Inflammation-induced formation of fat-associated lymphoid clusters.

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis.

Tuberculosis is the leading cause of death by an infectious disease worldwide1. However, the involvement of innate lymphoid cells (ILCs) in immune responses to infection with Mycobacterium tuberculosis (Mtb) is unknown. Here we show that circulating subsets of ILCs are depleted from the blood of participants with pulmonary tuberculosis and restored upon treatment. Tuberculosis increased accumulation of ILC subsets in the human lung, coinciding with a robust transcriptional response to infection, including a role in orchestrating the recruitment of immune subsets. Using mouse models, we show that group 3 ILCs (ILC3s) accumulated rapidly in Mtb-infected lungs and coincided with the accumulation of alveolar macrophages. Notably, mice that lacked ILC3s exhibited a reduction in the accumulation of early alveolar macrophages and decreased Mtb control. We show that the C-X-C motif chemokine receptor 5 (CXCR5)-C-X-C motif chemokine ligand 13 (CXCL13) axis is involved in Mtb control, as infection upregulates CXCR5 on circulating ILC3s and increases plasma levels of its ligand, CXCL13, in humans. Moreover, interleukin-23-dependent expansion of ILC3s in mice and production of interleukin-17 and interleukin-22 were found to be critical inducers of lung CXCL13, early innate immunity and the formation of protective lymphoid follicles within granulomas. Thus, we demonstrate an early protective role for ILC3s in immunity to Mtb infection.

Aging-associated and CD4 T-cell-dependent ectopic CXCL13 activation predisposes to anti-PD-1 therapy-induced adverse events.

Clinical success of immune-checkpoint blockade (ICB) cancer immunotherapy is compromised by increased risk of immune-related adverse events (irAEs). However, mechanistic action(s) of immune responses underlying development of irAE remain not fully explored. Here, we found that in tumor-bearing aged, but not young, mice, antiprogrammed death receptor (PD)-1 therapy elicited irAE-like multiorgan dysfunctions with ectopic accumulation of T and B cells in damaged organs. In this preclinical model, the organ toxicities were mediated by immunoglobulin G (IgG) deposition because administration of IG from ICB-treated aged mice induced the pathogenicity specifically in naïve aged hosts. Mechanistically, CD4 T-cell-derived interleukin (IL)-21 upregulated B-cell-homing chemokine, CXCL13, preferentially in irAE organs from aged mice treated with anti-PD-1 therapy. The ICB-induced pathogenicity was alleviated by B-cell depletion or by blockade of IL-21 or CXCL13 activity. These results suggest that age-associated immune regulatory milieu contributes to the formation of tertiary lymphoid structure-like lymphocytic aggregates in irAE organs and irAE-related toxicity employing IL-21-CXCL13-auto-antibody axis. Supporting this, a systemic increase in CXCL13 and Il21 expression in CD4 T cells correlated with irAE incidence in ICB-treated patients. These findings provide rationale for therapeutic usefulness of CXCL13 in irAE management.

Regulation of T(H)2 development by CXCR5+ dendritic cells and lymphotoxin-expressing B cells.

Although cognate encounters between antigen-bearing dendritic cells (DCs) that express the chemokine receptor CCR7 and CCR7(+) naive T cells take place in the T cell zone of lymph nodes, it is unknown whether the colocalization of DCs and T cells in the T cell area is required for the generation of effector cells. Here we found that after infection with an intestinal nematode, antigen-bearing DCs and CD4(+) T cells upregulated the chemokine receptor CXCR5 and localized together outside the T cell zone by a mechanism dependent on the chemokine CXCL13, B cells and lymphotoxin. Notably, lymphotoxin-expressing B cells, CXCR5-expressing DCs and T cells, and CXCL13 were also necessary for development of interleukin 4 (IL-4)-producing type 2 helper T cells (T(H)2 cells), which suggests that T(H)2 differentiation can initiate outside the T cell zone.

CXCL13 as a Novel Immune Checkpoint for Regulatory B Cells and Its Role in Tumor Metastasis.

