Tumor-Draining Lymph Node Reconstruction Promotes B Cell Activation During E0771 Mouse Breast Cancer Growth.

Lymph node metastasis is associated with tumor aggressiveness and poor prognosis in patients. Despite its significance in cancer progression, how immune cells in the tumor-draining lymph node (TDLN) participate in cancer immune regulation remains poorly understood. It has been reported that both anti-tumor and exhausted tumor-specific T cells can be induced in the TDLNs; however, B cell activation and maturation in the TDLN has received far less attention. In our studies using C57BL/6 mouse syngeneic E0771 breast cancer or B16F10 melanoma cell lines, tumor-associated antigens were found colocalized with the follicular dendritic cells (FDCs) in the germinal centers (GCs), where antigen-specific B cell maturation occurs. LN conduits and the subcapsular sinus (SCS) macrophages are two major routes of antigen trafficking to FDCs. Tumor growth induced LN conduit expansion in the B cell zone and disrupted the SCS macrophage layer, facilitating both the entry of tumor-associated antigens into the B cell zone and access to FDCs located in the GCs. Regional delivery of clodronate liposome specifically depleted SCS macrophages in the TDLN, increasing GC formation, and promoting tumor growth. Our study suggests that TDLN reconstruction creates a niche that favors B cell activation and maturation during tumor growth.

Absence of p21(WAF1/CIP1/SDI1) protects against osteopenia and minimizes bone loss after ovariectomy in a mouse model.

p21(WAF1/CIP1/SDI1) is a critical sentinel of the cell cycle that plays an important role in determining cell fate with respect to proliferation, differentiation and apoptosis. Recent studies have demonstrated that inhibition/loss of p21 promotes osteo-chondro differentiation in progenitor/stem cells, and that p21 knockout (p21-/-) mice demonstrate enhanced bone regeneration compared to wild-type controls after a non-critical size defect. It was therefore hypothesized that the absence of p21 may also protect against bone loss through enhancing bone formation, tilting the balance away from bone resorption, in an ovariectomy-induced osteopenia mouse model, investigated via microCT imaging. While p21-/- mice demonstrated significantly less bone loss after ovariectomy compared to wild-type controls, no increase in the number osteoclasts or osteoblasts in the bone or bone marrow was observed, nor was there an increase in osteoclast activity. Therefore, while the absence of p21 protected mice against estrogen mediated bone loss, the mechanisms/pathways responsible remained elusive. This study demonstrates that p21 may play a significant role in bone remodeling, and a better understanding of how the p21 pathway regulates bone anabolism and catabolism could lead to novel therapies for osteoporosis in the future.

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.

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.

Elastin Shapes Small Molecule Distribution in Lymph Node Conduits.

The spatial and temporal Ag distribution determines the subsequent T cell and B cell activation at the distinct anatomical locations in the lymph node (LN). It is well known that LN conduits facilitate small Ag distribution in the LN, but the mechanism of how Ags travel along LN conduits remains poorly understood. In C57BL/6J mice, using FITC as a fluorescent tracer to study lymph distribution in the LN, we found that FITC preferentially colocalized with LN capsule-associated (LNC) conduits. Images generated using a transmission electron microscope showed that LNC conduits are composed of solid collagen fibers and are wrapped with fibroblastic cells. Superresolution images revealed that high-intensity FITC is typically colocalized with elastin fibers inside the LNC conduits. Whereas tetramethylrhodamine isothiocyanate appears to enter LNC conduits as effectively as FITC, fluorescently-labeled Alexa-555-conjugated OVA labels significantly fewer LNC conduits. Importantly, injection of Alexa-555-conjugated OVA with LPS substantially increases OVA distribution along elastin fibers in LNC conduits, indicating immune stimulation is required for effective OVA traveling along elastin in LN conduits. Finally, elastin fibers preferentially surround lymphatic vessels in the skin and likely guide fluid flow to the lymphatic vessels. Our studies demonstrate that fluid or small molecules are preferentially colocalized with elastin fibers. Although the exact mechanism of how elastin fibers regulate Ag trafficking remains to be explored, our results suggest that elastin can be a potentially new target to direct Ag distribution in the LN during vaccine design.

p21-/- mice exhibit enhanced bone regeneration after injury.

BACKGROUND: p21(WAF1/CIP1/SDI1), a cyclin dependent kinase inhibitor has been shown to influence cell proliferation, differentiation and apoptosis; but more recently, p21 has been implicated in tissue repair. Studies on p21(-/-) knockout mice have demonstrated results that vary from complete regeneration and healing of tissue to attenuated healing. There have however been no studies that have evaluated the role of p21 inhibition in bone healing and remodeling. METHODS: The current study employs a burr-hole fracture model to investigate bone regeneration subsequent to an injury in a p21-/- mouse model. p21-/- and C57BL/6 mice were subjected to a burr-hole fracture on their proximal tibia, and their bony parameters were measured over 4 weeks via in vivo µCT scanning. RESULTS: p21-/- mice present with enhanced healing from week 1 through week 4. Differences in bone formation and resorption potential between the two mouse models are assessed via quantitative and functional assays. While the µCT analysis indicates that p21-/- mice have enhanced bone healing capabilities, it appears that the differences observed may not be due to the function of osteoblasts or osteoclasts. Furthermore, no differences were observed in the differentiation of progenitor cells (mesenchymal or monocytic) into osteoblasts or osteoclasts respectively. CONCLUSIONS: Therefore, it remains unknown how p21 is regulating enhanced fracture repair and further studies are required to determine which cell type(s) are responsible for this regenerative phenotype.