Lymphatic Endothelial Cells Control Initiation of Lymph Node Organogenesis.

Lymph nodes (LNs) are strategically situated throughout the body at junctures of the blood vascular and lymphatic systems to direct immune responses against antigens draining from peripheral tissues. The current paradigm describes LN development as a programmed process that is governed through the interaction between mesenchymal lymphoid tissue organizer (LTo) cells and hematopoietic lymphoid tissue inducer (LTi) cells. Using cell-type-specific ablation of key molecules involved in lymphoid organogenesis, we found that initiation of LN development is dependent on LTi-cell-mediated activation of lymphatic endothelial cells (LECs) and that engagement of mesenchymal stromal cells is a succeeding event. LEC activation was mediated mainly by signaling through receptor activator of NF-κB (RANK) and the non-canonical NF-κB pathway and was steered by sphingosine-1-phosphate-receptor-dependent retention of LTi cells in the LN anlage. Finally, the finding that pharmacologically enforced interaction between LTi cells and LECs promotes ectopic LN formation underscores the central LTo function of LECs.

Clonal Proliferation and Stochastic Pruning Orchestrate Lymph Node Vasculature Remodeling.

Lymph node (LN) expansion during an immune response relies on the transient remodeling of its vasculature. Although the mechanisms driving LN endothelial cell division are beginning to be understood, a comprehensive view of LN endothelial cell dynamics at the single-cell level is lacking. Here, we used multicolored fluorescent fate-mapping models to track the behavior of blood endothelial cells during LN expansion upon inflammation and subsequent return to homeostasis. We found that expansion of the LN vasculature relied on the sequential assembly of endothelial cell proliferative units. This segmented growth was sustained by the clonal proliferation of high endothelial venule (HEV) cells, which act as local progenitors to create capillaries and HEV neo-vessels at the periphery of the LN. Return to homeostasis was accompanied by the stochastic death of pre-existing and neo-synthesized LN endothelial cells. Thus, our fate-mapping studies unravel-at a single-cell level-the complex dynamics of vascular-tree remodeling during LN expansion and contraction.

Development of follicular dendritic cells in lymph nodes depends on retinoic acid-mediated signaling.

Specialized stromal cells occupy and help define B- and T-cell domains, which are crucial for proper functioning of our immune system. Signaling through lymphotoxin and TNF receptors is crucial for the development of different stromal subsets, which are thought to arise from a common precursor. However, mechanisms that control the selective generation of the different stromal phenotypes are not known. Using in vitro cultures of embryonic mouse stromal cells, we show that retinoic acid-mediated signaling is important for the differentiation of precursors towards the Cxcl13pos follicular dendritic cell (FDC) lineage, and also blocks lymphotoxin-mediated Ccl19pos fibroblastic reticular cell lineage differentiation. Accordingly, at the day of birth we observe the presence of Cxcl13posCcl19neg/low and Cxcl13neg/lowCcl19pos cells within neonatal lymph nodes. Furthermore, ablation of retinoic acid receptor signaling in stromal precursors early after birth reduces Cxcl13 expression, and complete blockade of retinoic acid signaling prevents the formation of FDC networks in lymph nodes.

Lymph node formation and B cell homeostasis require IKK-α in distinct endothelial cell-derived compartments.

Global inactivation of IκB kinase (IKK)-α results in defective lymph node (LN) formation and B cell maturation, and loss of IKK-α-dependent noncanonical NF-κB signaling in stromal organizer and hematopoietic cells is thought to underlie these distinct defects. We previously demonstrated that this pathway is also activated in vascular endothelial cells (ECs). To determine the physiologic function of EC-intrinsic IKK-α, we crossed IkkαF/F mice with Tie2-cre or Cdh5-cre mice to ablate IKK-α in ECs. Notably, the compound defects of global IKK-α inactivation were recapitulated in IkkαTie2 and IkkαCdh5 mice, as both lacked all LNs and mature follicular and marginal zone B cell numbers were markedly reduced. However, as Tie2-cre and Cdh5-cre are expressed in all ECs, including blood forming hemogenic ECs, IKK-α was also absent in hematopoietic cells (HC). To determine if loss of HC-intrinsic IKK-α affected LN development, we generated IkkαVav mice lacking IKK-α in only the hematopoietic compartment. While mature B cell numbers were significantly reduced in IkkαVav mice, LN formation was intact. As lymphatic vessels also arise during development from blood ECs, we generated IkkαLyve1 mice lacking IKK-α in lymphatic ECs (LECs) to determine if IKK-α in lymphatic vessels impacts LN development. Strikingly, while mature B cell numbers were normal, LNs were completely absent in IkkαLyve1 mice. Thus, our findings reveal that IKK-α in distinct EC-derived compartments is uniquely required to promote B cell homeostasis and LN development, and we establish that LEC-intrinsic IKK-α is absolutely essential for LN formation.

