Efferent Lymph Research Articles

Acute Neuroinflammation Alters the Transport of a Model Therapeutic Protein from the Brain into Lymph and Blood.

The drainage of fluid and solutes along lymphatic pathways from the brain has been found to be impaired in mouse models of multiple sclerosis, Alzheimer's disease, and Parkinson's disease where neuroinflammation is present. We recently demonstrated that 3H-albumin, a model therapeutic protein (∼65 kDa), undergoes preferential lymphatic transport from the brain using a cervical lymph cannulation model in healthy rats. We thus hypothesized that neuroinflammation would impede the lymphatic transport of 3H-albumin from the brain. Our aim was to quantify the impact of acute neuroinflammation on drainage of the model therapeutic protein (3H-albumin) from the rat brain into blood and deep cervical lymph. To establish the required neuroinflammation model, male Sprague-Dawley rats were administered an intraperitoneal (IP) dose of 0.5-2 mg/kg lipopolysaccharide (LPS, Escherichia coli) or a saline control. After 12 or 24 h, brain samples were collected and analyzed for concentrations of interferon gamma (IFN-γ) using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The impact of neuroinflammation on the drainage of 3H-albumin from the brain was determined via IP administration of 2 mg/kg LPS or saline followed by cannulation of the carotid artery for blood collection 24 h later with/without cannulation or ligation at the efferent deep cervical lymph trunk. Rats were then administered 3H-albumin via direct injection into the brain striatum or via intravenous (IV) injection (lymph-intact group only). Blood ± lymph samples were collected for up to 8 h following dosing. At the end of the study, brain and lymph node samples were harvested for biodistribution analysis, with samples analyzed for radioactivity levels via scintillation counting. Brain concentrations of the pro-inflammatory cytokine IFN-γ were only significantly elevated 24 h after IP administration of 2 mg/kg LPS compared to saline control. Therefore, this induction regimen was utilized for subsequent studies. The plasma concentrations of 3H-albumin over time were elevated in LPS-induced rats compared to saline-injected rats in the lymph-intact and lymph-ligated groups but not in the lymph-cannulated group. In the deep cervical lymph-cannulated animals, the lymph transport of 3H-albumin was not increased and appeared to be slower in the LPS-administered rats. Acute LPS-induced neuroinflammation therefore led to an enhanced overall transport of 3H-albumin from the brain into the systemic circulation. This appeared to be primarily due to increased transport of 3H-albumin from the brain directly into the blood circulation as 3H-albumin transport from the brain via the lymphatics was not increased in the LPS-induced neuroinflammation model. Such changes in the clearance of therapeutic proteins from the brain in the setting of neuroinflammation may impact the therapeutic efficacy and safety.

Spatial Characterization of the Immune Microenvironment within HIV+ Lymph Nodes

Abstract Background: Persistent HIV replication is hypothesized to induce chronic inflammation in ART-suppressed people living with HIV (PLWH), however defining this chronic inflammation within tissues is limited. Molecular and spatial characterization of chronic inflammation within human lymph nodes from PLWH provide valuable information on the immunological microenvironment that limits viral eradication. Methods: We spatially characterized the transcriptomic and proteomic profiles of the lymph node microenvironment from 6 human lymph nodes: three from ART-suppressed PLWH, two from chronically infected PLWH, and one from a male HIV- control donor. For spatial transcriptomics analysis, lymph nodes were either from fresh-frozen or FFPE tissue. Lymph node sections (10μm) were placed onto the 10X Visium slides, stained with H&E, and imaged prior to RNA-Seq library prep. Following sequencing cluster identification was done with single cell RNA-seq datasets and downstream bioinformatics analyses were performed. Results: Following the bioinformatic integration of spatial transcriptomic datasets, cluster analysis identified pro-inflammatory T cells within the HIV+ tissues compared to the HIV- tissue within the T cell zone. Additionally, two sets of proinflammatory myeloid cells were identified that were spatially located within the afferent and efferent lymphatics in the HIV+ tissues. These proinflammatory clusters further characterize chronic inflammation that occurs in PLWH.

