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Durable serological memory following vaccination is critically dependent on the production and survival of long-lived plasma cells (LLPCs). Yet, the factors that control LLPC specification and survival remain poorly resolved. Using intra-vital two-photon imaging, we find that in contrast to most plasma cells in the bone marrow, LLPCs are uniquely sessile and organized into clusters that are dependent on April, an important survival factor. Using deep, bulk RNA sequencing, and surface protein flow-based phenotyping, we find that LLPCs express a unique transcriptome and proteome compared to bulk PCs, fine tuning expression of key cell surface molecules, CD93, CD81, CXCR4, CD326, CD44 and CD48, important for adhesion and homing, and phenotypically label LLPCs within mature PC pool. Conditional deletion ofCxcr4in PCs following immunization leads to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and ultimately accelerated decay of antibody titer. In naïve mice, the endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting LLPC specification is non-random. As mice age, the BM PC compartment becomes enriched in LLPCs, which may outcompete and limit entry of new PC into the LLPC niche and pool.HIGHLIGHTS•LLPCs have reduced motility and increased clustering in the BM•LLPCs accumulate in the BM PC pool, with mouse age•LLPCs have unique surfaceome, transcriptome, and BCR clonality•CXCR4 controls maintenance of PCs and antibody titers

Plasmacytoid dendritic cells (pDCs) are the most potent producer of type I interferon (IFN), but how pDC is primed in vivo is poorly defined. Using a mouse model of severe malaria, we have previously established that upon priming by CD169+ macrophages (MPs), pDC initiates type I IFN-I secretion in the bone marrow (BM) of infected mice via cell-intrinsic TLR7 sensing and cell-extrinsic STING sensing. Herein we show that CD169+ MP and TLR7 sensing are both required for pDC arrest during priming, suggesting CD169+ MP are the source of TLR7 ligands. We establish that TLR7 sensing in pDC and chemotaxis are both required for pDC arrest and functional communication with CD169+ MP in the BM. Lastly, we demonstrate that STING sensing in CD169+ MP control pDC initiation of type I IFN production while also regulating pDC clustering and retention/egress from the BM. Collectively, these results link pDC acquisition of type I IFN-secreting capacity with changes in their motility, homing and interactions with CD169+ MP during infection. Thus, targeting this cellular interaction may help modulate type I IFN to improve outcomes of microbial infections and autoimmune diseases.

Plasmacytoid dendritic cells (pDC) are the most potent producer of type I interferon (IFN), but how pDC are primed in vivo is poorly defined. Using a mouse model of severe malaria, we have previously established that upon priming by CD169+ macrophages (MP), pDC initiate type I IFN-I secretion in the bone marrow (BM) of infected mice via cell-intrinsic TLR7 sensing and cell-extrinsic STING sensing. Herein we show that CD169+ MP and TLR7-sensing are both required for pDC arrest during priming, suggesting CD169+ MP are the source of TLR7 ligands. We establish that TLR7 sensing in pDC and chemotaxis are both required for pDC arrest and functional clustering with CD169+ MP in the BM. Lastly, we demonstrate that STING-sensing in CD169+ MP control pDC initiation of type I IFN production while also regulating pDC clustering and egress from the BM. Collectively, these results link pDC acquisition of type I IFN secreting capacity with changes in their motility, homing and interactions with CD169+ MP during infection. Thus, targeting this cellular interaction may help modulate type I IFN to improve outcomes of microbial infections and autoimmune diseases.

SUMMARY Using intravital imaging, we report that bone marrow (BM) plasma cells (PCs) are motile. BM PCs exhibit a unique migration pattern, characterized by intermittent periods of high motility and longer stretches of confined migration or arrest. BM PCs accumulate into clusters, which have reduced cell motility. APRIL promotes cluster formation and overall PC motility in the BM. Although CXCL12 and its receptor, CXCR4, promote PC motility in the BM, VLA4 activity promotes arrest. However, blocking either pathway promotes PC egress from the BM. Under steady-state conditions, BM PCs recirculate to other bones and spleen. In older mice, overall PC motility and recirculation increase, and this is correlated with increased CXCR4 expression, which depends on PC age or maturation rather than mouse age. Altogether, these results suggest that changes in PC motility and CXCR4 expression are linked with survival of long-lived PCs in the BM., Graphical Abstract, In Brief Benet et al. report that bone marrow (BM) plasma cells (PCs) are motile and found in clusters organized by the survival factor APRIL. They identify CXCR4 and VLA4 as key factors regulating PC motility and retention in the BM. PCs recirculate to other bones. PC motility increases with age.

The proline iminopeptidase (PchPiPA) of Phanerochaete chrysosporium catalyze specifically hydrolysis of N-terminal proline from peptides. The substrate Pro-pNA was docked into the catalytic pocket and several amino acid residues were identified to interact or associate with the substrate. Eight residues were selected for site-directed mutagenesis. The wild-type and mutant proteins were expressed in Escherichia coli and purified. Kinetic parameters were calculated by hydrolyzing Pro-pNA for these enzymes. Substitution of two Glu residues (Glu198 and Glu227) which interact with the substrate via formation of hydrogen bond, led to deleterious effect on catalytic efficiency (k(cat)/K(m)) due to decrease of k(cat) and increase of K(m). Four Phe residues consisting of catalytic pocket and surrounding the docked substrate, were substituted with Ala, resulting in decrease in k(cat)/K(m) to various extents. Substitution of two residues (Val267 and Cys267) localized at the deep end of the catalytic pocket also yielded negative influence on the substrate hydrolysis. Besides, all the mutants except E227Q exhibited lower thermostability than the wild-type did, indicating that these mutations may modulate the local structure. In conclusion, these amino acid residues may play an important role in maintaining local environment of the impacted catalytic pocket and be involved in recognizing or positioning the substrate.

Germinal center (GC) selection is critical for generating affinity-matured antibodies in T-cell-dependent immune responses. Despite the essential help follicular T cells provide to GC B cells, it is unclear how GC B cell fate decisions are made. Although cognate peptide-MHCII (pMHCII) is critical for T cell help, how pMHCII density directly contributes to B cell selection and cell fate remain unclear. The a-DEC205-OVA (dec) model system has been widely used to interrogate GC B selection, by enhancing selection of WT (Ly75+/+) B cells over untargeted Ly75−/− (KO) B cells. However, under these conditions, it is unclear what the fate of B cells presenting intermediate levels of pMHCII is and whether T cell help is indeed limited. To investigate these questions, we used Ly75+/− (DEC-het) B cells, which expressed intermediate levels (~50%) of surface DEC205 protein compared to WT B cells, and presented proportional amount of OVA peptide after dec treatment. Using competitive transfers, we found that WT B cells expanded two-fold more than DEC-het B cells. This 2-fold difference in expansion was maintained at a wide range of dec administration, indicating that in vivo, T cells can distinguish different densities of pMHCII on multiple B cells in the same GC and respond proportionally. Surprisingly, we found that upon dec stimulation, both WT and DEC-het GC B cells were equally capable of differentiating into plasma cells, suggesting that pMHCII density does not control B cell fate directly. Blocking of CD40 signaling removed the two-fold advantage of WT over DEC-het B cells during selection after dec treatment, suggesting CD40 signaling is proportional to pMHCII density. These results provide new interpretations of how GC selection occurs in vivo.