Subcapsular Sinus Research Articles

Low Molecular Weight Cytokeratin Immunostaining for Extrafollicular Reticulum Cells is an Effective Means of Separating Salivary Gland Tumor-Associated Lymphoid Proliferation from True Lymph Node Involvement

Tumor-associated lymphoid proliferation (TALP) is a well-recognized lymphocytic reaction that is commonly associated with certain salivary gland tumors. A salivary carcinoma with TALP may be confused for true lymph node involvement by that tumor, constituting a potential pitfall in tumor staging that may result in unnecessary therapeutic intervention or erroneous prognostication for patients. True lymph nodes harbor populations of extrafollicular reticulum cells (ERCs), which can be highlighted by low molecular weight cytokeratin immunohistochemistry. We sought to determine whether low molecular weightcytokeratin Cam5.2 immunostaining may be utilized to differentiate true lymph node involvement by salivary gland tumors from TALP. The surgical pathology archives of the University of Texas Southwestern Medical Center was searched forcases of salivary gland neoplasms exhibiting either TALP or true lymph node involvement. Hematoxylin and eosin-stained sections were examined. Cases were classified on the basis of a definitive lymph node capsule and subcapsular sinus, as seen on routine histologic evaluation. Low molecular weight cytokeratin Cam5.2 immunostaining was performed and evaluated on all cases. Twenty-three salivary gland carcinomas with TALP and 16 carcinomas involving a lymph node (14 carcinomas metastatic to regional lymph nodes and 2 carcinomas arising from benign lymph node inclusions) were identified. Numerous Cam5.2-positive ERCs were identified within the nodal tissue of all true lymph nodes involved by carcinoma (16 of 16 cases), while Cam5.2-positive ERCs were completely absent in all cases of salivary gland lesions with TALP (0 of 23 cases) (100% vs. 0%, p < .0001, Fisher's Exact). Utilization of low molecular weight cytokeratin Cam 5.2 immunostaining for ERCs is a highly useful tool for distinguishing true lymph node involvement by salivary gland carcinomas from TALP. This strategy may be useful in identifying genuine nodal metastasis in histologically ambiguous cases, and to avoid erroneously upstaging tumor with TALP as nodal metastasis with the resulting prognostic and therapeutic implications. Moreover, low molecular weight cytokeratin immunostaining may be useful in confirming the rare examples of salivary gland tumors arising from intranodal salivary gland inclusions.

Single-Cell Survey of Human Lymphatics Unveils Marked Endothelial Cell Heterogeneity and Mechanisms of Homing for Neutrophils

Lymphatic vessels form a critical component in the regulation of human health and disease. While their functional significance is increasingly being recognized, the comprehensive heterogeneity of lymphatics remains uncharacterized. Here, we report the profiling of 33,000 lymphatic endothelial cells (LECs) in human lymph nodes (LNs) by single-cell RNA sequencing. Unbiased clustering revealed six major types of human LECs. LECs lining the subcapsularsinus (SCS) of LNs abundantly expressed neutrophil chemoattractants, whereas LECs lining the medullary sinus (MS) expressed a C-type lectin CD209. Binding of a carbohydrate Lewis X (CD15) to CD209 mediated neutrophil binding to the MS. The neutrophil-selective homing by MS LECs may retain neutrophils in the LN medulla and allow lymph-borne pathogens to clear, preventing their spread through LNs in humans. Our study provides a comprehensive characterization of LEC heterogeneity and unveils a previously undefined role for medullary LECs in human immunity.

Transcytosis route mediates rapid delivery of intact antibodies to draining lymph nodes.