Supermicrosurgery lymphaticovenous and lymphaticolymphatic anastomosis: Technical detail and short-term follow-up for immediate lymphatic reconstruction in breast cancer treatment-related lymphedema prevention

ObjectiveWe describe the feasibility and short-term outcome of our surgical technique to repair the lymph vessel disruption directly after axillary lymph node dissection during breast cancer surgery. This procedure is called immediate lymphatic reconstruction to prevent breast cancer treatment-related lymphedema (BCRL), which frequently occurs after axillary lymph node dissection. The surgical technique consisted of lymphaticovenous anastomosis (LVA) or lymphaticolymphatic anastomosis. We named the procedure lymphatic bypass supermicrosurgery (LBS). MethodsThis study used a retrospective cohort design of patients with breast cancer between May 2020 and February 2023. LBS was performed by making an intima-to-intima coaptation between afferent lymph vessels and the recipient's veins (LVA) or efferent lymph vessels lymphaticolymphatic anastomosis. ResultsA total of 82 patients underwent lymphatic bypass. The mean age of patients was 50 ± 12 years, and most had stage III breast cancer (n = 59 [72%]). LVA was the most common type of lymphatic bypass (94.6%). The median number of LVA was 1 (range, 1-4) and 1 (range, 1-3) for lymphaticolymphatic anastomosis. The median follow-up time was 12.5 months (range, 1-33 months). The 50 patients who had postoperative indocyanine green lymphography described arm dermal backflow stage 0 in 20 (40%), stage 1 in 19 (38%), stage 2 in 2 (4%), and stage 3 in 9 (18%) cases. The proportion of BCRL was 11 (22%), and subclinical lymphedema was 19 (38%) in this period. Most cases were in stable subclinical lymphedema (10, 58.8%). The 1-year and 2-year BCRL rates were 14% (95% confidence interval, 4%-23.9%) and 22% (95% confidence interval, 10.1%-33.9%), respectively. ConclusionsAlong with the emerging immediate lymphatic reconstruction, LBS is a feasible supermicrosurgery technique that may have a potential role in BCRL prevention. A randomized controlled study would confirm the effectiveness of the technique.

Plasma Volume Oscillations during Fluid Therapy in Humans

Introduction: Oscillations are frequently observed on plasma dilution curves during intravenous fluid therapy. This study aimed to examine how common these oscillations are and what they represent. Methods: Fourier transforms were used to analyze the residuals obtained during fitting of a volume kinetic model to 269 plasma dilution curves. Oscillating patterns were identified in two-thirds of the fluid infusion experiments. Results: The wave frequency usually had a dominating frequency of 1 h or multiples thereof. The wave amplitudes varied between 1% and 4% of the plasma volume. The “peak-to-peak” amplitudes were then twice as large, which corresponded to blood volume changes of 60–240 mL. A population kinetic analysis of the distribution of infused fluid between body fluid compartments was then applied to search for clues that could explain the oscillations. This analysis showed that amplitudes >1.5% were associated with doubled turnover of fluid in a fast-exchange interstitial fluid compartment and, together with data on plasma albumin, suggested that oscillations might represent bursts of efferent lymph. Conclusions: Oscillations with very low frequency were often observed on plasma dilution-time curves obtained during fluid therapy. They were associated with fast turnover of interstitial fluid and can possibly have resulted from accelerated lymphatic flow.

Naive T lymphocytes chemotax long distance to CCL21 but not to a source of bioactive S1P

Naive T lymphocytes traffic through the organism in search for antigen, alternating between blood and secondary lymphoid organs. Lymphocyte homing to lymph nodes relies on CCL21 chemokine sensing by CCR7 receptors, while exit into efferent lymphatics relies on sphingolipid S1P sensing by S1PR1 receptors. While both molecules are claimed chemotactic, a quantitative analysis of naive T lymphocyte migration along defined gradients is missing. Here, we used a reductionist approach to study the real-time single-cell response of naive T lymphocytes to CCL21 and serum rich in bioactive S1P. Using microfluidic and micropatterning ad hoc tools, we show that CCL21 triggers stable polarization and long-range chemotaxis of cells, whereas S1P-rich serum triggers a transient polarization only and no significant displacement, potentially representing a brief transmigration step through exit portals. Our invitro data thus suggest that naive T lymphocyte chemotax long distances to CCL21 but not toward a source of bioactive S1P.

