Protocol to Isolate Germinal Centers by Laser Microdissection.

During adaptive immune responses, germinal centers (GC) appear as transient microstructures, in which antigen-specific B and T cells interact with each other. Because only the antigen-activated B and T cells, such as Plasmablasts or follicular T helper (Tfh) cells, are present in GC, the in depth-analysis of GC is of great interest. To identify the cells that reside within GC, the majority of studies use the expression of specific surface molecules for analysis by flow cytometry. To do so, the tissue has to be disrupted for the preparation of single-cell suspensions. Thereby, the local information regarding neighborhoods of B cells and T cells and their potential interaction is lost. To study GC in vivo within their original microenvironment, we established a protocol for the isolation of GC by laser microdissection. To enable the identification of GC for subsequent transcriptomic analysis, the degradation of mRNA was diminished by using frozen tissues and by establishing a rapid staining protocol. This procedure enables histological and transcriptomic analysis of individual GC even within one lymphoid organ.

Protocol to Induce Follicular T Helper Cells, Germinal Centers, and Skin Lesions in Mouse Models for Skin Blistering Diseases.

Autoreactive T cells in autoantibody-mediated autoimmune diseases can be divided into two major subsets: (i) follicular T helper cells (Tfh) that provide T cell help in germinal centers (GC) and (ii) effector T (Teff) cells that immigrate into peripheral tissue sites such as the skin and mediate local inflammation. To study the sequence of events leading to the loss of tolerance in autoantibody-mediated autoimmune diseases it is required to investigate both T cell subsets simultaneously. This approach is hampered mainly because the appearance of skin inflammation in mouse models is a random process, which makes it difficult to define the location of inflammation at the right time point. To overcome this problem, we developed a scratching technique for ear skins that leads to the establishment of chronic autoimmune wounds in the mouse model for the pemphigoid-like disease epidermolysis bullosa acquisita. By defining the exact place where the skin wounds should form, this protocol enables a detailed analysis of skin-immigrating Teff cells. Of note, this protocol induces GC in draining lymph nodes in parallel so that Tfh cells in GC can be investigated concurrently. This protocol is not restricted to T cells and can be used for any other skin-immigrating inflammatory cells.

Receptor repertoires of murine follicular T helper cells reveal a high clonal overlap in separate lymph nodes in autoimmunity.

Follicular T helper cells (Tfh) are a specialized subset of CD4 effector T cells that are crucial for germinal center (GC) reactions and for selecting B cells to undergo affinity maturation. Despite this central role for humoral immunity, only few data exist about their clonal distribution when multiple lymphoid organs are exposed to the same antigen (Ag) as it is the case in autoimmunity. Here, we used an autoantibody-mediated disease model of the skin and injected one auto-Ag into the two footpads of the same mouse and analyzed the T cell receptor (TCR)ß sequences of Tfh located in GCs of both contralateral draining lymph nodes. We found that over 90% of the dominant GC-Tfh clonotypes were shared in both lymph nodes but only transiently. The initially dominant Tfh clonotypes especially declined after establishment of chronic disease while GC reaction and autoimmune disease continued. Our data demonstrates a dynamic behavior of Tfh clonotypes under autoimmune conditions and emphasizes the importance of the time point for distinguishing auto-Ag-specific Tfh clonotypes from potential bystander activated ones.