Affinity-coupled CCL22 promotes positive selection in germinal centres.

Antibody affinity maturation depends on positive selection in germinal centres (GCs) of rare B cell clones that acquire higher-affinity B cell receptors via somatic hypermutation, present more antigen to follicular helper T (TFH) cells and, consequently, receive more contact-dependent T cell help1. As these GC B cells and TFH cells do not maintain long-lasting contacts in the chaotic GC environment2-4, it is unclear how sufficient T cell help is cumulatively focused onto those rare clones. Here we show that, upon stimulation of CD40, GC B cells upregulate the chemokine CCL22 and to a lesser extent CCL17. By engaging the chemokine receptor CCR4 on TFH cells, CCL22 and CCL17 can attract multiple helper cells from a distance, thus increasing the chance of productive help. During a GC response, B cells that acquire higher antigen-binding affinities express higher levels of CCL22, which in turn 'highlight' these high-affinity GC B cells. Acute increase or blockade of TFH cells helps to rapidly increase or decrease CCL22 expression by GC B cells, respectively. Therefore, a chemokine-based intercellular reaction circuit links the amount of T cell help that individual B cells have received recently to their subsequent ability to attract more help. When CCL22 and CCL17 are ablated in B cells, GCs form but B cells are not affinity-matured efficiently. When competing with wild-type B cells in the same reaction, B cells lacking CCL22 and CCL17 receive less T cell help to maintain GC participation or develop into bone-marrow plasma cells. By uncovering a chemokine-mediated mechanism that highlights affinity-improved B cells for preferential help from TFH cells, our study reveals a principle of spatiotemporal orchestration of GC positive selection.

Transcriptional profiling identifies novel regulators of macrophage polarization.

Macrophages are key inflammatory immune cells that display dynamic phenotypes and functions in response to their local microenvironment. Major advances have occurred in understanding the transcriptional, epigenetic, and functional differences in various macrophage subsets by in vitro modeling and gene expression and epigenetic profiling for biomarker discovery. However, there is still no standardized protocol for macrophage polarization largely due to the lack of thorough validation of macrophage phenotypes following polarization. In addition, transcriptional regulation is recognized as a major mechanism governing differential macrophage polarization programs and as such, many genes have been identified to be associated with each macrophage subset. However, the functional role of many of these genes in macrophage polarization is still unknown. Moreover, the role of other regulatory mechanisms, such as DNA methylation, in macrophage polarization remains poorly understood. Here, we employed an optimized model of human M1 and M2 macrophage polarization which we used for large-scale transcriptional and DNA methylation profiling. We were unable to demonstrate a role for DNA methylation in macrophage polarization, as no significant changes were identified. However, we observed significant changes in the transcriptomes of M1 and M2 macrophages. Additionally, we identified numerous novel differentially regulated genes involved in macrophage polarization, including CYBB and DHCR7 which we show as important regulators of M1 and M2 macrophage polarization, respectively. Taken together, our improved in vitro human M1 and M2 macrophage model provides new understandings of the regulation of macrophage polarization and candidate macrophage biomarkers.

Requirement of CCL17 for CCR7- and CXCR4-dependent migration of cutaneous dendritic cells.

Chemokines are known to regulate the steady-state and inflammatory migration of cutaneous dendritic cells (DCs). The beta-chemokine CCL17, a ligand of CCR4, is inducibly expressed in a subset of DCs and is strongly up-regulated in atopic diseases. Using an atopic dermatitis model, we show that CCL17-deficient mice develop acanthosis as WT mice, whereas dermal inflammation, T helper 2-type cytokine production, and the allergen-specific humoral immune response are significantly decreased. Notably, CCL17-deficient mice retained Langerhans cells (LCs) in the lesional skin after chronic allergen exposure, whereas most LCs emigrated from the epidermis of allergen-treated WT controls into draining lymph nodes (LNs). Moreover, CCL17-deficient LCs showed impaired emigration from the skin after exposure to a contact sensitizer. In contrast, the absence of CCR4 had no effect on cutaneous DC migration and development of atopic dermatitis symptoms. As an explanation for the major migratory defect of CCL17-deficient DCs in vivo, we demonstrate impaired mobility of CCL17-deficient DCs to CCL19/21 in 3D in vitro migration assays and a blockade of intracellular calcium release in response to CCR7 ligands. In addition, responsiveness of CCL17-deficient DCs to CXCL12 was impaired as well. We demonstrate that the inducible chemokine CCL17 sensitizes DCs for CCR7- and CXCR4-dependent migration to LN-associated homeostatic chemokines under inflammatory conditions and thus plays an important role in cutaneous DC migration.