Utilization of endogenous albumin trafficking pathways in the lungs has potential to modestly increase the lung interstitial access and absorption of drug delivery systems after inhaled administration

ABSTRACT Objectives Drug delivery systems typically show limited access to the lung interstitium and absorption after pulmonary delivery. The aim of this work was to undertake a proof-of-concept investigation into the potential of employing endogenous albumin and albumin absorption mechanisms in the lungs to improve lung interstitial access and absorption of inhaled drug delivery systems that bind albumin. Methods The permeability of human albumin (HSA) through monolayers of primary human alveolar epithelia, small airway epithelia, and microvascular endothelium were investigated. The pulmonary pharmacokinetics of bovine serum albumin (BSA) was also investigated in efferent caudal mediastinal lymph duct-cannulated sheep after inhaled aerosol administration. Results Membrane permeability coefficient values (Papp) of HSA increased in the order alveolar epithelia<small airway epithelia<microvascular endothelium, where the permeability of HSA through small airway and microvascular endothelia were approximately 4- and 28-fold higher than alveolar epithelia, respectively. Only 6.5% of the delivered BSA aerosol dose was absorbed from the lungs of sheep over 5 days, although half of the absorbed dose was absorbed via the lung lymph. Conclusion Drug delivery systems that bind endogenous albumin may show a modest increase in lung permeability and absorption after inhaled delivery compared to systems that do not efficiently bind albumin.

Recruitment of efferent lymph during infusion of 20% albumin.

Intravenous infusion of hyper-oncotic 20% albumin expands the plasma volume by approximately twice the infused volume. We investigated whether the recruited fluid stems from accelerated flow of efferent lymph, which would add protein to the plasma, or from reversed transcapillary solvent filtration, where the solvent is expected to be low in protein. We analyzed data from 27 intravenous infusions of 20% albumin (3mL/kg; approximately 200mL) over 30min given to 27 volunteers and patients. Twelve of the volunteers were also given a 5% solution and served as controls. The pattern of blood hemoglobin, colloid osmotic pressure, and the plasma concentrations of two immunoglobulins (IgG and IgM) were studied over 5h. A decrease of the difference between the plasma colloid osmotic pressure and plasma albumin occurred during the infusions and was almost four times greater for 5% albumin than for 20% albumin at 40min (P<0.0036), which indicates that non-albumin protein enriched the plasma when 20% was infused. Moreover, the difference between the infusion-derived dilution of the blood plasma based on hemoglobin and the two immunoglobulins amounted to -1.9% (-6 to +0.2) for 20% albumin and to -4.4% (25th-75th percentile range-8.5 to +0.2) during experiments with 5% albumin (P<0.001). This supports that the plasma was enriched by immunoglobulins, probably via the lymph, when 20% was infused. Between half and two-thirds of the extravascular fluid that was recruited during infusion of 20% albumin in humans consisted of protein-containing fluid consistent with efferent lymph.

Isolation of lymph shows dysregulation of STAT3 and CREB pathways in the spleen and liver during leukemia development in a rat model.

Acute myeloid leukemia (AML) is a heterogeneous malignant condition characterized by massive infiltration of poorly differentiated white blood cells in the blood stream, bone marrow, and extramedullary sites. During leukemic development, hepatosplenomegaly is expected to occur because large blood volumes are continuously filtered through these organs. We asked whether infiltration of leukemic blasts initiated a response that could be detected in the interstitial fluid phase of the spleen and liver. We used a rat model known to mimic human AML in growth characteristics and behavior. By cannulating efferent lymphatic vessels from the spleen and liver, we were able to monitor the response of the microenvironment during AML development. Flow cytometric analysis of lymphocytes showed increased STAT3 and CREB signaling in spleen and depressed signaling in liver, and proteins related to these pathways were identified with a different profile in lymph and plasma in AML compared with control. Additionally, several proteins were differently regulated in the microenvironment of spleen and liver in AML when compared with control. Interstitial fluid, and its surrogate efferent lymph, can be used to provide unique information about responses in AML-infiltered organs and substances released to the general circulation during leukemia development.

