Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.     

Respiratory Syncytial Virus-Induced Activation and Migration of Respiratory Dendritic Cells and Subsequent Antigen Presentation in the Lung-Draining Lymph Node

In the respiratory tract, different dendritic cell (DC) populations guard a tight balance between tolerance and immunity to infectious or harmless materials to which the airways are continuously exposed. For infectious and noninfectious antigens administered via different routes, different subsets of DC might contribute during the induction of T-cell tolerance and immunity. We studied the impact of primary respiratory syncytial virus (RSV) infection on respiratory DC composition in C57BL/6 mice. We also tracked the migration of respiratory DC to the lymph nodes and studied antigen presentation by lung-derived and lymph node-resident DC to CD4⁺ and CD8⁺ T cells. We observed a massive influx of mainly CD103⁻ CD11b^high CD11c⁺ conventional DC (cDC) and plasmacytoid DC during the first 7 days of RSV infection, while CD103⁺ CD11b^low CD11c⁺ cDC disappeared from the lung. The two major subsets of lung tissue DC, CD103⁺ CD11b^low CD11c⁺ and CD103⁻ CD11b^high CD11c⁺ cDC, both transported RSV RNA to the lung-draining lymph node. Furthermore, these lung-derived cDC subsets as well as resident LN DC, which did not contain viral RNA, displayed viral antigen by major histocompatibility complex class I and class II to CD8⁺ and CD4⁺ T cells. Taken together, our data indicate that during RSV infections, at least three DC subsets might be involved during the activation of lymph node-homing naïve and memory CD4⁺ and CD8⁺ T cells.Respiratory syncytial virus (RSV) constitutes a major health burden for infants, elderly people, and immunocompromised individuals (16, 19). The virus infects most children in their first year of life and is the main cause of severe lower respiratory tract infections in infants (19). Despite many decades of research, the immune response to RSV is still not completely understood. Infection with RSV leads to poor development of immunity, and recurrent infections are common (23). In mice, it was found that RSV induces virus-specific CD8⁺ T-cell responses in the lung that are functionally impaired (10). It has been suggested that a functional inactivation of CD8⁺ T cells by RSV could be a reason for the short-lived immune response. Furthermore, we and others have previously shown that human monocyte-derived dendritic cells (DC) can be infected with RSV, which results in a strong inhibition of their ability to support proliferative responses and induction of effector function in naïve T cells (11, 12). An early vaccine trial with formalin-inactivated RSV in alum administered intramuscularly elicited a memory immune response that caused a strong aberrant secondary immune response in vaccinees upon natural exposure with live virus. This resulted in a high rate of morbidity in the vaccinated children (31). These observations underscore the necessity to understand the components of the immune response that are protective during RSV infections and the need to understand the mechanism by which protective immunity can be elicited for the development of an effective and safe vaccine.DC play an important role in the initiation of both the innate and adaptive immune responses to pathogens including RSV (3). They are a heterogeneous population of cells represented by two main subsets, the myeloid or “conventional” CD11c⁺ DC (cDC) and the CD11c^low/mPDCA-1⁺ plasmacytoid DC (pDC) (47, 52). cDC can be further divided based on the expression of surface markers and anatomic location. cDC in the tissue and cDC in lymph nodes (LN) appear to be different subsets arising from different pools of progenitor cells and with specialized functions (13, 17, 30, 33, 46). In the mouse lung, two major cDC populations are derived from blood monocytes. CD11c⁺ major histocompatibility complex class II (MHC-II)-positive (MHC-II⁺) CD103⁻ CD11b^high cDC (CD11b^hi cDC) are localized in the parenchyma. These cells are the main producers of chemokines and are important for the recruitment of leukocytes (4). A second cDC population, CD11c⁺ MHC-II⁺ CD103⁺ CD11b^low cDC (CD103⁺ cDC), is located directly underneath the airway epithelium. These CD103⁺ cDC express the integrin α_Eβ₇; therefore, they are found mainly at the basal lamina of the bronchial epithelia and arterioles, which express E-cadherin, the ligand for α_Eβ₇. Furthermore, CD103⁺ cDC express the tight-junction proteins ZO-2 and claudin-7, which enables them to sample the airways with their extensions (45). In the lung-draining LN, in addition to pDC, at least two steady-state populations of cDC are present, which are characterized by the expression or absence of CD8α. In contrast to the lung tissue DC, these cells enter the LN from the blood, and they are directly derived from a bone marrow precursor (38, 39, 41). In addition, minor fractions of tissue-derived cDC also access draining LN in the steady state (28). Several studies have addressed the roles of different DC subsets that are present in the tissue and LN draining the infection site. In spleen and skin-draining LN, the role of CD8α⁺ cDC seems to be important for the initiation of anti-ovalbumin and antiviral CD8⁺ T-cell responses (6, 26, 35). In mice exposed to innocuous (ovalbumin) or infectious (influenza virus) antigen, functional specialization was described for CD103⁺ and CD11b^hi lung cDC subsets. CD11b^hi cDC presented intranasally administered ovalbumin or influenza virus antigen mainly to naïve CD4⁺ T cells, while CD103⁺ cDC were important for the induction of CD8⁺ T-cell responses (14, 32).The ability of DC to present or cross-present antigens depends on the type of antigenic materials and the uptake mechanism used by antigen-presenting cells. Hence, different pathogens and innocuous antigens might be differently presented by different DC subsets. We studied the kinetics of lung DC migration and repopulation during primary RSV infection in C57BL/6 mice. We found that upon RSV infection, CD103⁺ cDC disappeared from the lung, while there was a net increase in numbers of CD11b^hi cDC, pDC, and macrophages. Within the first 48 h after virus exposure, both CD103⁺ and CD11b^hi cDC rapidly migrated to the lung-draining mediastinal LN (MLN), while this accumulation was absent in the non-lung-draining axillary LN. The migrating cDC showed the highest level of expression of the costimulatory molecules CD40, CD80, and CD86, which are necessary for T-cell stimulation, compared to the MLN-resident cDC. Furthermore, the migrating cDC transported viral RNA to the MLN and were capable of stimulating RSV-specific CD4⁺ and CD8⁺ T-cell responses. Resident cDC in the LN were uniformly negative for viral RNA. However, resident cDC in the LN did present viral antigen to CD8⁺ and CD4⁺ T cells via MHC-I and MHC-II, respectively.     