Tumor metastasis is the primary cause of mortality in patients with cancer. Several chemokines are identified as important mediators of tumor growth and/or metastasis. The level of CXCL13 has been reported to be elevated in serum or tumor tissues in patients, which mainly functions to attract B cells and follicular B helper T cells. However, the role of CXCL13 in cancer growth and metastasis is not fully explored. In the current study, we found that CXCL13 is not a strong mediator to directly promote tumor growth; however, the mice deficient in CXCL13 had far fewer pulmonary metastatic foci than did the wild-type mice in experimental pulmonary metastatic models. In addition, Cxcl13 -/- mice also had fewer IL-10-producing B cells (CD45+CD19+IL-10+) in the metastatic tumor immune microenvironment than those of wild-type C57BL/6 mice, resulting in an enhanced antitumor immunity. Notably, CXCL13 deficiency further improved the efficacy of a traditional chemotherapeutic drug (cyclophosphamide), as well as that of anti-programmed death receptor-1 immunotherapy. These results suggested that CXCL13 has an important role in regulating IL-10-producing B cells in tumor metastasis and might be a promising target for improving therapeutic efficiency and stimulating tumor immunity in future cancer therapy.

The development of inducible bronchus-associated lymphoid tissue depends on IL-17.

Ectopic or tertiary lymphoid tissues, such as inducible bronchus-associated lymphoid tissue (iBALT), form in nonlymphoid organs after local infection or inflammation. However, the initial events that promote this process remain unknown. Here we show that iBALT formed in mouse lungs as a consequence of pulmonary inflammation during the neonatal period. Although we found CD4(+)CD3(-) lymphoid tissue-inducer cells (LTi cells) in neonatal lungs, particularly after inflammation, iBALT was formed in mice that lacked LTi cells. Instead, we found that interleukin 17 (IL-17) produced by CD4(+) T cells was essential for the formation of iBALT. IL-17 acted by promoting lymphotoxin-α-independent expression of the chemokine CXCL13, which was important for follicle formation. Our results suggest that IL-17-producing T cells are critical for the development of ectopic lymphoid tissues.

Autoreactivity of Peripheral Helper T Cells in the Joints of Rheumatoid Arthritis.

Autoreactive CD4 T cells are thought to play pivotal roles in the pathogenesis of rheumatoid arthritis (RA). Recently, a subset of CD4 T cells that express high levels of programmed death-1 (PD-1) but are distinct from follicular helper T cells have been identified in the joints of RA patients and named peripheral helper T (Tph) cells. Because PD-1 is expressed on T cells chronically stimulated with the Ags, we tested a hypothesis that Tph cells are the pathogenic autoreactive CD4 T cells in RA. We found that human Tph cells in RA joints produce proinflammatory effector cytokines, including IFN-γ, TNF-α, and GM-CSF, in addition to B cell-helping cytokines, such as IL-21 and CXCL13. Flow cytometric analysis showed different bias of TCR Vß usage between PD-1high Tph cells and PD-1low/neg CD4 T cells, including Th1 cells, in the joint or memory CD4 T cells in the peripheral blood, whereas there was little difference between the latter two subsets. In line with this, deep sequencing of TCR demonstrated an overlap of expanded clones between peripheral blood memory CD4 T cells and PD-1low/neg CD4 T cells but not Tph cells in the joint. Interestingly, Tph cells preferentially exhibited autologous MLR in vitro, which required recognition of self-MHC class II and was pronounced by blocking PD-1 signaling. Taken together, these results suggest that Tph cells are the pathogenic autoreactive CD4 T cells in RA, which expand locally in the joints and are regulated by PD-1 signaling.

A "big data" view of the tumor "immunome".

Tumors are complex masses containing not just neoplastic cells but also stromal cells, neovasculature, and a gamut of immune cells. In this issue, Bindea et al. (2013) identify a surprising new "immune landscape" associated with prolonged disease-free survival.

Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer.