Development and application of a novel cervical lymph collection method to assess lymphatic transport in rats.

Background: Fluids, solutes and immune cells have been demonstrated to drain from the brain and surrounding structures to the cervical lymph vessels and nodes in the neck via meningeal lymphatics, nasal lymphatics and/or lymphatic vessels associated with cranial nerves. A method to cannulate the efferent cervical lymph duct for continuous cervical lymph fluid collection in rodents has not been described previously and would assist in evaluating the transport of molecules and immune cells from the head and brain via the lymphatics, as well as changes in lymphatic transport and lymph composition with different physiological challenges or diseases. Aim: To develop a novel method to cannulate and continuously collect lymph fluid from the cervical lymph duct in rats and to analyze the protein, lipid and immune cell composition of the collected cervical lymph fluid. Methods: Male Sprague-Dawley rats were cannulated at the carotid artery with or without cannulation or ligation at the cervical lymph duct. Samples of blood, whole lymph and isolated lipoprotein fractions of lymph were collected and analyzed for lipid and protein composition using commercial kits. Whole lymph samples were centrifuged and isolated pellets were stained and processed for flow cytometry analysis of CD3+, CD4+, CD8a+, CD45R+ (B220) and viable cell populations. Results: Flow rate, phospholipid, triglyceride, cholesterol ester, free cholesterol and protein concentrations in cervical lymph were 0.094 ± 0.014mL/h, 0.34 ± 0.10, 0.30 ± 0.04, 0.07 ± 0.02, 0.02 ± 0.01 and 16.78 ± 2.06mg/mL, respectively. Protein was mostly contained within the non-lipoprotein fraction but all lipoprotein types were also present. Flow cytometry analysis of cervical lymph showed that 67.1 ± 7.4% of cells were CD3+/CD4+ T lymphocytes, 5.8 ± 1.6% of cells were CD3+/CD8+ T lymphocytes, and 10.8 ± 4.6% of cells were CD3-/CD45R+ B lymphocytes. The remaining 16.3 ± 4.6% cells were CD3-/CD45- and identified as non-lymphocytes. Conclusion: Our novel cervical lymph cannulation method enables quantitative analysis of the lymphatic transport of immune cells and molecules in the cervical lymph of rats for the first time. This valuable tool will enable more detailed quantitative analysis of changes to cervical lymph composition and transport in health and disease, and could be a valuable resource for discovery of biomarkers or therapeutic targets in future studies.

Leukocyte Trafficking in Lymphatic Vessels.

To ensure proper immune function, most leukocytes constantly move within tissues or between them using the blood and lymphatic vessels as transport routes. While afferent lymphatic vessels transfer leukocytes from peripheral tissues to draining lymph nodes (dLNs), efferent lymphatics return lymphocytes from LNs back into the blood vascular circulation. Over the last decades, great progress has been made in our understanding of leukocyte migration into and within the lymphatic compartment, leading to the approval of new drugs targeting this process. In this review, we first introduce the anatomy of the lymphatic vasculature and the main cell types migrating through lymphatics. We primarily focus on dendritic cells (DCs) and T cells, the most prominent lymph-borne cell types, and discuss the functional significance as well as the main molecules and steps involved in their migration. Additionally, we provide an overview of the different techniques used to study lymphatic trafficking.

Antigen-Presenting B Cells Program the Efferent Lymph T Helper Cell Response.

