Abstract
Understanding cellular processes occurring in vivo on time scales of days to weeks requires repeatedly interrogating the same tissue without perturbing homeostasis. We describe a novel setup for longitudinal intravital imaging of murine peripheral lymph nodes (LNs). The formation and evolution of single germinal centers (GCs) was visualized over days to weeks. Naïve B cells encounter antigen and form primary foci, which subsequently seed GCs. These experience widely varying rates of homogenizing selection, even within closely confined spatial proximity. The fluidity of GCs is greater than previously observed with large shifts in clonality over short time scales; and loss of GCs is a rare, observable event. The observation of contemporaneous, congruent shifts in clonal composition between GCs within the same animal suggests inter-GC trafficking of memory B cells. This tool refines approaches to resolving immune dynamics in peripheral LNs with high temporospatial resolution and minimal perturbation of homeostasis.
Highlights
The advent of intravital imaging techniques allowing sequential, continuous imaging of tissues in the live organism for up to 8–12 hr has significantly advanced several fields over the past decade, not least within immunology (Allen et al, 2007; Miller et al, 2002; Roozendaal et al, 2009; Schwickert et al, 2007)
To allow longitudinal studies of cellular dynamics in the lymph nodes (LNs), we adopted a modification of the chronic mammary fat pad window model referred to as the chronic lymph node window (Jeong et al, 2015)
Window chambers were surgically implanted over the inguinal LN (iLN), and a small incision made in the skin to expose the underlying node (Figure 1)
Summary
The advent of intravital imaging techniques allowing sequential, continuous imaging of tissues in the live organism for up to 8–12 hr has significantly advanced several fields over the past decade, not least within immunology (Allen et al, 2007; Miller et al, 2002; Roozendaal et al, 2009; Schwickert et al, 2007). A significant gap has remained between the capability for continuous imaging of individual animals on time scales of less than half a day, and the statistically based analysis of timed cohorts of experimental animals on longer time scales This gap has narrowed in the field of neurobiology by the advent of techniques such as the cranial (Holtmaat et al, 2009) and the thinned skull (Yang et al, 2010) windows, and similar methodologies have been harnessed for long-term imaging of tumors, such as the mammary window (Kedrin et al, 2008), and the abdominal imaging window (Ritsma et al, 2013). Cognate T cells recognize antigen-derived peptides presented by DCs and they take on the characteristics of T follicular helper cells (Tfh), allowing them to provide a necessary second signal to begin an adaptive immune response (Barnett et al, 2014). We measured the dynamics of B cell clonality at the single GC level over time
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