Integrin-Alpha IIb Identifies Murine Lymph Node Lymphatic Endothelial Cells Responsive to RANKL

Microenvironment and activation signals likely imprint heterogeneity in the lymphatic endothelial cell (LEC) population. Particularly LECs of secondary lymphoid organs are exposed to different cell types and immune stimuli. However, our understanding of the nature of LEC activation signals and their cell source within the secondary lymphoid organ in the steady state remains incomplete. Here we show that integrin alpha 2b (ITGA2b), known to be carried by platelets, megakaryocytes and hematopoietic progenitors, is expressed by a lymph node subset of LECs, residing in medullary, cortical and subcapsular sinuses. In the subcapsular sinus, the floor but not the ceiling layer expresses the integrin, being excluded from ACKR4⁺ LECs but overlapping with MAdCAM-1 expression. ITGA2b expression increases in response to immunization, raising the possibility that heterogeneous ITGA2b levels reflect variation in exposure to activation signals. We show that alterations of the level of receptor activator of NF-κB ligand (RANKL), by overexpression, neutralization or deletion from stromal marginal reticular cells, affected the proportion of ITGA2b⁺ LECs. Lymph node LECs but not peripheral LECs express RANK. In addition, we found that lymphotoxin-β receptor signaling likewise regulated the proportion of ITGA2b⁺ LECs. These findings demonstrate that stromal reticular cells activate LECs via RANKL and support the action of hematopoietic cell-derived lymphotoxin.     