Lymph nodes (LNs) filter lymph to mount effective immune responses. Small soluble lymph-borne molecules from the periphery enter the draining LNs via a reticular conduit system. Intact antibodies and other larger molecules, in contrast, are physically unable to enter the conduits, and they are thought to be transported to the LNs only within migratory DCs after proteolytic degradation. Here, we discovered that lymph-borne antibodies and other large biomolecules enter within seconds into the parenchyma of the draining LN in an intact form. Mechanistically, we found that the uptake of large molecules is a receptor-independent, fluid-phase process that takes place by dynamin-dependent vesicular transcytosis through the lymphatic endothelial cells in the subcapsular sinus of the LN. Physiologically, this pathway mediates a very fast transfer of large protein antigens from the periphery to LN-resident DCs and macrophages. We show that exploitation of the transcytosis system allows enhanced whole-organ imaging and spatially controlled lymphocyte activation by s.c. administered antibodies in vivo. Transcytosis through the floor of the subcapsular sinus thus represents what we believe to be a new physiological and targetable mode of lymph filtering.

Macrophage-B Cell Interactions in the Inverted Porcine Lymph Node and Their Response to Porcine Reproductive and Respiratory Syndrome Virus.

Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the center to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros, and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN MΦ to their murine counterparts: subcapsular sinus MΦ, medullary cord MΦ and medullary sinus MΦ. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of MΦ, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (MΦ). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN MΦ populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning.

Lymphatic Endothelial Cells Are Essential Components of the Subcapsular Sinus Macrophage Niche

In lymph nodes, subcapsular sinus macrophages (SSMs) form an immunological barrier that monitors lymph drained from peripheral tissues. Upon infection, SSMs activate B and natural killer T(NKT) cells while secreting inflammatory mediators. Here, we investigated the mechanisms regulating development and homeostasis of SSMs. Embryonic SSMs originated from yolk sac hematopoiesis and were replaced by a postnatal wave of bone marrow (BM)-derived monocytes that proliferated to establish the adult SSM network. The SSM network self-maintained by proliferation with minimal BM contribution. Upon pathogen-induced transient deletion, BM-derived cells contributed to restoring the SSM network. Lymphatic endothelial cells (LECs) were the main source of CSF-1 within the lymph node and conditional deletion of Csf1 in adult LECs decreased the network of SSMs and medullary sinus macrophages (MSMs). Thus, SSMs have a dual hematopoietic origin, and LECs are essential to the niche supporting these macrophages.

TLR7 Controls VSV Replication in CD169+ SCS Macrophages and Associated Viral Neuroinvasion.

Vesicular stomatitis virus (VSV) is an insect-transmitted rhabdovirus that is neurovirulent in mice. Upon peripheral VSV infection, CD169+ subcapsular sinus (SCS) macrophages capture VSV in the lymph, support viral replication, and prevent CNS neuroinvasion. To date, the precise mechanisms controlling VSV infection in SCS macrophages remain incompletely understood. Here, we show that Toll-like receptor-7 (TLR7), the main sensing receptor for VSV, is central in controlling lymph-borne VSV infection. Following VSV skin infection, TLR7−/− mice display significantly less VSV titers in the draining lymph nodes (dLN) and viral replication is attenuated in SCS macrophages. In contrast to effects of TLR7 in impeding VSV replication in the dLN, TLR7−/− mice present elevated viral load in the brain and spinal cord highlighting their susceptibility to VSV neuroinvasion. By generating novel TLR7 floxed mice, we interrogate the impact of cell-specific TLR7 function in anti-viral immunity after VSV skin infection. Our data suggests that TLR7 signaling in SCS macrophages supports VSV replication in these cells, increasing LN infection and may account for the delayed onset of VSV-induced neurovirulence observed in TLR7−/− mice. Overall, we identify TLR7 as a novel and essential host factor that critically controls anti-viral immunity to VSV. Furthermore, the novel mouse model generated in our study will be of valuable importance to shed light on cell-intrinsic TLR7 biology in future studies.

Lymph Node Subcapsular Sinus Macrophages as the Frontline of Lymphatic Immune Defense.

