in Th1-polarized lymph nodes of epidermolysis bullosa acquisita-susceptible mice.

The environment encountered in secondary lymphoid organs (e.g., lymph nodes) influences the outcome of immune responses. Immunization of mice with type VII collagen, an adhesion protein expressed at the cutaneous basement membrane, induces experimental epidermolysis bullosa acquisita (EBA). In this model, clinical disease is associated with the H2s haplotype of the MHC found in SJL/J mice. Most other strains (e.g., BALB/c, C57BL/6, NZM2410/J) are resistant to clinical disease, despite autoantibody production. Comparison of autoantibody response in EBA-resistant and -susceptible mice showed an IgG2-dominated response in the latter. We hypothesized that EBA susceptibility is due to specific cytokine gene expression in draining lymph nodes (dLN). To challenge this hypothesis, EBA-susceptible (SJL/J) and -resistant (BALB/c, C57BL/6) mice were immunized with type VII collagen, followed by analysis of clinical phenotype, subclasses of circulating and tissue-bound autoantibodies, complement activation, and cytokine gene expression in dLN. Disease manifestation was associated with induction of complement-fixing autoantibodies, confirming previous observations. Furthermore, however, IFN-γ/IL-4 ratio in dLN of EBA-susceptible mice was significantly increased compared with EBA-resistant strains, suggesting a Th1 polarization. Immunization of H2s-congenic C57BL/6 mice (B6.SJL-H2s) led to Th1 polarization in dLN and clinical disease. In addition to their cytokine milieu, EBA-susceptible and -resistant mice also differed regarding the expression of FcγR on peripheral leukocytes, in which a higher FcγRIV expression in SJL/J and B6.SJL-H2s mice, compared with C57BL/6, was associated with skin lesions. In summary, blistering in experimental EBA is regulated by both adaptive (divergent class switch recombination due to polarized cytokine expression) and innate (FcγR expression) immune mechanisms.