Chemokines and other mediators in the development and functional organization of lymph nodes.

Secondary lymphoid organs like lymph nodes (LNs) are the main inductive sites for adaptive immune responses. Lymphocytes are constantly entering LNs, scanning the environment for their cognate antigen and get replenished by incoming cells after a certain period of time. As only a minor percentage of lymphocytes recognizes cognate antigen, this mechanism of permanent recirculation ensures fast and effective immune responses when necessary. Thus, homing, positioning, and activation as well as egress require precise regulation within LNs. In this review we discuss the mediators, including chemokines, cytokines, growth factors, and others that are involved in the formation of the LN anlage and subsequent functional organization of LNs. We highlight very recent findings in the fields of LN development, steady-state migration in LNs, and the intranodal processes during an adaptive immune response.

Prognostic impact of lymph node micrometastasis in patients with gastric cancer.

PURPOSE: The clinical significance of lymph node micrometastasis (LNMM) remains controversial in gastric cancer (GC). In this study, we investigated the prognostic impact of LNMM in patients with GC. METHODS: A total of 624 patients with pathologically lymph node metastasis-negative (pN0) and N1 status (pN1) who underwent gastrectomy between 2004 and 2018 were enrolled in this retrospective study. The diameter of tumor cell clusters in metastatic lymph nodes was measured in 120 patients with pN1 GC. RESULTS: Patients with lymph node tumors < 1500 µm in diameter (LNMM) had a significantly better prognosis than those with tumors ≥ 1500 µm in diameter (p = 0.012; log-rank test). Cox's proportional hazards model revealed that LNMM (p = 0.016), several dissected lymph nodes (p = 0.049), and the provision of adjuvant chemotherapy (p = 0.002) were independent prognostic factors for the overall survival of patients with pN1 GC. There was no significant difference in the overall survival between patients with LNMM who received chemotherapy and those who did not (p = 0.332). CONCLUSIONS: LNMM is associated with a favorable prognosis and maybe an independent prognostic marker in patients with pN1 GC. LNMM in GC may be considered a factor preventing adjuvant chemotherapy.

Monocytic myeloid-derived suppressor cells home to tumor-draining lymph nodes via CCR2 and locally modulate the immune response.

Efficient priming of anti-tumor T cells requires the uptake and presentation of tumor antigens by immunogenic dendritic cells (DCs) and occurs mainly in lymph nodes draining the tumor (tdLNs). However, tumors expand and activate myeloid-derived suppressor cells (MDSCs) that inhibit CTL functions by several mechanisms. While the immune-suppressive nature of the tumor microenvironment is largely documented, it is not known whether similar immune-suppressive mechanisms operate in the tdLNs. In this study, we analyzed MDSC characteristics within tdLNs. We show that, in a metastasis-free context, MO-MDSCs are the dominant MDSC population within tdLNs, that they are highly suppressive and that tumor proximity enhances their recruitment to tdLN via a CCR2/CCL2-dependent pathway. Altogether our results uncover a mechanism by which tumors evade the immune system that involves MDSC-mediated recruitment to the tdLN and the inhibition of T-cell activation even before reaching the highly immunosuppressive tumor microenvironment.

A Fresh View on Lymph Node Organogenesis.