Lymphatic vessels collect and transport lymph and pathogens to the draining lymph node (LN) to generate proper immune protection. A layer of macrophages that strategically line the LN subcapsular sinus (SCS) is directly exposed to the afferent lymph and are denoted as SCS macrophages. These macrophages are the frontline of immune defense that interact with lymph-borne antigens. The importance of these macrophages in limiting the spread of pathogens has been demonstrated in both viral and bacterial infection. In anti-microbial responses, these macrophages can directly or indirectly activate other LN innate immune cells to fight against pathogens, as well as activate T cells or B cells for adaptive immunity. As the first layer of immune cells embracing the tumor-derived antigens, SCS macrophages also actively participate in cancer immune regulation. Recent studies have shown that the LNs' SCS macrophage layer is interrupted in disease models. Despite their importance in fighting the spread of pathogens and in activating anti-tumor immunity, the mechanism and the immunological functional consequences for their disruption are not well-understood. Understanding the mechanism of these macrophages will enhance their capability for therapeutic targeting.

Loss of CD169+ Subcapsular Macrophages during Metastatic Spread of Head and Neck Squamous Cell Carcinoma.

The purpose of this study is to assess CD169 expression in metastatic and nearby tumor-free lymph nodes of patients with head and neck squamous cell carcinoma (SCC). Retrospective analysis based on immunohistochemistry. Tertiary care center. The abundance of CD169+ cells in the subcapsular sinuses (SCSs) of lymph nodes was assessed immunohistochemically in paraffin-embedded tissue samples derived from 22 patients with oral cavity and oropharyngeal SCC. SCSs of lymph nodes harboring metastatic SCC contained significantly fewer CD169+ macrophages (106.5 ± 113.6 cells/mm2) compared to nearby tumor-free lymph nodes (321.3 ± 173.4 cells/mm2, P < .001). This observation extended to 21 of the 22 cases investigated. In addition, 6 patients who later developed recurrent disease contained lower numbers of CD169+ cells (268.6 ± 169.5 cells/mm2) in nearby tumor-free lymph nodes compared to 341.0 ± 176.1 cells/mm2 in those who remained disease free (P = .399). Human papillomavirus (HPV)-positive patients (n = 4) had a 6-fold lower number of CD169+ cells in metastatic nodes (61.2 ± 85.5 cells/mm2) compared to nearby tumor-free lymph nodes (369.5 ± 175.5 cells/mm2, P = .028). In comparison, HPV-negative patients had only a 3-fold reduction (116.6 ± 118.5 cells/mm2 vs 310.6 ± 176.2 cells/mm2, P < .001). Metastatic spread of SCC to regional lymph nodes is associated with lower abundance of CD169+ macrophages in the SCSs of draining lymph nodes. These results set the stage for an in-depth investigation into the mechanism(s) by which metastatic SCC controls CD169+ macrophage abundance and its significance as it relates to prognosis and treatment response.

Enzyme Histochemistry of Hemal Nodes of Indian Buffalo (Bubalus bubalis)

The present study was conducted on hemal nodes of buffalo to study the histoenzymic distribution of phosphatases and oxidoreductases. The fresh unfixed buffalo hemal nodes were immediately collected after sacrifice and were subjected to cryostat sectioning at -200 C with cryostat microtome. The sections were incubated with substrates for demonstration of various enzymes. The study revealed that in both the groups, weak alkaline phosphatase (AKPase) activity was observed in capsule of the hemal node. The AKPase activity was moderate in subcapsular sinus, periphery of lymphoid follicles and diffused lymphocytes. Uniform weak Glucose 6-phosphate activity was observed in entire hemal node. Succinic dehydrogenase (SDH) activity was moderate in capsule and lymphoid follicles and weak in subcapsular sinuses. Lactic dehydrogenase activity was moderate to strong in capsule, sub-capsular sinus and peripheral area of lymphoid follicles. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-D) and reduced Nicotinamide adenine dinucleotide diaphorase (NADH-D) was weak to moderate in the capsule and moderate to strong in lymphoid follicles. The presence of variable activity of different enzymes in hemal nodes was correlated with maturation of lymphocytes and development of different metabolic pathways.

