Definition of Germinal-Center B Cell Migration In Vivo Reveals Predominant Intrazonal Circulation Patterns

Abstract

Proliferation, mutation, and selection in the germinal center (GC) are thought to occur in distinct microanatomical compartments-the dark zone (DZ) and the light zone (LZ). Thus, affinity maturation has been posited to require frequent trafficking between zones. Here we report the use of multiphoton in vivo microscopy to determine migration patterns of GC B cells. Analysis of time-resolved images revealed unexpected patterns of movement as well as GC B cell morphology. Though frequent movement between the DZ and LZ was anticipated, few cells were observed to cross the interface between the two compartments. Moreover, cell-track trajectories indicated that cell movement in this region is predominantly parallel to the interface, suggesting that B cells circulate within individual LZ and DZ compartments. The results suggest a revision to our views of B cell circulation within GCs and the functional relationship of its two major compartments.

Lymphocyte-rich classical Hodgkin's lymphoma: distinctive tumor and microenvironment markers

“…The outer zone was described as containing small lymphoid cells, blasts, and plasmacytoid cells, with many cells of the outer zone strongly expressing CDw75. Hauser et al 21 described the tendency of germinal-center B cells to migrate toward the outer zone of the germinal center, and Brink 22 questioned whether this reflected a need for interactions with T cells or antigen-bearing cells bordering the germinal center. Indeed, although the specific function of the outer zone is still essentially unknown, antibodies for follicular T-cell markers such as PD-1 clearly show that the PD-1-positive cells in reactive follicles very characteristically occupy this outer zone.…”

3D and 4D imaging of immune cells in vitro and in vivo

“…It is very much dependent on the type of question being asked, what type of analysis is required and to what degree it can be automated. While calcium waves in neuronal networks or optical colocalizations in Wxed or non-moving tissues can be readily analyzed with existing software tools (Crepel et al 2007;Gobel et al 2007), the analysis of cell migration or cellcell contact formation is still not done properly by existing software and, in fact, is done manually or at least with heavy manual veriWcation till today (Hauser et al 2007). …”

“…The Wrst way is the injection of anesthetic drugs like ketamine/rompun intraperitoneally (Hauser et al 2007;Mempel et al 2004). The advantage of this strategy is the simple application, but the disadvantage is the necessity for repeated applications of anesthetic to maintain a deep level of anesthesia during preparation and imaging.…”

“…Transferring a population of naive antigen-speciWc B cells that all express GFP into a host leads to completely green GC several days after the injection of the B cell-speciWc antigen which does not allow distinguishing individual migrating cells within the GC. In this case, it is necessary to "dilute" the GFPtransgenic cells up to 90% with GFP-non-transgenic counterparts (Hauser et al 2007). …”

“…For a long time, it was a matter of debate whether there was the possibility for centrocytes to become centroblasts again and go back from the light-to the dark zone in a process called recirculation or recycling (Meyer-Hermann and Maini 2005). It was only possible to address by direct imaging of the GC reaction, what is actually happening and, consequently, one of the prime targets of the Wrst set of three imaging studies on the germinal center response was the analysis of just this parameter (Allen et al 2007b;Hauser et al 2007;Schwickert et al 2007). However, this example also demonstrates the diYculties in coming to generally accepted conclusions.…”