Lymph nodes (LNs) are strategically positioned outposts of the immune system that underpin regional immune surveillance. The current model describing LN formation in mice is based on a two cell-type interaction scheme with lymphoid tissue inducer cells regulating the activation of mesenchymal lymphoid tissue organizer cells. We highlight here the key role of lymphatic endothelial cells during the initiation of LN formation. The involvement of lymphatic endothelial cells as an additional organizer cell type in LN organogenesis unveils multiple control levels that govern the generation of lymphoid organs. Moreover, the linkage between lymphangiogenic and lymphvasculogenic processes and guidance of the accumulation and activation of lymphoid tissue inducer cells in the embryo suggests that LN formation may be driven on demand by developing organ systems.

Transdermal entry of yeast components elicits transient B cell-associated responses in skin-draining lymph nodes.

Immune responses to non-pathogenic yeasts induced within the draining lymph node remain to be understood. In this study, we have investigated the changes in lymphocytes and their activity in skin-draining lymph nodes in response to transdermally injected zymosan (component of the yeast cell wall). Zymosan elicited the transient increase of B cell number and activation status without affecting the capacity for proliferation. The increased B cell content in the regional lymph nodes was likely due to the reduction of B cell egress from the tissue and in part the increase of homing from the circulation. Zymosan also upregulated the inflammatory cytokines, such as IL-1ß, IL-6, IL-12, and IFNγ, regulatory cytokines IL-10 and TGFß, and lymphoid chemokine CXCL13. Among these, the expression of IL-12 and IL-10 was markedly high in B cells. Altogether, these findings demonstrate a unique B cell-associated response to non-pathogenic yeast component in the draining lymph nodes. This will provide insights into the clinical and healthcare applications of non-pathogenic beneficial microbes.

PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

C-C chemokine receptor type 7 (CCR7) is essential for migration of dendritic cells (DCs) to draining lymph nodes. PU.1/Spi1 is a transcription factor playing a critical role in the gene regulation of DCs. PU.1 knockdown decreased the expression of CCR7 in bone marrow-derived DCs and subsequently attenuated migration in vitro and in vivo. Reporter assays, EMSA, and chromatin immunoprecipitation assays revealed that PU.1 binds to the most proximal Ets motif of the Ccr7 promoter, which is involved in transcriptional activation. The CCR7 expression level, which was higher in the programmed cell death 1 ligand 2 (PD-L2)+ population than in the PD-L2- population and was markedly suppressed by TGF-ß treatment, coincided with the binding level of PU.1 to the Ccr7 promoter. The PU.1 binding level in CCR7high mesenteric lymph nodes DCs was higher than in other DC subtypes. The involvement of PU.1 in the expression of the CCR7 gene was also observed in human DCs. We conclude that PU.1 plays a pivotal role in DC migration by transactivating the CCR7 gene via the Ets motif in the promoter in both humans and mice.-Yashiro, T., Takeuchi, H., Nakamura, S., Tanabe, A., Hara, M., Uchida, K., Okumura, K., Kasakura, K., Nishiyama, C. PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

Intravital microscopy: Imaging host-parasite interactions in lymphoid organs.

Intravital microscopy allows imaging of biological phenomena within living animals, including host-parasite interactions. This has advanced our understanding of both, the function of lymphoid organs during parasitic infections, and the effect of parasites on such organs to allow their survival. In parasitic research, recent developments in this technique have been crucial for the direct study of host-parasite interactions within organs at depths, speeds and resolution previously difficult to achieve. Lymphoid organs have gained more attention as we start to understand their function during parasitic infections and the effect of parasites on them. In this review, we summarise technical and biological findings achieved by intravital microscopy with respect to the interaction of various parasites with host lymphoid organs, namely the bone marrow, thymus, lymph nodes, spleen and the mucosa-associated lymphoid tissue, and present a view into possible future applications.

Neutrophil trafficking to lymphoid tissues: physiological and pathological implications.

