Santiago F. Gonzalez

Conduits mediate transport of low-molecular-weight antigen to lymph node follicles.

To track drainage of lymph-borne small and large antigens (Ags) into the peripheral lymph nodes and subsequent encounter by B cells and follicular dendritic cells, we used the approach of multiphoton intravital microscopy. We find a system of conduits that extend into the follicles and mediate delivery of small antigens to cognate B cells and follicular dendritic cells. The follicular conduits provide an efficient and rapid mechanism for delivery of small antigens and chemokines such as CXCL13 to B cells that directly contact the conduits. By contrast, large antigens were bound by subcapsular sinus macrophages and subsequently transferred to follicular B cells as previously reported. In summary, the findings identify a unique pathway for the channeling of small lymph-borne antigens and chemoattractants from the subcapsular sinus directly to the B cell follicles. This pathway could be used for enhancing delivery of vaccines or small molecules for improvement of humoral immunity.

Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.

Lymph node stromal cells (LNSCs) closely regulate immunity and self-tolerance, yet key aspects of their biology remain poorly elucidated. Here, comparative transcriptomic analyses of mouse LNSC subsets demonstrated the expression of important immune mediators, growth factors and previously unknown structural components. Pairwise analyses of ligands and cognate receptors across hematopoietic and stromal subsets suggested a complex web of crosstalk. Fibroblastic reticular cells (FRCs) showed enrichment for higher expression of genes relevant to cytokine signaling, relative to their expression in skin and thymic fibroblasts. LNSCs from inflamed lymph nodes upregulated expression of genes encoding chemokines and molecules involved in the acute-phase response and the antigen-processing and antigen-presentation machinery. Poorly studied podoplanin (gp38)-negative CD31− LNSCs showed similarities to FRCs but lacked expression of interleukin 7 (IL-7) and were identified as myofibroblastic pericytes that expressed integrin α7. Together our data comprehensively describe the transcriptional characteristics of LNSC subsets.

Podoplanin-Rich Stromal Networks Induce Dendritic Cell Motility via Activation of the C-type Lectin Receptor CLEC-2

Summary To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.

Endocytosis and Recycling of Immune Complexes by Follicular Dendritic Cells Enhances B Cell Antigen Binding and Activation

Stromal-derived follicular dendritic cells (FDCs) are a major reservoir for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate. A long-standing question is how FDCs retain antigen in its native form for extended periods and how they display it to specific B cells. Here we found that FDCs acquired complement-coated immune complexes (ICs) from noncognate B cells via complement receptors 1 and 2 (CD35 and CD21, respectively) and rapidly internalized them by an actin-dependent pathway. ICs were retained intact within a nondegradative cycling compartment and were displayed periodically on the cell surface where they were accessible to antigen-specific B cells. This would explain how antigens are protected from damage and retained over long periods of time, while remaining accessible for B cells.

Simultaneous Detection of Marine Fish Pathogens by Using Multiplex PCR and a DNA Microarray

ABSTRACT We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (cyt, rpoN, gyrB, toxR, ureC, dly, and vapA) and two plasmid-borne loci (fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with <20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans.

Trafficking of B Cell Antigen in Lymph Nodes

The clonal selection theory first proposed by Macfarlane Burnet is a cornerstone of immunology (1). At the time, it revolutionized the thinking of immunologists because it provided a simple explanation for lymphocyte specificity, immunological memory, and elimination of self-reactive clones (2). The experimental demonstration by Nossal & Lederberg (3) that B lymphocytes bear receptors for a single antigen raised the central question of where B lymphocytes encounter antigen. This question has remained mostly unanswered until recently. Advances in techniques such as multiphoton intravital microscopy (4, 5) have provided new insights into the trafficking of B cells and their antigen. In this review, we summarize these advances in the context of our current view of B cell circulation and activation.

Complement-Dependent Transport of Antigen into B Cell Follicles

Since the original proposal by Fearon and Locksley (Fearon and Locksley. 1996. Science 272: 50–53) that the complement system linked innate and adaptive immunity, there has been a rapid expansion of studies on this topic. With the advance of intravital imaging, a number of recent papers revealed an additional novel pathway in which complement C3 and its receptors enhance humoral immunity through delivery of Ag to the B cell compartment. In this review, we discuss this pathway and highlight several novel exceptions recently found with a model influenza vaccine, such as mannose-binding lectin opsonization of influenza and uptake by macrophages, and the capture of virus by dendritic cells residing in the medullary compartment of peripheral lymph nodes.

Cochlin Produced by Follicular Dendritic Cells Promotes Antibacterial Innate Immunity

Cochlin, an extracellular matrix protein, shares homologies with the Factor C, a serine protease found in horseshoe crabs, which is critical for antibacterial responses. Mutations in the COCH gene are responsible for human DFNA9 syndrome, a disorder characterized by neurodegeneration of the inner ear that leads to hearing loss and vestibular impairments. The physiological function of cochlin, however, is unknown. Here, we report that cochlin is specifically expressed by follicular dendritic cells and selectively localized in the fine extracellular network of conduits in the spleen and lymph nodes. During inflammation, cochlin was cleaved by aggrecanases and secreted into blood circulation. In models of lung infection with Pseudomonas aeruginosa and Staphylococcus aureus, Coch(-/-) mice show reduced survival linked to defects in local cytokine production, recruitment of immune effector cells, and bacterial clearance. By producing cochlin, FDCs thus contribute to the innate immune response in defense against bacteria.

Epithelial‐mesenchymal transition in cancer metastasis through the lymphatic system

It was already in the 18th century when the French surgeon LeDran first noted that breast cancer patients with spread of tumor cells to their axillary lymph nodes had a drastically worse prognosis than patients without spread (LeDran et al., ). Since then, metastatic spread of cancer cells to regional lymph nodes has been established as the most important prognostic factor in many types of cancer (Carter et al., ; Elston and Ellis, ). However, despite its clinical importance, lymph metastasis remains an underexplored area of tumor biology. Fundamental questions, such as when, how, and perhaps most importantly, why tumor cells disseminate through the lymphatic system, remain largely unanswered. Accordingly, no treatment strategies exist that specifically target lymph metastasis. The identification of epithelial‐mesenchymal transition (EMT) as a mechanism, which allows cancer cells to dedifferentiate and acquire enhanced migratory and invasive properties, has been a game changer in cancer research. Conceptually, EMT provides an explanation for why epithelial cancers with poor differentiation status are generally more aggressive and prone to metastasize than more differentiated cancers. Inflammatory cytokines, such as TGF‐β, which are produced and secreted by tumor‐infiltrating immune cells, are potent inducers of EMT. Thus, reactivation of EMT also links cancer‐related inflammation to invasive and metastatic disease. Recently, we found that breast cancer cells undergoing TGF‐β‐induced EMT acquire properties of immune cells allowing them to disseminate in a targeted fashion through the lymphatic system similar to activated dendritic cells during inflammation. Here, we review our current understanding of the mechanisms by which cancer cells spread through the lymphatic system and the links to inflammation and the immune system. We also emphasize how imaging techniques have the potential to further expand our knowledge of the mechanisms of lymph metastasis, and how lymph nodes serve as an interface between cancer and the immune system.