A Protective Role for the Lectin CD169/Siglec-1 against a Pathogenic Murine Retrovirus

SummaryLymph- and blood-borne retroviruses exploit CD169/Siglec-1-mediated capture by subcapsular sinus and marginal zone metallophilic macrophages for trans-infection of permissive lymphocytes. However, the impact of CD169-mediated virus capture on retrovirus dissemination and pathogenesis in vivo is unknown. In a murine model of the splenomegaly-inducing retrovirus Friend virus complex (FVC) infection, we find that while CD169 promoted draining lymph node infection, it limited systemic spread to the spleen. At the spleen, CD169-expressing macrophages captured incoming blood-borne retroviruses and limited their spread to the erythroblasts in the red pulp where FVC manifests its pathogenesis. CD169-mediated retroviral capture activated conventional dendritic cells 1 (cDC1s) and promoted cytotoxic CD8+ T cell responses, resulting in efficient clearing of FVC-infected cells. Accordingly, CD169 blockade led to higher viral loads and accelerated death in susceptible mouse strains. Thus, CD169 plays a protective role during FVC pathogenesis by reducing viral dissemination to erythroblasts and eliciting an effective cytotoxic T lymphocyte response via cDC1s.

Lymph node mass transport

Immunology Cytokines are transported around the body via a network of lymph ducts and lymph nodes. Conduits within lymph nodes convey cytokines from the subcapsular sinus (SCS) into the deep parenchyma. From there, they discharge into high endothelial venules. In the SCS, sinus-lining cells prevent molecules greater than 70 kilodaltons from entering conduits. Nevertheless, Thierry et al. found that the massive 970-kilodalton pentamers of immunoglobulin M antibodies can cross into conduits. Transport is mediated by transient, activated, antigen-specific B cells, thus enabling rapid mobilization of the first wave of antibodies produced during an acute infection. J. Exp. Med. 215 , 2972 (2018).

Expression profiles of interferon-stimulated gene 15 and prostaglandin synthases in the ovine lymph nodes during early pregnancy.

Lymph nodes are distributed all over the body and are part of the lymphatic system. The interferon-stimulated gene 15 kDa protein (ISG15) and prostaglandins (PGs) are involved in the establishment of pregnancy and are expressed in the uterus during early pregnancy in sheep. In this study, the ovine lymph nodes were obtained on Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX-1), COX-2, prostaglandin E (PGE) synthase (PTGES), and a PGF synthase (aldo-keto reductase family 1, member B1, AKR1B1) were detected by quantitative real-time polymerase chain reaction, western blot analysis, and immunohistochemistry analysis. Our results showed that there were peaks in the expression of mRNAs and the proteins of ISG15, COX-1, COX-2, PTGES, and AKR1B1 in the lymph nodes during early pregnancy and that the COX-2 and AKR1B1 proteins were limited to the subcapsular sinus and lymph sinuses. In conclusion, the ISG15, COX-1, COX-2, PTGES, and AKR1B1 were upregulated in the maternal lymph nodes, which may be beneficial for the development of conceptus, maternal systemic immunoregulation, and anti-luteolysis during early pregnancy in sheep.

A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo.

Dendritic cell activation of CD4 T cells in the lymph node draining a site of infection or vaccination is widely considered the central event in initiating adaptive immunity. The accepted dogma is that this occurs by stimulating local activation and antigen acquisition by dendritic cells, with subsequent lymph node migration, however the generalizability of this mechanism is unclear. Here we show that in some circumstances antigen can bypass the injection site inflammatory response, draining freely and rapidly to the lymph nodes where it interacts with subcapsular sinus (SCS) macrophages resulting in their death. Debris from these dying SCS macrophages is internalized by monocytes recruited from the circulation. This coordinated response leads to antigen presentation by monocytes and interactions with naïve CD4 T cells that can drive the initiation of T cell and B cell responses. These studies demonstrate an entirely novel pathway leading to initiation of adaptive immune responses in vivo.