Recent advances have provided evidence for the involvement of neutrophils in both innate and adaptive immunity, robustly challenging the old dogma that neutrophils are short-lived prototypical innate immune cells solely involved in acute responses to microbes and exerting collateral tissue damage. There is now ample evidence showing that neutrophils can migrate into different compartments of the lymphoid system where they contribute to the orchestration of the activation and/or suppression of lymphocyte effector functions in homeostasis and during chronic inflammation, such as autoimmune disorders and cancer. In support of this notion, neutrophils can generate a wide range of cytokines and other mediators capable of regulating the survival, proliferation and functions of both T and B cells. In addition, neutrophils can directly engage with lymphocytes and promote antigen presentation. Furthermore, there is emerging evidence of the existence of distinct and diverse neutrophil phenotypes with immunomodulatory functions that characterise different pathological conditions, including chronic and autoimmune inflammatory conditions. The aim of this review is to discuss the mechanisms implicated in neutrophil trafficking into the lymphoid system and to provide an overview of the immuno-regulatory functions of neutrophils in health and disease in the context of adaptive immunity. Copyright © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Perinodal Adipose Tissue Participates in Immune Protection through a Lymphatic Vessel-Independent Route.

Lymphatic vessels remove and transport excess interstitial fluid to lymph nodes (LNs) for fluid balance and immune protection. LNs are typically surrounded by perinodal adipose tissue (PAT). However, PAT is a blood vessel-rich but lymphatic-rare tissue; therefore, how excess fluid in PAT is removed remains unclear. Using C57BL/6 mice, fluorescent dye tracing and transmission electron microscopy results suggest that fluid in PAT can travel to the LN via collagen I+ channels (PAT-LN conduits), merge into a collagen-rich space between the PAT and LN capsule (PAT-LN sinus), and may enter the LN via the LN capsule-associated conduits. This newly identified route of fluid flow allows fluid to enter the draining LN even when the afferent lymphatic vessels are blocked, indicating that fluid trafficking in PAT-LN conduits is not dependent on functional lymphatic vessels. Similar to lymphatic vessels, PAT-LN conduits can deliver Ags to the LN for immune protection. Additionally, Staphylococcus aureus from intradermal or i.v. infection may use PAT-LN conduits to infect PAT and stimulate PAT immune protection. Our studies revealed a new route of material exchange between PAT and the LN. Ag accumulation and bacterial infection in PAT demonstrate that PAT not only provides energy and regulatory factors, but can also directly participate in immune protection, indicating a new immune function of PAT for host immunity.

Nasal-associated lymphoid tissues (NALTs) support the recall but not priming of influenza virus-specific cytotoxic T cells.

The lymphoid tissue that drains the upper respiratory tract represents an important induction site for cytotoxic T lymphocyte (CTL) immunity to airborne pathogens and intranasal vaccines. Here, we investigated the role of the nasal-associated lymphoid tissues (NALTs), which are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage, in the initial priming and recall expansion of CD8+ T cells following an upper respiratory tract infection with a pathogenic influenza virus and immunization with a live attenuated influenza virus vaccine. Whereas NALTs served as the induction site for the recall expansion of memory CD8+ T cells following influenza virus infection or vaccination, they failed to support activation of naïve CD8+ T cells. Strikingly, NALTs, unlike other lymphoid tissues, were not routinely surveyed during the steady state by circulating T cells. The selective recruitment of memory T cells into these lymphoid structures occurred in response to infection-induced elevation of the chemokine CXCL10, which attracted CXCR3+ memory CD8+ T cells. These results have significant implications for intranasal vaccines, which deliver antigen to mucosal-associated lymphoid tissue and aim to elicit protective CTL-mediated immunity.

The effect of surgery on lymphoscintigraphy drainage patterns from the canine brachium in a simulated tumor model.

OBJECTIVE: To determine the effect of surgery on lymphoscintigraphy drainage patterns from the canine brachium. STUDY DESIGN: Experimental study. ANIMALS: Eight healthy research beagles. METHODS: A predefined area of skin measuring 2 × 1.5 cm in dimension was designated on either the right or left brachium. Preoperative lymphoscintigraphy was performed with technetium sulfur colloid injected into the subcutaneous tissues around the predefined anatomic location in a four-quadrant technique. Dogs underwent surgery for excision of the predefined area of skin, subcutis, and fascia of the lateral head of the triceps muscle with 1-cm margins. Eighteen days after surgery, lymphoscintigraphy was again performed with technetium sulfur colloid injected into the subcutaneous tissues around the surgical scar in a four-quadrant technique. RESULTS: Sentinel lymph nodes were identified in eight of eight dogs preoperatively and in eight of eight dogs postoperatively. Agreement between the results of the preoperative and postoperative lymphoscintigraphy studies was identified as complete in four of eight dogs and partial in four of eight dogs. Sentinel lymph node identification occurred immediately in three of eight dogs preoperatively and in eight of eight dogs postoperatively. CONCLUSION: Sentinel lymph node identification occurred faster postoperatively. Agreement or partial agreement between the results of the preoperative and postoperative lymphoscintigraphy studies was observed in eight of eight dogs. CLINICAL SIGNIFICANCE: Surgery appears to have an effect on lymphoscintigraphy drainage patterns. Additional studies are required to compare preoperative and postoperative sentinel lymph node mapping patterns in tumor-bearing dogs. However, this study provides preliminary information regarding the effect of surgery on sentinel lymph node identification.