B cells interact with T follicular helper (Tfh) cells in germinal centers (GCs) to generate high-affinity antibodies. Much less is known about how cognate T–B-cell interactions influence Th cells that enter circulation and peripheral tissues. Therefore, we generated mice lacking MHC-II expressing B cells and, by thoracic duct cannulation, analyzed Th cells in the efferent lymph at defined intervals post-immunization. Focusing on gut-draining mesenteric lymph nodes (MLNs), we show that antigen-specific α4β7 + gut-homing effector Th cells enter the circulation prior to CXCR5+PD-1+ Tfh-like cells. B cells appear to have no or limited impact on the early generation and egress of gut-homing Th cells but are critical for the subsequent appearance of Tfh-like cells that peak in the lymph before GCs have developed. At this stage, antigen-presenting B cells also reduce the proportion of α4β7 + Th cells in the MLN and efferent lymph. Furthermore, cognate B-cell interaction drives a broad transcriptional program in Th cells, including IL-4 that is confined to the Tfh cell lineage. The IL-4-producing Tfh-like cells originate from Bcl6+ precursors in the LNs and have gut-homing capacity. Hence, B cells program the efferent lymph Th cell response within a limited window of time after antigenic challenge.

In Sickness and in Health: The Immunological Roles of the Lymphatic System.

The lymphatic system plays crucial roles in immunity far beyond those of simply providing conduits for leukocytes and antigens in lymph fluid. Endothelial cells within this vasculature are distinct and highly specialized to perform roles based upon their location. Afferent lymphatic capillaries have unique intercellular junctions for efficient uptake of fluid and macromolecules, while expressing chemotactic and adhesion molecules that permit selective trafficking of specific immune cell subsets. Moreover, in response to events within peripheral tissue such as inflammation or infection, soluble factors from lymphatic endothelial cells exert “remote control” to modulate leukocyte migration across high endothelial venules from the blood to lymph nodes draining the tissue. These immune hubs are highly organized and perfectly arrayed to survey antigens from peripheral tissue while optimizing encounters between antigen-presenting cells and cognate lymphocytes. Furthermore, subsets of lymphatic endothelial cells exhibit differences in gene expression relating to specific functions and locality within the lymph node, facilitating both innate and acquired immune responses through antigen presentation, lymph node remodeling and regulation of leukocyte entry and exit. This review details the immune cell subsets in afferent and efferent lymph, and explores the mechanisms by which endothelial cells of the lymphatic system regulate such trafficking, for immune surveillance and tolerance during steady-state conditions, and in response to infection, acute and chronic inflammation, and subsequent resolution.

The Identity of Human Tissue-Emigrant CD8+ T Cells

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were Tcells. Cytolytic CD8+ Tcell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8+ Tcell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8+ Tcells that recirculate via TDL.

Extracellular bacterial lymphatic metastasis drives Streptococcus pyogenes systemic infection

Unassisted metastasis through the lymphatic system is a mechanism of dissemination thus far ascribed only to cancer cells. Here, we report that Streptococcus pyogenes also hijack lymphatic vessels to escape a local infection site, transiting through sequential lymph nodes and efferent lymphatic vessels to enter the bloodstream. Contrasting with previously reported mechanisms of intracellular pathogen carriage by phagocytes, we show S. pyogenes remain extracellular during transit, first in afferent and then efferent lymphatics that carry the bacteria through successive draining lymph nodes. We identify streptococcal virulence mechanisms important for bacterial lymphatic dissemination and show that metastatic streptococci within infected lymph nodes resist and subvert clearance by phagocytes, enabling replication that can seed intense bloodstream infection. The findings establish the lymphatic system as both a survival niche and conduit to the bloodstream for S. pyogenes, explaining the phenomenon of occult bacteraemia. This work provides new perspectives in streptococcal pathogenesis with implications for immunity.

Neutrophils Recirculate through Lymph Nodes to Survey Tissues for Pathogens.

The adaptive immune function of lymph nodes is dependent on constant recirculation of lymphocytes. In this article, we identify neutrophils present in the lymph node at steady state, exhibiting the same capacity for recirculation. In germ-free mice, neutrophils still recirculate through lymph nodes, and in mice cohoused with wild microbiome mice, the level of neutrophils in lymph nodes increases significantly. We found that at steady state, neutrophils enter the lymph node entirely via L-selectin and actively exit via efferent lymphatics via an S1P dependent mechanism. The small population of neutrophils in the lymph node can act as reconnaissance cells to recruit additional neutrophils in the event of bacterial dissemination to the lymph node. Without these reconnaissance cells, there is a delay in neutrophil recruitment to the lymph node and a reduction in swarm formation following Staphylococcus aureus infection. This ability to recruit additional neutrophils by lymph node neutrophils is initiated by LTB4. This study establishes the capacity of neutrophils to recirculate, much like lymphocytes via L-selectin and high endothelial venules in lymph nodes and demonstrates how the presence of neutrophils at steady state fortifies the lymph node in case of an infection disseminating through lymphatics.

