In this study, we imaged the differentiation and migratory behavior of nascent plasma cells (PCs) in mouse lymph nodes by intravital microscopy. we suggest pre-PCs undergo a persistent random walk to find the medullary cords, where localized chemokines help retain these cells until they undergo differentiation and arrest in situ. Introduction Plasma cells (PCs) play important roles in the acute response to infection and the long-term protection of the host by acting as antibody factories (Calame et al., 2003; Tarlinton et al., 2008). These terminally differentiated B cells can be divided into two subsets. Short-lived PCs are produced early in the immune responses, starting on day 3 after immunization providing a first wave of lower affinity antibodies, but die shortly thereafter (Ho et al., 1986) . Meanwhile, long-lived late PCs are produced by T cell-dependent germinal centers (GCs) that coalesce around day 6 post immunization and can continue to generate PCs for weeks. These late PCs generate higher affinity antibodies through affinity maturation (Radbruch et al., 2006). Acute ablation of the germinal center by ETV4 anti-CD40 treatment halts generation of new PCs and prevents further improvements of antibody affinity (Takahashi et al., 1998). PCs are identified by syndecan-1 surface expression, have an extensive rough endoplasmic reticulum (ER), and are enriched within the red pulp of the spleen, medullary cords of lymph nodes, and in the bone marrow (Smith et al., 1996). The precursors of PCs (pre-PCs, also called plasmablasts), are dividing cells that are found in B cell follicles in addition to red pulp and medullary cords, but not in the bone marrow. Plasma cells and pre-PC are collectively referred to as antibody secreting cells (ASCs) or antibody forming cells (AFCs) based on their ability to secrete antibody, which is often class-switched. PC differentiation is dependent on a key transcriptional repressor, Blimp-1 (Calame et al., 2003), which inhibits many B-cell lineage (locus to generate a Blimp-1 reporter mouse (Blimp-1-GFP) and showed Blimp-1 expression as an early marker of PC development (Kallies et al., 2004). Pre-PCs expressed lower levels of Blimp-1-GFP than fully differentiated PCs, and were heterogeneous for syndecan-1 expression (Kabashima 243967-42-2 et al., 2006; Kallies et al., 2004). Within secondary lymphoid organs, both short and long-lived PCs localize to medullary cords in lymph nodes and red-pulp regions of the spleen where they are thought to secrete antibody (Smith et al., 1996). Within these anatomic locations, PCs are largely sessile (Allen et al., 2007; Schwickert et al., 2007). PC migration to these regions has never been visualized directly, but chemokine receptors are thought to play a role because expression of CXCR4, CCR6 and EBI2 increases while CXCR5 is reduced during PC differentiation (Hargreaves et al., 2001; Pereira et al., 2009; Wehrli et al., 2001). In addition, transwell assays have shown chemotaxis of spleen red pulp PCs to the chemokines S1p and CXCL12, which are ligands of S1p1/S1p3 and CXCR4 receptors, respectively (Hargreaves et al., 2001; Kabashima et al., 2006). Consistent with this idea, CXCL12 is expressed in red pulp and medullary cords, and genetic ablation experiments showed that CXCR4-deficient PCs failed to accumulate in red pulp or bone marrow, but were enriched in blood and normal in lymph nodes compared to CXCR4-sufficent cells (Hargreaves et al., 2001). These results suggest a role for CXCL12 in PC homing (Hargreaves et al., 2001; Wehrli et al., 2001). Long-lived PC egress from lymph nodes and homing to the bone marrow is critical for their long-term survival. In both s1p1- and 2 integrin-deficient conditions (Kabashima et al., 2006; Pabst et al., 2005), PCs are unable to exit the lymph nodes. S1p expression is high in blood and low in secondary lymphoid organs providing a gradient that may be used for egress (Rosen and Goetzl, 2005; Schwab and Cyster, 2007). Intercellular adhesion molecule-1 (ICAM-1) is highly expressed in medullary cords, 243967-42-2 which are the exit sites 243967-42-2 of PCs from lymph nodes. From these reports and others, an image of newly-minted pre-PCs leaving the germinal center to the medullary cords along a chemokine highway has emerged. However, the current model of pre-PC chemotaxis to the medullary cords poses a few conceptual challenges. While CXCL12 is a candidate for attracting PCs to the medullary cords, it is not clear how pre-PCs would first escape from the GC, where CXCL12 is also used to partition GC B 243967-42-2 cells between the light and dark zones (Allen et al., 2004). After exit from the GC, a long and stable chemokine gradient would have to be produced to attract cells to structures.