The complex interactions between tumors and their microenvironment remain to be elucidated. Combining large-scale approaches, we examined the spatio-temporal dynamics of 28 different immune cell types (immunome) infiltrating tumors. We found that the immune infiltrate composition changed at each tumor stage and that particular cells had a major impact on survival. Densities of T follicular helper (Tfh) cells and innate cells increased, whereas most T cell densities decreased along with tumor progression. The number of B cells, which are key players in the core immune network and are associated with prolonged survival, increased at a late stage and showed a dual effect on recurrence and tumor progression. The immune control relevance was demonstrated in three endoscopic orthotopic colon-cancer mouse models. Genomic instability of the chemokine CXCL13 was a mechanism associated with Tfh and B cell infiltration. CXCL13 and IL21 were pivotal factors for the Tfh/B cell axis correlating with survival. This integrative study reveals the immune landscape in human colorectal cancer and the major hallmarks of the microenvironment associated with tumor progression and recurrence.

Comparison of the Euroimmun Borrelia 'antibody index' with Virotech immunoblot-based detection of intrathecal Borrelia antibody production for the diagnosis of Lyme neuroborreliosis.

For diagnosis of neuroborreliosis, calculation of the antibody index, based on Euroimmun Anti-Borrelia plus VlsE ELISA was compared to Virotech Borrelia Europe plus TpN17 immunoblot-based detection of Borrelia-specific intrathecal antibody production. CXCL13 results in cerebrospinal fluid were used to evaluate discordant results. A total of 64 serum/CSF pairs were analysed. Patients were classified according to European Federation of Neurological Societies criteria incorporating Virotech results. For the Euroimmun assay, a sensitivity of 100% and specificity of 94% was found. Agreement between the both tests was almost perfect (κ 0.81). Both methods are appropriate for the detection of Borrelia-specific intrathecal antibody production.

Characteristics of B lymphocyte infiltration in HPV+ head and neck squamous cell carcinoma.

Human papillomavirus (HPV) is an important etiological factor of head and neck squamous cell carcinoma (HNSCC). HPV+ HNSCC patients usually have a better prognosis, which probably results from the higher infiltration of B lymphocytes. This study was purposed to detect the infiltration of B lymphocyte subsets and the correlation between B lymphocyte subsets and the prognosis in HPV-related HNSCC. In this study, 124 HPV+ and 513 HPV- HNSCC samples were obtained from the Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA) database for transcriptomic analysis. Infiltration of B lymphocytes subsets was detected with 7 HPV+ HNSCC and 13 HPV- HNSCC tissues through immunohistochemistry and immunofluorescence. One HPV- HNSCC sample was detected with single-cell sequencing for chemokine analysis. In the results, the infiltration of plasma cells (CD19+ CD38+ ) and memory B cells (MS4A1+ CD27+ ) was higher in HPV+ HNSCC samples. High infiltration of plasma cells and memory B cells was related to a better prognosis. High density of B lymphocytes was positively correlated with high CXCL13 production mainly from CD4+ T lymphocytes in HNSCC. These results indicated that a high density of plasma cells and memory B cells could predict excellent prognosis. CD4+ T lymphocytes might affect B lymphocytes and their subsets through the CXCL13/CXCR5 axis in HNSCC.

Bioinformatics-based study to identify immune infiltration and inflammatory-related hub genes as biomarkers for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease whose principal pathological change is aggressive chronic synovial inflammation; however, the specific etiology and pathogenesis have not been fully elucidated. We downloaded the synovial tissue gene expression profiles of four human knees from the Gene Expression Omnibus database, analyzed the differentially expressed genes in the normal and RA groups, and assessed their enrichment in functions and pathways using bioinformatics methods and the STRING online database to establish protein-protein interaction networks. Cytoscape software was used to obtain 10 hub genes; receiver operating characteristic (ROC) curves were calculated for each hub gene and differential expression analysis of the two groups of hub genes. The CIBERSORT algorithm was used to impute immune infiltration. We identified the signaling pathways that play important roles in RA and 10 hub genes: Ccr1, Ccr2, Ccr5, Ccr7, Cxcl5, Cxcl6, Cxcl13, Ccl13, Adcy2, and Pnoc. The diagnostic value of these 10 hub genes for RA was confirmed using ROC curves and expression analysis. Adcy2, Cxcl13, and Ccr5 are strongly associated with RA development. The study also revealed that the differential infiltration profile of different inflammatory immune cells in the synovial tissue of RA is an extremely critical factor in RA progression. This study may contribute to the understanding of signaling pathways and biological processes associated with RA and the role of inflammatory immune infiltration in the pathogenesis of RA. In addition, this study shows that Adcy2, Cxcl13, and Ccr5 have the potential to be biomarkers for RA treatment.