Multiple roles of lymphatic vessels in peripheral lymph node development.

The mammalian lymphatic system consists of strategically located lymph nodes (LNs) embedded into a lymphatic vascular network. Mechanisms underlying development of this highly organized system are not fully understood. Using high-resolution imaging, we show that lymphoid tissue inducer (LTi) cells initially transmigrate from veins at LN development sites using gaps in venous mural coverage. This process is independent of lymphatic vasculature, but lymphatic vessels are indispensable for the transport of LTi cells that egress from blood capillaries elsewhere and serve as an essential LN expansion reservoir. At later stages, lymphatic collecting vessels ensure efficient LTi cell transport and formation of the LN capsule and subcapsular sinus. Perinodal lymphatics also promote local interstitial flow, which cooperates with lymphotoxin-β signaling to amplify stromal CXCL13 production and thereby promote LTi cell retention. Our data unify previous models of LN development by showing that lymphatics intervene at multiple points to assist LN expansion and identify a new role for mechanical forces in LN development.

Comparison of Th1 and Th2 cytokines production in ovine lymph nodes during early pregnancy

As a fetal allograft to the mother, early conceptus regulates the intrauterine immune and systemic immune responses during early pregnancy in sheep. However, expression of T helper 1 (Th1) and Th2 cytokines in maternal lymph nodes is unclear during early pregnancy in sheep. In this study, inguinal lymph nodes were obtained on day 16 of the estrous cycle and on days 13, 16 and 25 of pregnancy (n = 4 for each group) in ewes, and qRT-PCR, western blot and immunohistochemistry were used to compare the expression of Th1 and Th2 cytokines in the lymph nodes. Our results showed that there were the highest levels of Th1 cytokines (IFN-γ, TNF-β and IL-2) and Th2 cytokines (IL-4, IL-5, IL-6 and IL-10) in the lymph nodes on day 13 or 16 of pregnancy. Furthermore, there were a downregulation of TNF-β and IL-2 and an upregulation of IL-5 and IL-10 on day 25 of pregnancy compared with that in nonpregnancy, with no significant difference in the expression of IFN-γ, IL-4, and IL-6 between the ewes on day 25 of pregnancy and nonpregnancy. The immunohistochemistry results showed that the IL-2 and IL-10 proteins were limited to the subcapsular sinus and trabeculae in the cortex, lymph sinus. In conclusion, early pregnancy exerted its effects on the lymph node and induced a Th2-biased response, which was essential for a normal pregnancy in sheep.

The localization of CD3, CD79a, CD68 and S100 protein immunoreactive cells in hemal nodes of Saanen goat (Capra hircus)

We investigated the structure of hemal nodes in Saanen goats using immunohistochemical staining. We examined the distribution of CD3 positive T lymphocytes, CD79a positive B lymphocytes, CD68 positive macrophages and S100 protein positive follicular dendritic cells. Hemal nodes of six healty adult female goats were used. Hemal nodes were removed from the thoracic and abdominal cavities. The oval to round hemal nodes were observed especially between the abdominal aorta and vena cava, and near the kidneys and adrenal glands. Tissue sections were stained with Crossmon’s modified triple stain to demonstrate general histological structure. The avidin-biotin-peroxidase technique using anti-CD3, anti-CD79a, anti-CD68 and anti-S100 primary antibodies was used for immunohistochemistry. Many CD3 positive T lymphocytes were found in the germinal center of the lymph follicles and in the lymphatic cords of hemal nodes; CD3 positive cells also were observed in the sinuses. CD79a and CD68 positive cells were found at the germinal center of the lymph follicles. In the lymph follicles near the subcapsular sinuses, CD79a and CD68 positive cells were found especially in e areas bordering the mantle zone. S100 positive cells were found in the lymph follicles, lymphatic cords and sinuses.