Mechanism of sphingosine 1-phosphate clearance from blood.

The interplay of sphingosine 1-phosphate (S1P) synthetic and degradative enzymes as well as S1P exporters creates concentration gradients that are a fundamental to S1P biology. Extracellular S1P levels, such as in blood and lymph, are high relative to cellular S1P. The blood-tissue S1P gradient maintains endothelial integrity while local S1P gradients influence immune cell positioning. Indeed, the importance of S1P gradients was recognized initially when the mechanism of action of an S1P receptor agonist used as a medicine for multiple sclerosis was revealed to be inhibition of T-lymphocytes’ recognition of the high S1P in efferent lymph. Furthermore, the increase in erythrocyte S1P in response to hypoxia influences oxygen delivery during high altitude acclimatization. However, understanding of how S1P gradients are maintained is incomplete. For example, S1P is synthesized but is only slowly metabolized by blood yet circulating S1P turns over quickly by an unknown mechanism. Prompted by the counterintuitive observation that blood S1P increases markedly in response to inhibition S1P synthesis (by sphingosine kinase 2 (SphK2)), we studied mice wherein several tissues were made deficient in either SphK2 or S1P degrading enzymes. Our data reveal a mechanism whereby S1P is de-phosphorylated at the hepatocyte surface and the resulting sphingosine is sequestered by SphK phosphorylation and in turn degraded by intracellular S1P lyase. Thus, we identify the liver as the primary site of blood S1P clearance and provide an explanation for the role of SphK2 in this process. Our discovery suggests a general mechanism whereby S1P gradients are shaped.

Immune Profiling of Human Gut-Associated Lymphoid Tissue Identifies a Role for Isolated Lymphoid Follicles in Priming of Region-Specific Immunity

The intestine contains some of the most diverse and complex immune compartments in the body. Here we describe a method for isolating human gut-associated lymphoid tissues (GALTs) that allows unprecedented profiling of the adaptive immune system in submucosal and mucosal isolated lymphoid follicles (SM-ILFs and M-ILFs, respectively) as well as in GALT-free intestinal lamina propria (LP). SM-ILF and M-ILF showed distinct patterns of distribution along the length of the intestine, were linked to the systemic circulation through MAdCAM-1+ high endothelial venules and efferent lymphatics, and had immune profiles consistent with immune-inductive sites. IgA sequencing analysis indicated that human ILFs are sites where intestinal adaptive immune responses are initiated in an anatomically restricted manner. Our findings position ILFs as key inductive hubs for regional immunity in the human intestine, and the methods presented will allow future assessment of these compartments in health and disease.

SPECT/CT and fusion ultrasound to target the efferent groin lymph node for lymphatic surgery.

Pelvic lymphadenectomy (PL) causes changes to the inguinal lymph nodes with progressive loss of immune and lymphatic pump function. Efferent lymphatic vessel-to-venous anastomosis (ELVA) has been reported to address this problem. The aim of this report was to describe the feasibility of the SPECT/CT combined with ultrasound fusion imaging (UFI) to target the groin efferent lymph node (GELN) for ELVA. Twelve patients with lower limb lymphedema after PL were scheduled for peripheric lymphaticovenular anastomosis (LVA) combined with ELVA. All-patients were clinically ISL-stage1, with good visualization of the inguinal lymph nodes at preoperative lymphoscintigraphy. The mean patient age was 55.4 years and the mean BMI was 25.5.The mean limb circumference (MLC) was calculated before surgery and 1 year after surgery. The LymQoL-Leg questionnaire was administered before surgery and 6 months after surgery. Before surgery, the GELN was identified by SPECT/CT and its location was marked on the skin by UFI virtual navigation. Peripheric LVA sites were planned by ultrasound and indocyanine green (ICG) lymphography. Pre and postoperative MLC and LymQoL-Leg scores were compared. In all-patients, the SPECT/CT succeeded at detecting and targeting the GELN. In all-patients, real-time anatomical coregistration with US was feasible, and it was possible to mark on the groin skin the depth and position of the GELN on the skin at the groin. During surgery, in every patient, we found the GELN marked before surgery and performed ELVA. We also performed two or three peripheric LVAs in every patient. The mean value of MLC decreased (38.2 ± 2.13 cm vs. 36.33 ± 2.14 cm; p = .04) and the mean score of the LymQoL-Leg questionnaire improved (9.3 ± 1.7 vs. 7.7 ± 1.1; p = .02). SPECT/CT combined with UFI is feasible for the preoperative identification of GELN for ELVA.