Natural Killer Cell Transcript 4 promotes the development of SjÓ§gren's syndrome via activation of Rap1 on B cells.

Autoimmune disorders are the third most common diseases in the United States, and affect the daily lives of millions of people. In this study, we analyzed patient samples, utilized a transgenic mouse model and human B cells to reveal Natural Killer Cell Transcript 4 (NK4) as a novel regulator that promotes the development of autoimmune disorders. NK4 was significantly elevated in samples from patients with SjÓ§gren's Syndrome (SS). SS patients show elevated NK4 levels. There is a strong and positive correlation between the increased levels of NK4 and the duration of SS. Interestingly, transgenic expression of NK4 in a mouse model led to the development of autoantibodies and lymphocytic infiltration in salivary glands similar to those in SS patients. Those phenotypes were associated with increased B1a cells in the peritoneum, plasma cells in the spleen, and increased IgM, IgA, and IgG2a in serum of the NK4 transgenic mice. The autoimmune phenotypes became more severe in older mice. Moreover, after NK4 transfection, human naïve B cells were activated and memory B cells differentiation into IgG and IgA-plasmablasts, resulting in an increased production of autoantibodies.NK4 regulated the differentiation and activation of B cells through activating Rap1 activity. NK4 also promoted B cell migration in a paracrine fashion through an induction of CXCL13 in endothelial cells. Collectively, these findings identify NK4 as a promoter of the development of autoimmune disorders through its roles on B cells. Therefore, NK4 may be a novel therapeutic target for the treatment of autoimmune diseases.

Unique Sjögren's syndrome patient subsets defined by molecular features.

OBJECTIVE: To address heterogeneity complicating primary SS (pSS) clinical trials, research and care by characterizing and clustering patients by their molecular phenotypes. METHODS: pSS patients met American-European Consensus Group classification criteria and had at least one systemic manifestation and stimulated salivary flow of ⩾0.1 ml/min. Correlated transcriptional modules were derived from gene expression microarray data from blood (n = 47 with appropriate samples). Patients were clustered based on this molecular information using an unbiased random forest modelling approach. In addition, multiplex, bead-based assays and ELISAs were used to assess 30 serum cytokines, chemokines and soluble receptors. Eleven autoantibodies, including anti-Ro/SSA and anti-La/SSB, were measured by Bio-Rad Bioplex 2200. RESULTS: Transcriptional modules distinguished three clusters of pSS patients. Cluster 1 showed no significant elevation of IFN or inflammation modules. Cluster 2 showed strong IFN and inflammation modular network signatures, as well as high plasma protein levels of IP-10/CXCL10, MIG/CXCL9, BLyS (BAFF) and LIGHT. Cluster 3 samples exhibited moderately elevated IFN modules, but with suppressed inflammatory modules, increased IP-10/CXCL10 and B cell-attracting chemokine 1/CXCL13 and trends toward increased MIG/CXCL9, IL-1α, and IL-21. Anti-Ro/SSA and anti-La/SSB were present in all three clusters. CONCLUSION: Molecular profiles encompassing IFN, inflammation and other signatures can be used to separate patients with pSS into distinct clusters. In the future, such profiles may inform patient selection for clinical trials and guide treatment decisions.

PD-1-expressing MAIT cells from patients with tuberculosis exhibit elevated production of CXCL13.