Homing of Immunologically Committed Lymph Node Cells to Germinal Centres in Rabbits

IT is well established that B-lymphocytes are the precursors of antibody forming cells^1,2. Germinal centres of peripheral lymphoid tissues are considered to represent foci of proliferating B-lymphocytes arising in response to antigen injection, particularly since neonatal bursectomy but not thymectomy results in an absence of these centres in adult chickens³.     

Marine Sediment Bacteria Harbor Antibiotic Resistance Genes Highly Similar to Those Found in Human Pathogens

The ocean is a natural habitat for antibiotic-producing bacteria, and marine aquaculture introduces antibiotics into the ocean to treat infections and improve aquaculture production. Studies have shown that the ocean is an important reservoir of antibiotic resistance genes. However, there is a lack of understanding and knowledge about the clinical importance of the ocean resistome. We investigated the relationship between the ocean bacterial resistome and pathogenic resistome. We applied high-throughput sequencing and metagenomic analyses to explore the resistance genes in bacterial plasmids from marine sediments. Numerous putative resistance determinants were detected among the resistance genes in the sediment bacteria. We also found that several contigs shared high identity with transposons or plasmids from human pathogens, indicating that the sediment bacteria recently contributed or acquired resistance genes from pathogens. Marine sediment bacteria could play an important role in the global exchange of antibiotic resistance.     

Dendritic Cells Route Human Immunodeficiency Virus to Lymph Nodes after Vaginal or Intravenous Administration to Mice

We have developed a murine model to study the involvement of dendritic cells (DC) in human immunodeficiency virus (HIV) routing from an inoculation site to the lymph nodes (LN). Murine bone marrow-derived DC migrate to the draining LN within 24 h after subcutaneous injection. After incubation of these cells with heat-inactivated (Hi) HIV type 1 (HIV-1), HIV RNA sequences were detected in the draining LN only. Upon injection of DC pulsed with infectious HIV, the virus recovered in the draining LN was still able to productively infect human T cells. After a vaginal challenge with Hi HIV-1, the virus could be detected in the iliac and sacral draining LN at 24 h after injection. After an intravenous challenge, the virus could be detected in peripheral LN as soon as 30 min after injection. The specific depletion of a myeloid-related LN DC population, previously shown to take up blood macromolecules and to translocate them into the LN, prevented HIV transport to LN. Together, our data demonstrate the critical role of DC for HIV routing to LN after either a vaginal or an intravenous challenge, which does not require their infection. Therefore, despite the fact that the mouse is not infectable by HIV, this small animal model might be useful to test preventive strategies against HIV.     

T Cells Kill Bacteria Captured by Transinfection from Dendritic Cells and Confer Protection in Mice

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Lymph Node Stromal Cells Generate Antigen-Specific Regulatory T Cells and Control Autoreactive T and B Cell Responses

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病理分期在颌颈部淋巴结核治疗中的价值

目的:探讨病理分期在颌颈部淋巴结核治疗中的价值,以期寻找颌颈部淋巴结核的最佳治疗方案。方法:收集我科近5年的临床资料,对颌颈部淋巴结核患者的发病及治疗情况作一临床统计,并结合淋巴结核的病理分期对之加以分析,寻找治疗规律。结果:所有颌颈部淋巴结核患者按病理分期采取不同治疗方案,均达到较佳治疗效果,处于病理初期和中期的患者,临床治疗周期明显小于常规化疗。结论:遵从病理分期治疗颌颈部淋巴结核是一种较为科学合理的治疗思路和方法。     

从灾害救援特点看新时期心理卫生支援分队建设

根据自然灾害医学救援要求及组织体制,结合灾害救援过程中的心理压力来源及心理需求,提出心理卫生支援分队建设指导:人员需具备专业技能,兼顾全面技术;采取的工作方式主要为团体合作、小组协作和单人干预等;日常训练重点在于做好实战训练、预案、心理和物资方面的准备,力求使心理卫生支援分队满足新时期非战争军事任务的需要。     

Migration Inhibition Studies on Peripheral Leukocytes and Lymph Node Cells from Ruminants with Fascioliasis

Delayed type hypersensitivity against antigens of Fasciola hepatica has been repeatedly documented in infected hosts. Evidence has been presented to suggest that the delayed reactivity may develop earlier in the regional lymph nodes of the parasitized organ than in other lymph nodes of the body (Soulsby 1971).     

