B cells within germinal centers migrate preferentially from dark to light zone

Abstract

One of the main questions in the field of imaging immune cell migration in living tissues is whether cells fulfill their functionality via random or nonrandom migration processes. For some applications, this issue has remained controversial even after publication of various imaging studies. A prime example is B-cell migration in germinal centers (GCs) where somatic hypermutation and clonal selection of B cells are thought to occur within morphologically distinct regions termed dark zone (DZ) and light zone (LZ). Here, we reanalyze a previously published dataset on GC B-cell migration, applying a sensitive analysis technique to detect directed migration and using a procedure to correct for a number of artifacts that frequently occur in time-lapse imaging experiments. Although B cells roughly perform a persistent random walk, we present evidence that they have a small preference (of on average about 0.2−0.3 μm min −1 ) to migrate from DZ to LZ, which is consistent with classical views of the GC reaction. This preference is most pronounced among a large subset of almost half of the B-cell population migrating along relatively straight tracks. Using a computational model to generate long-lasting B-cell tracks based on the experimental motility data (including the small directional preference), we predict a time course to travel from DZ to LZ of a few hours. This is consistent with experimental observations, and we show that at the observed cellular motility such a time course cannot be explained without the small preferential migration from DZ to LZ.B-cell motility | cyclic reentry | two-photon microscopy T hanks to the application of two-photon microscopy to living lymphoid tissues, an exciting glimpse of how the migration of various types of immune cells takes place was recently obtained (1-5). One of the central questions in this field is whether migration is best described as a (persistent) random walk or whether directed or confined migration is involved, because this is important for our understanding of how cells manage to carry out their functions. Examples of persistent random walk include the migration of T cells, B cells, and plasma cells in lymph nodes (2, 6-8) and of effector T cells migrating in tumors (9), whereas nonrandom migration has for instance been discovered in antigen-engaged B cells moving toward the T zone boundary early in a B-cell response (10), CD8 + T cells being attracted toward "licensed" dendritic cells (11)(12)(13)(14), neutrophil movement in skin (15, 16), and during thymocyte maturation (17,18).In some applications, it is controversial whether migration is best described as random or not, and a prime example is B-cell migration in germinal centers (GCs). At these sites, B lymphocytes mature by somatic hypermutation and clonal selection of the mutants that produce antibody of high affinity. These two processes are thought to occur in morphologically distinct regions termed dark zone (DZ) and light zone (LZ), but in the literature competing views exist on the functional meaning of D...

Defining the quantitative limits of intravital two-photon lymphocyte tracking

“…Thus, we next investigated how the sensitivity of this method compares with that of more established ones using computer generated cell tracks with and without taxis. We limit our discussion here to two important alternative methods: displacement analysis, which is widely used (14,15,18), and angle analysis, because it has recently received much attention (13,15). For displacement analysis, we generated mean square displacement (MSD) plots, which are expected to be linear for random migration and curve up in case of directed migration (14).…”

“…Without knowing the quantitative limits of two-photon cell tracking with regard to taxis detection, this question is hard to answer, and doors remain wide open for speculation. For example, although the migration of B cells in the germinal center was first described as random (10), it was then argued that two-photon data are consistent with both random and directed migration (11,12), and very recently, a small directional bias to the light zone was revealed (13). In light of such controversy, we ask here the following two questions.…”

Subtle CXCR3-Dependent Chemotaxis of CTLs within Infected Tissue Allows Efficient Target Localization

“…4, lower panels), thereby explaining why directional migration is not apparent from visual inspection of in vivo imaging data (Supplemental Videos 1-3). To determine to what extent T cell arrival at sites of infection is influenced by this small directional preference, we generated realistic, long-lasting tracks based on experimental data, using a computational approach similar to that in previously published work (10,25,26). We reconstructed cellular migration paths using the experimentally measured distributions of speed, angle to infection, and turning angle.…”

“…A large number of studies have described the migration patterns of T and B lymphocytes within lymph nodes (reviewed in Ref. 9), and evidence for such directed migration has been obtained in several of them (10)(11)(12)(13)(14). Within peripheral tissues (i.e., subsequent to extravasation), it was recently proposed that the observed CD8 + T cell clustering around malaria-infected hepatocytes is consistent with directed migration (15), but direct evidence for chemotaxis was not obtained.…”

The Ectopic Germinal Centre Response in Autoimmune Disease and Cancer

“…Detailed studies of the kinetics and cellular interactions within germinal centres using multiphoton microscopy of living tissue in combination with B & T-lymphocytes expressing defined antigen receptors from transgenic animals have revealed much more dynamic activity than was previously suspected. It is now recognised that there is less distinction between the dark and light zones than suggested by static immunohistological examination, and there is continual recycling of B-cells both between and within the two zones, although there is net migration from the dark zone to the light zone (Beltman et al, 2011) (Figure 1B). Centrocytes move rapidly through the network of follicular dendritic cell processes, apparently sampling the immune complexes attached to their membranes and some of these cells return to the dark zone for further rounds of proliferation and somatic hypermutation.…”