Dendritic cells in remodeling of lymph nodes during immune responses.

A critical hallmark of adaptive immune responses is the rapid and extensive expansion of lymph nodes. During this process, the complex internal structure of the organs is maintained revealing the existence of mechanisms able to balance lymph node integrity with structural flexibility. This article reviews the extensive architectural remodeling that occurs within lymph nodes during adaptive immune responses and how it is regulated by dendritic cells (DCs). In particular we focus on previously unappreciated functions of DCs in coordinating remodeling of lymph node vasculature, expansion of the fibroblastic reticular network and maintenance of lymphoid stromal phenotypes. Our increased understanding of these processes indicates that DCs need to be viewed not only as key antigen-presenting cells for lymphocytes but also as broad-acting immune sentinels that convey signals to lymphoid organ stroma and thereby facilitate immune response initiation at multiple levels.

[Laparoscopic radical resection of rectal cancer with preservation of the left colic artery: anatomical basis and surgical experience].

In order to increase the blood supply of anastomosis, surgeons choose to preserve the left colon artery (LCA) during the laparoscopic radical resection of rectal cancer. However, surgeons are always ailed by hemorrhage and incompletely dissection of No. 253 lymph nodes. One reason is the shortage of understanding the relationship between inferior mesenteric artery (IMA), LCA, and inferior mesenteric vein before surgery. Another reason is that surgeon always remove the lymph nodes around LCA, while don't normatively resect No. 253 lymph nodes, which affect the overall survival rate. Therefore, the "medial-to-lateral approach" for laparoscopic preservation with LCA radical resection in rectal cancer was suggested in this article. The CT technique could be used to analyze the IMA classification, which contribuated to the standard conservation of LCA. Laparoscopic radical resection of rectal cancer could be completed of high quality, through accurate definition and exactly dissection of the No. 235 lymph nodes.

A Novel Computational Model Predicts Key Regulators of Chemokine Gradient Formation in Lymph Nodes and Site-Specific Roles for CCL19 and ACKR4.

The chemokine receptor CCR7 drives leukocyte migration into and within lymph nodes (LNs). It is activated by chemokines CCL19 and CCL21, which are scavenged by the atypical chemokine receptor ACKR4. CCR7-dependent navigation is determined by the distribution of extracellular CCL19 and CCL21, which form concentration gradients at specific microanatomical locations. The mechanisms underpinning the establishment and regulation of these gradients are poorly understood. In this article, we have incorporated multiple biochemical processes describing the CCL19-CCL21-CCR7-ACKR4 network into our model of LN fluid flow to establish a computational model to investigate intranodal chemokine gradients. Importantly, the model recapitulates CCL21 gradients observed experimentally in B cell follicles and interfollicular regions, building confidence in its ability to accurately predict intranodal chemokine distribution. Parameter variation analysis indicates that the directionality of these gradients is robust, but their magnitude is sensitive to these key parameters: chemokine production, diffusivity, matrix binding site availability, and CCR7 abundance. The model indicates that lymph flow shapes intranodal CCL21 gradients, and that CCL19 is functionally important at the boundary between B cell follicles and the T cell area. It also predicts that ACKR4 in LNs prevents CCL19/CCL21 accumulation in efferent lymph, but does not control intranodal gradients. Instead, it attributes the disrupted interfollicular CCL21 gradients observed in Ackr4-deficient LNs to ACKR4 loss upstream. Our novel approach has therefore generated new testable hypotheses and alternative interpretations of experimental data. Moreover, it acts as a framework to investigate gradients at other locations, including those that cannot be visualized experimentally or involve other chemokines.