Plasmacytoid Dendritic Cells Accumulate and Secrete Interferon Alpha in Lymph Nodes of HIV-1 Patients

Circulating plasmacytoid dendritic cells (pDC) decline during HIV-1 infection, but at the same time they express markedly higher levels of interferon alpha (IFNα), which is associated with HIV-1 disease progression. Here we show an accumulation of pDC in lymph nodes (LN) of treatment-naïve HIV-1 patients. This phenomenon was associated with elevated expression of the LN homing marker, CCR7, on pDC in peripheral blood of HIV-1 patients, which conferred increased migratory capacity in response to CCR7 ligands in ex vivo functional assays. LN-homed pDC of HIV-1 patients presented higher CD40 and lower BDCA2 levels, but unchanged CD83 and CD86 expression. In addition, these cells expressed markedly higher amounts of IFNα compared to uninfected individuals, and were undergoing faster rates of cell death. These results demonstrate for the first time that in asymptomatic, untreated HIV-1 patients circulating pDC up-regulate CCR7 expression, accumulate in lymph nodes, and express high amounts of IFNα before undergoing cell death. Since IFNα inhibits cell proliferation and modulates immune responses, chronically high levels of this cytokine in LN of HIV-1 patients may impair differentiation and immune function of bystander CD4⁺ T cells, thus playing into the mechanisms of AIDS immunopathogenesis.     

Macrophage Death following Influenza Vaccination Initiates the Inflammatory Response that Promotes Dendritic Cell Function in the Draining Lymph Node

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Adoptive Transfer of Immunity to Plasmodium berghei by Spleen and Lymph Node Cells from Young and Old Mice*

SYNOPSIS. Different numbers of spleen and lymph node cells of 6-week and 6–8 month A/J mice, immune to Plasmodium berghei, were transferred into normal 4-week old mice. Better protection was observed with 2.5 × 10^s than with 10⁷ spleen cells, and spleen cells afforded better protection than an equal number of lymph node cells. Further, spleen cells from older mice were more effective than those from young animals. Possible mechanisms of immunity transfer are discussed.     

Laminin and Fibronectin Treatment Leads to Generation of Dendritic Cells with Superior Endocytic Capacity

Background

Sampling the microenvironment at sites of microbial exposure by dendritic cells (DC) and their subsequent interaction with T cells in the paracortical area of lymph nodes are key events for initiating immune responses. Most of our knowledge of such events in human is based on in vitro studies performed in the absence of extracellular matrix (ECM) proteins. ECM in basement membranes and interstitial spaces of different tissues, including lymphoid organs, plays an important role in controlling specific cellular functions such as migration, intracellular signalling and differentiation. The aim of this study was, therefore, to investigate the impact of two abundant ECM components, fibronectin and laminin, on the phenotypical and functional properties of DC and how that might influence DC induced T-cell differentiation.

Methodology/Principal Findings

Human monocyte derived DC were treated with laminin and fibronectin for up to 48 hours and their morphology and phenotype was analyzed using scanning electron microscopy, flow cytometry and real time PCR. The endocytic ability of DC was determined using flow cytometry. Furthermore, co-culture of DC and T cells were established and T cell proliferation and cytokine profile was measured using H³-thymidine incorporation and ELISA respectively. Finally, we assessed formation of DC-T cell conjugates using different cell trackers and flow cytometry. Our data show that in the presence of ECM, DC maintain a ‘more immature’ phenotype and express higher levels of key endocytic receptors, and as a result become significantly better endocytic cells, but still fully able to mature in response to stimulation as evidenced by their superior ability to induce antigen-specific T cell differentiation.

Conclusion

These studies underline the importance of including ECM components in in vitro studies investigating DC biology and DC-T cell interaction. Within the context of antigen specific DC induced T cell proliferation, inclusion of ECM proteins could lead to development of more sensitive assays.