Estimating Residence Times of Lymphocytes in Ovine Lymph Nodes.

The ability of lymphocytes to recirculate between blood and secondary lymphoid tissues such as lymph nodes (LNs) and spleen is well established. Sheep have been used as an experimental system to study lymphocyte recirculation for decades and multiple studies document accumulation and loss of intravenously (i.v.) transferred lymphocytes in efferent lymph of various ovine LNs. Yet, surprisingly little work has been done to accurately quantify the dynamics of lymphocyte exit from the LNs and to estimate the average residence times of lymphocytes in ovine LNs. In this work we developed a series of mathematical models based on fundamental principles of lymphocyte recirculation in the body under non-inflammatory (resting) conditions. Our analysis suggested that in sheep, recirculating lymphocytes spend on average 3 h in the spleen and 20 h in skin or gut-draining LNs with a distribution of residence times in LNs following a skewed gamma (lognormal-like) distribution. Our mathematical models also suggested an explanation for a puzzling observation of the long-term persistence of i.v. transferred lymphocytes in the efferent lymph of the prescapular LN (pLN); the model predicted that this is a natural consequence of long-term persistence of the transferred lymphocytes in circulation. We also found that lymphocytes isolated from the skin-draining pLN have a 2-fold increased entry rate into the pLN as opposed to the mesenteric (gut-draining) LN (mLN). Likewise, lymphocytes from mLN had a 3-fold increased entry rate into the mLN as opposed to entry rate into pLN. In contrast, these cannulation data could not be explained by preferential retention of cells in LNs of their origin. Taken together, our work illustrates the power of mathematical modeling in describing the kinetics of lymphocyte migration in sheep and provides quantitative estimates of lymphocyte residence times in ovine LNs.

T follicular helper cells in human efferent lymph retain lymphoid characteristics.

T follicular helper cells (Tfh), a subset of CD4+ T cells, provide requisite help to B cells in the germinal centers (GC) of lymphoid tissue. GC Tfh are identified by high expression of the chemokine receptor CXCR5 and the inhibitory molecule PD-1. Although more accessible, blood contains lower frequencies of CXCR5+ and PD-1+ cells that have been termed circulating Tfh (cTfh). However, it remains unclear whether GC Tfh exit lymphoid tissues and populate this cTfh pool. To examine exiting cells, we assessed the phenotype of Tfh present within the major conduit of efferent lymph from lymphoid tissues into blood, the human thoracic duct. Unlike what was found in blood, we consistently identified a CXCR5-bright PD-1-bright (CXCR5BrPD-1Br) Tfh population in thoracic duct lymph (TDL). These CXCR5BrPD-1Br TDL Tfh shared phenotypic and transcriptional similarities with GC Tfh. Moreover, components of the epigenetic profile of GC Tfh could be detected in CXCR5BrPD-1Br TDL Tfh and the transcriptional imprint of this epigenetic signature was enriched in an activated cTfh subset known to contain vaccine-responding cells. Together with data showing shared TCR sequences between the CXCR5BrPD-1Br TDL Tfh and cTfh, these studies identify a population in TDL as a circulatory intermediate connecting the biology of Tfh in blood to Tfh in lymphoid tissue.