Temporal dynamics of immune response following prolonged myocardial ischemia/reperfusion with and without cyclosporine A.

Understanding the dynamics of the immune response following late myocardial reperfusion is critical for the development of immunomodulatory therapy for myocardial infarction (MI). Cyclosporine A (CSA) possesses multiple therapeutic applications for MI, but its effects on the inflammation caused by acute MI are not clear. This study aimed to determine the dynamics of the immune response following myocardial ischemia/reperfusion (I/R) and the effects of CSA in a mouse model of prolonged myocardial ischemia designated to represent the human condition of late reperfusion. Adult C57BL/6 mice were subjected to 90 min of closed-chest myocardial I/R, which induced severe myocardial injury and excessive inflammation in the heart. Multicomponent analysis of the immune response caused by prolonged I/R revealed that the peak of cytokines/chemokines in the systemic circulation was synchronized with the maximal influx of neutrophils and T-cells in the heart 1 day after MI. The peak of cytokine/chemokine secretion in the infarcted heart coincided with the maximal macrophage and natural killer cell infiltration on day 3 after MI. The cellular composition of the mediastinal lymph nodes changed similarly to that of the infarcted hearts. CSA (10 mg/kg/day) given after prolonged I/R impaired heart function, enlarged the resulting scar, and reduced heart vascularization. It did not change the content of immune cells in hearts exposed to prolonged I/R, but the levels of MCP-1 and MIP-1α (hearts) and IL-12 (hearts and serum) were significantly reduced in the CSA-treated group in comparison to the untreated group, indicating alterations in immune cell function. Our findings provide new knowledge necessary for the development of immunomodulatory therapy targeting the immune response after prolonged myocardial ischemia/reperfusion.

Immune cell populations residing in mesenteric adipose depots and mesenteric lymph nodes of lean dairy cows.

Inconsistent evidence of inflammatory immune cell infiltrates in adipose tissues with extensive triglyceride mobilization raises the possibility that regulatory or anti-inflammatory immune cell populations reside within the mesenteric adipose tissue (MAT) and mesenteric lymph nodes (MLN). These resident immune cell populations may be involved in attenuating the inflammatory response. We explored the immune cell population of MAT and MLN collected from lean, lactating Holstein cows without apparent disease in an abattoir (n = 42). Lean cows had a body condition score of 2.6 ± 0.1 (mean ± SD) with a greater frequency of adipocyte area occurring in small rather than large adipocytes. Cells were labeled with monoclonal antibodies specific to bovine leukocyte antigens for enumeration by flow cytometry. Within both lymph node and adipose tissues, relatively large subpopulations of cells expressed the ß2 integrins CD11b and CD11c, class II major histocompatibility antigens (MHCII), and the SIIRP-1α receptor (CD172a) typical of dendritic cells and macrophages. Macrophage/dendritic cell heterogeneity was marked by ß2 integrin expression alone or in conjunction with CD172a or MHCII across subpopulations from both tissues; CD209, the DC-SIGN c-type lectin receptor of dendritic cells, was not detected by fluorescence-activated cell sorting in either tissue. Lymphocytes comprised 74.1 ± 3.7% and 13.7 ± 3.7% of the MLN and MAT cell populations, respectively, and CD3+CD4+ lymphocytes accounted for 49.8 ± 9.9% of the MLN and 6.13 ± 1.23% of the MAT cells. Fox P3+ regulatory lymphocytes comprised 15.3 ± 1.1% and 6.73 ± 0.52% of the MLN and MAT cells, whereas γδ+ lymphocytes accounted for 6.65 ± 0.74% and 3.91 ± 0.43% of the MLN and MAT cells, respectively. Subpopulations of CD3+CD8+ cytotoxic T cells and CD3+CD11c+ innate lymphocytes were present in MLN but not MAT. These results show that subpopulations of resident tissue macrophages, dendritic cells, T helper lymphocytes, regulatory T lymphocytes (Tregs), and γδ lymphocytes reside in mesenteric lymph nodes and adipose tissues. Balance in the innate and adaptive immune functions embedded in these tissues could support metabolic health.