To understand functional role of PD-1-expressing MAIT cells during tuberculosis infection in humans, sorted PD-1+ and PD-1- MAIT cells from pleural effusions of patients with pleural tuberculosis were subjected to transcriptome sequencing. PD-1-expressing MAIT cells were analysed by flow cytometry and their phenotypic and functional features were investigated. Transcriptome sequencing identified 144 genes that were differentially expressed between PD-1+ and PD-1- MAIT cells from tuberculous pleural effusions and CXCL13 was the gene with highest fold difference. The level of PD-1-expressing MAIT cells was associated with extent of TB infection in humans. PD-1-expressing MAIT cells had increased production of CXCL13 and IL-21 as determined by flow cytometry. PD-1high CXCR5- MAIT cells were significantly expanded in pleural effusions from patients with pleural tuberculosis as compared with those from peripheral blood of both patients with tuberculosis and healthy controls. Although PD-1high CXCR5- MAIT cells from tuberculous pleural effusions had reduced IFN-γ level and increased expression of Tim-3 and GITR, they showed activated phenotype and had higher glucose uptake and lipid content. It is concluded that PD-1-expressing MAIT cells had reduced IFN-γ level but increased production of both CXCL13 and IL-21.

CXCL13 is a differentiation- and hypoxia-induced adipocytokine that exacerbates the inflammatory phenotype of adipocytes through PHLPP1 induction.

Hypoxia in adipose tissue is regarded as a trigger that induces dysregulation of the secretory profile in adipocytes. Similarly, local dysregulation of adipocytokine secretion is an initial event in the deleterious effects of obesity on metabolism. We previously reported that CXCL13 is highly produced during adipogenesis, however little is known about the roles of CXCL13 in adipocytes. Here, we found that hypoxia, as modeled by 1% O2 or exposure to the hypoxia-mimetic reagent desferrioxamine (DFO) has strong inductive effects on the expression of CXCL13 and CXCR5, a CXCL13 receptor, in both undifferentiated and differentiated adipocytes and in organ-cultured white adipose tissue (WAT). CXCL13 was also highly expressed in WAT from high fat diet-fed mice. Hypoxic profile, typified by increased expression of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) and decreased expression of adiponectin, was significantly induced by CXCL13 treatment during adipogenic differentiation. Conversely, the treatment of adipocytes with a neutralizing-antibody against CXCL13 as well as CXCR5 knockdown by specific siRNA effectively inhibited DFO-induced inflammation. The phosphorylation of Akt2, a protective factor of adipose inflammation, was significantly inhibited by CXCL13 treatment during adipogenic differentiation. Mechanistically, CXCL13 induces the expression of PHLPP1, an Akt2 phosphatase, through focal adhesion kinase (FAK) signaling; and correspondingly we show that CXCL13 and DFO-induced IL-6 and PAI-1 expression was blocked by Phlpp1 knockdown. Furthermore, we revealed the functional binding sites of PPARγ2 and HIF1-α within the Cxcl13 promoter. Taken together, these results indicate that CXCL13 is an adipocytokine that facilitates hypoxia-induced inflammation in adipocytes through FAK-mediated induction of PHLPP1 in autocrine and/or paracrine manner.

Multi-dimensional analysis identified rheumatoid arthritis-driving pathway in human T cell.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease accompanied by lymphocyte infiltration into joint synovium. While T cells are considered to be important for its pathogenesis, the features that are the most relevant to disease and how they change after treatment remain unclear. The aim of this study was to clarify the characteristics of T cells in RA, comprehensively. METHODS: We enrolled a total of 311 patients with RA and 73 healthy participants, and carefully classified them by disease state, constructed multiple cohorts and analysed clinical samples from them in a stepwise manner. We performed immunophenotyping with multiple evaluation axes, and two independent transcriptome analyses complementary to each other. RESULTS: We identified that 'effector memory-Tfh' subset was specifically expanded in the peripheral blood (PB) of patients with RA in correlation with disease activity, and reverted after treatment. Besides, we revealed distinct features of T cells in synovial fluid (SF) that the expression of Tfh/Tph-related genes and pro-inflammatory cytokines and chemokines, including CXCL13, were significantly enriched, whereas these phenotype were Th1-like. Finally, we identified specific pathways, such as mTORC1, IL-2-stat5, E2F, cell cycle and interferon-related genes, that were significantly enriched in SF, in particular, as well as PB of untreated patients with RA, and notably, these features reverted after treatment. CONCLUSION: Our multi-dimensional investigation identified disease relevant T-cell subsets and gene signatures deeply involved in pathogenesis of RA. These findings could aid in our understanding of essential roles of T cells in RA and will facilitate to development better diagnostic and therapeutic interventions.

