The transcription factor Bcl6 is essential for the development of germinal center (GC) B cells and follicular helper T (Tfh) cells. However, little is known about in vivo dynamics of Bcl6 protein expression during and after development of these cells. By using a Bcl6 reporter mouse strain, we found that antigen-engaged B cells upregulated Bcl6 before clustering in GCs. Two-photon microscopic analysis indicated that Bcl6 upregulation in pre-GC B cells contributed to sustaining their interactions with helper T cells and was required for their entry to GC clusters. Our data also suggested that Tfh cells gradually downmodulated Bcl6 protein over weeks after development. The Bcl6-low Tfh cells rapidly terminated proliferation and upregulated IL-7 receptor. These results clarify the role of Bcl6 in pre-GC B cell dynamics and highlight the modulation of Bcl6 expression in Tfh cells that persist in the late phase of the antibody response.

Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response 1,2 . Rab5 is known as a key regulator of the early endocytic pathway 3 and we have recently shown that Rab5 is also implicated in apoptotic cell engulfment 4 ; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominantnegative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.To make Rab5 activity visible in living cells, we developed a genetically encoded fluorescence resonance energy transfer (FRET) probe, designated Raichu-Rab5. The probe comprised a modified yellow fluorescent protein (YFP) called Venus, the amino-terminal Rab5-binding domain of EEA1, a modified cyan fluorescent protein (CFP) called SECFP, and Rab5 (Fig. 1a). In this probe design, an increase in Rab5-GTP results in an increase in FRET, which can be represented by the 525 nm/475 nm emission ratio. Characterization of Raichu-Rab5 was conducted similarly to that of other Raichu probes reported previously 5,6 . In comparison with the wild-type Rab5 probe, Raichu-Rab5-Q79L-which lacks GTPase activity-had an increased FRET efficiency, whereas Raichu-Rab5-S34N-which shows a reduced affinity for guanine nucleotides-had a decreased FRET efficiency, as expected ( Supplementary Fig. 1a). The GTP loading of the Rab5 probes correlated well with that of the authentic Rab5 proteins ( Supplementary Fig. 1b), and the GTP loadings obtained here were similar to those reported previously 7 . Next, we examined the sensitivity of Raichu-Rab5 to guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) (Supplementary Fig. 1c). Rab5 GEFs such as Rabex-5, Rin1 and Gapex-5 induced a dose-dependent increase in the FRET efficiency of Raichu-Rab5. In contrast, the expression of Rab5 GAPs such as RabGAP-5 and RN-tre decreased the FRET efficiency in a dose-dependent manner. These results indicate that Raichu-Rab5 is capable of monitoring the balance between GEF and GA...

Dendritic cells (DCs) are antigen-presenting cells specialized for activating T cells to elicit effector T-cell functions. Cross-presenting DCs are a DC subset capable of presenting antigens to CD8+ T cells and play critical roles in cytotoxic T-cell-mediated immune responses to microorganisms and cancer. Although their importance is known, the spatiotemporal dynamics of cross-presenting DCs in vivo are incompletely understood. Here, we study the T-cell zone in skin-draining lymph nodes (SDLNs) and find it is compartmentalized into regions for CD8 + T-cell activation by cross-presenting DCs that express the chemokine (C motif) receptor 1 gene, Xcr1 and for CD4 + T-cell activation by CD11b + DCs. Xcr1-expressing DCs in the SDLNs are composed of two different populations: migratory (CD103 hi ) DCs, which immigrate from the skin, and resident (CD8α hi ) DCs, which develop in the nodes. To characterize the dynamic interactions of these distinct DC populations with CD8 + T cells during their activation in vivo, we developed a photoconvertible reporter mouse strain, which permits us to distinctively visualize the migratory and resident subsets of Xcr1-expressing DCs. After leaving the skin, migratory DCs infiltrated to the deep T-cell zone of the SDLNs over 3 d, which corresponded to their half-life in the SDLNs. Intravital two-photon imaging showed that after soluble antigen immunization, the newly arriving migratory DCs more efficiently form sustained conjugates with antigen-specific CD8 + T cells than other Xcr1-expressing DCs in the SDLNs. These results offer in vivo evidence for differential contributions of migratory and resident cross-presenting DCs to CD8 + T-cell activation.dendritic cell | CD8 + T cell | cross-presentation | intravital two-photon imaging | photoconversion