Deep inferior epigastric lymph node basin: Analysis of novel donor site for vascularized lymph node transfer among 10 consecutive patients.

Breast cancer-related extremity lymphedema is a potentially devastating condition. Vascularized lymph node transfer (VLNT) has shown benefit in lymphedema treatment. Due to concerns over potential iatrogenic complications, various donor sites have been described. The current study aims at defining the deep inferior epigastric lymph node basin as a novel donor site for VLNT. A retrospective study was performed on patients undergoing routine abdominal-based breast reconstruction. Resection of all perivascular adipose and lymphatic tissue surrounding the proximal deep inferior epigastric pedicle was performed at the time of pedicle dissection and submitted for Pathologic evaluation. Patient demographics and pertinent medical/surgical history was obtained from medical records. Specimens were obtained from 10 consecutive patients. Seven patients underwent bilateral reconstruction for a total of 17 specimens obtained. Mean patient age and BMI were 48 years ± 9.4 and 27 ± 4.2, respectively. Fourteen out of 17 (82%) specimens contained viable lymph nodes displaying a thin fibrous connective tissue capsule overlying an unremarkable subcapsular sinus with a cortex and paracortex containing germinal centers composed of B lymphocytes, tangible body macrophages, and T-cells. The medullary sinus space displayed a fatty unremarkable hilum. The mean number and size of lymph nodes were 2.6 ±1.2 nodes/specimen and 3.67 mm ± 2.3, respectively. All patients experienced an uneventful postoperative course without evidence any of compromised flap viability. Lacking previous description, the deep inferior epigastric lymph node basin is a readily accessible donor site with significant anatomic advantages for potential VLNT during autologous breast reconstruction.

The Chemokine Receptor CCR8 Promotes the Migration of Dendritic Cells into the Lymph Node Parenchyma to Initiate the Allergic Immune Response

The migration of mature dendritic cells (DCs) into thedraining lymph node (dLN) is thought to depend solely on the chemokine receptor CCR7. CD301b+ DCs migrate into the dLN after cutaneous allergen exposure and are required for T helper 2 (Th2) differentiation. We found that CD301b+ DCs poorly upregulated CCR7 expression after allergen exposure and required a second chemokine signal, mediated by CCR8 on CD301b+ DCs and its ligand CCL8, toexit the subcapsular sinus (SCS) and enter the lymph node (LN) parenchyma. After allergen exposure, CD169+SIGN-R1+ macrophages in interfollicular regions produced CCL8, which synergized with CCL21 in a Src-kinase-dependent manner to promote CD301b+ DC migration. In CCR8-deficient mice, CD301b+ DCs remained in the SCS and wereunable to enter the LN parenchyma, resulting in defective Th2 differentiation. We have defined aCCR8-dependent stepwise mechanism of DC-subset-specific migration through which LN CD169+SIGN-R1+ macrophages control the polarization of the adaptive immune response.

CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo.

To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca2+ sensor GCaMP6S to visualize in real-time chemokine-induced Ca2+ signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo.

Memory B cells are reactivated in subcapsular proliferative foci of lymph nodes

Vaccine-induced immunity depends on the generation of memory B cells (MBC). However, where and how MBCs are reactivated to make neutralising antibodies remain unknown. Here we show that MBCs are prepositioned in a subcapsular niche in lymph nodes where, upon reactivation by antigen, they rapidly proliferate and differentiate into antibody-secreting plasma cells in the subcapsular proliferative foci (SPF). This novel structure is enriched for signals provided by T follicular helper cells and antigen-presenting subcapsular sinus macrophages. Compared with contemporaneous secondary germinal centres, SPF have distinct single-cell molecular signature, cell migration pattern and plasma cell output. Moreover, SPF are found both in human and mouse lymph nodes, suggesting that they are conserved throughout mammalian evolution. Our data thus reveal that SPF is a seat of immunological memory that may be exploited to rapidly mobilise secondary antibody responses and improve vaccine efficacy.