Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC, and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza.

SUMMARY During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell cycle progression itself, and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection.

T follicular helper cells (TFH) are a specialized subset of effector T cells that provide help to and thereby select high-affinity B cells in germinal centers (GCs). To examine the dynamic behavior of TFH cells in GCs in mice we combined two-photon microscopy and optical highlighting using a photoactivatable fluorescent reporter. Unlike GC B cells, which are clonally restricted, TFH distributed among all GCs in lymph nodes and continually emigrated into the follicle and neighboring GCs. Moreover, newly activated TFH cells invaded pre-existing GCs, where they contributed to B cell selection and plasmablast differentiation. Our data suggest that dynamic exchange of TFH between GCs ensures maximal diversification of T cell help and their ability to enter ongoing GCs accommodates antigenic variation during the immune response.

Interaction between different cell types is essential for multiple biological processes, including immunity, embryonic development, and neuronal signaling. While the dynamics of cell-cell interactions can be monitored in vivo by intravital microscopy19, this approach provides no information on the receptors and ligands involved, and does not allow isolation of interacting cells for downstream analysis. Here, we present a complementary approach that uses bacterial sortase labeling across immune synapses to identify receptor-ligand interactions between cells within living animals, generating a signal that can be readily detected by flow cytometry. We call this approach to labeling “kiss-and-run” interactions between immune cells Labeling Immune Partnerships by SorTagging Intercellular Contacts (LIPSTIC). Using LIPSTIC, we show that interactions between dendritic cells (DCs) and CD4+ T cells during T cell priming in vivo occur in two distinct modalities: an early, cognate stage when CD40-CD40L interactions occur specifically between T cells and antigen-loaded DCs, and a later, non-cognate stage when these interactions no longer require T cell receptor (TCR) engagement. Thus, LIPSTIC allows direct measurement of dynamic cell-cell interactions both in vitro and in vivo. Given its flexibility for use with different receptor ligand pairs and a range of detectable labels, we expect that this approach will be of use to any field of biology interested in quantifying intercellular communication.

The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

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A crucial step in the arenavirus life cycle is the biosynthesis of the viral envelope glycoprotein (GP) responsible for virus attachment and entry. Processing of the GP precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexin-isozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infection and production of infectious virus. Here, we sought to evaluate arenavirus GPC processing by S1P as a target for antiviral therapy using a recently developed peptide-based S1P inhibitor, decanoyl (dec)-RRLL-chloromethylketone (CMK), and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). To control for off-target effects of dec-RRLL-CMK, we employed arenavirus reverse genetics to introduce a furin recognition site into the GPC of LCMV. The rescued mutant virus grew to normal titers, and the processing of its GPC critically depended on cellular furin, but not S1P.

A crucial step in the life cycle of arenaviruses is the biosynthesis of the mature fusion-active viral envelope glycoprotein (GP) that is essential for virus-host cell attachment and entry. The maturation of the arenavirus GP precursor (GPC) critically depends on proteolytic processing by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we undertook a molecular characterization of the SKI-1/S1P processing of the GPCs of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the pathogenic Lassa virus (LASV). Previous studies showed that the GPC of LASV undergoes processing in the endoplasmic reticulum (ER)/cis-Golgi compartment, whereas the LCMV GPC is cleaved in a late Golgi compartment. Herein we confirm these findings and provide evidence that the SKI-1/S1P recognition site RRLL, present in the SKI-1/S1P prodomain and LASV GPC, but not in the LCMV GPC, is crucial for the processing of the LASV GPC in the ER/cisGolgi compartment. Our structure-function analysis revealed that the cleavage of arenavirus GPCs, but not cellular substrates, critically depends on the autoprocessing of SKI-1/S1P, suggesting differences in the processing of cellular and viral substrates. Deletion mutagenesis showed that the transmembrane and intracellular domains of SKI-1/S1P are dispensable for arenavirus GPC processing. The expression of a soluble form of the protease in SKI-I/S1P-deficient cells resulted in the efficient processing of arenavirus GPCs and rescued productive virus infection. However, exogenous soluble SKI-1/S1P was unable to process LCMV and LASV GPCs displayed at the surface of SKI-I/S1P-deficient cells, indicating that GPC processing occurs in an intracellular compartment. In sum, our study reveals important differences in the SKI-1/S1P processing of viral and cellular substrates.