CXCL13 is a plasma biomarker of germinal center activity.

Significantly higher levels of plasma CXCL13 [chemokine (C-X-C motif) ligand 13] were associated with the generation of broadly neutralizing antibodies (bnAbs) against HIV in a large longitudinal cohort of HIV-infected individuals. Germinal centers (GCs) perform the remarkable task of optimizing B-cell Ab responses. GCs are required for almost all B-cell receptor affinity maturation and will be a critical parameter to monitor if HIV bnAbs are to be induced by vaccination. However, lymphoid tissue is rarely available from immunized humans, making the monitoring of GC activity by direct assessment of GC B cells and germinal center CD4(+) T follicular helper (GC Tfh) cells problematic. The CXCL13-CXCR5 [chemokine (C-X-C motif) receptor 5] chemokine axis plays a central role in organizing both B-cell follicles and GCs. Because GC Tfh cells can produce CXCL13, we explored the potential use of CXCL13 as a blood biomarker to indicate GC activity. In a series of studies, we found that plasma CXCL13 levels correlated with GC activity in draining lymph nodes of immunized mice, immunized macaques, and HIV-infected humans. Furthermore, plasma CXCL13 levels in immunized humans correlated with the magnitude of Ab responses and the frequency of ICOS(+) (inducible T-cell costimulator) Tfh-like cells in blood. Together, these findings support the potential use of CXCL13 as a plasma biomarker of GC activity in human vaccine trials and other clinical settings.

Heightened TLR7/9-Induced IL-10 and CXCL13 Production with Dysregulated NF-ҝB Activation in CD11chiCD11b+ Dendritic Cells in NZB/W F1 Mice.

Systemic lupus erythematosus (SLE) is a chronic, multifactorial autoimmune disease that predominantly affects young females. Dysregulation of different immune cell populations leads to self-tolerance breakdown and subsequent multiple organ damage as the disease develops. Plasmacytoid dendritic cells (pDCs) are potent producers of type I interferon (IFN), while myeloid dendritic cells (mDCs) are more specialized in antigen presentations. We have previously reported that bone-marrow (BM)-derived pDCs from the murine lupus model New Zealand black/white F1 (BWF1) possess abnormalities. Therefore, this study continues to investigate what aberrant properties peripheral pDCs and mDCs possess in BWF1 and how they mediate SLE progression, by comparing their properties in pre-symptomatic and symptomatic mice. Results showed that CD11chiCD11b+ myeloid DCs expanded during the disease state with down-regulation of co-stimulatory molecules and major histocompatibility complex class II molecules (MHC II), but their capacity to stimulate T cells was not hampered. During the disease state, this subset of mDCs displayed heightened toll-like receptors 7 and 9 (TLR 7/9) responses with increased interleukin 10 (IL-10) and C-X-C motif chemokine ligand 13 (CXCL13) expressions. Moreover, the expressions of myeloid differentiation primary response 88 (Myd88) and nuclear factor kappa B subunit 1 (Nfkb1) were higher in CD11chiCD11b+ DCs at the disease stage, leading to higher nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 phosphorylation activity. In summary, we reported aberrant phenotypic properties with enhanced TLR7/9 responses of CD11chiCD11b+ DCs in SLE mediated by aberrant NF-κB signaling pathway. Our findings add additional and novel information to our current understanding of the role of DCs in lupus immunopathogenesis. Lastly, molecular candidates in the NF-κB pathway should be exploited for developing therapeutic targets for SLE.

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.