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Background Aims: Manufacturing of potent chimeric antigen receptor (CAR) T cells requires phenotypically naive and early memory T cells. We hypothesized lymphatic fluid collected from the thoracic duct of children would serve as a unique reservoir for early T cells, which could then be used for CAR T-cell therapy., Methods: We evaluated lymphatic fluid collected from 25 pediatric patients undergoing thoracic duct cannulation for other clinical indications., Results: Lymphatic fluid in the thoracic duct was rich in T cells, with higher percentage of naive and stem central memory T-cell subsets compared with paired blood samples. T cells from lymphatic fluid showed decreased negative checkpoint regulators on the surface and increased rapid expansion with bead activation. Creation of CD19-directed CAR T cells from blood and lymphatic T cells showed similar lentiviral transduction properties, but CAR T cells generated from lymphatic fluid produced superior cytotoxicity in a murine leukemia model because they were able to achieve equivalent tumor eradication at lower doses., Conclusions: These results are the first characterization of T cells from the thoracic duct of pediatric patients and suggest an alternative approach for manufacturing of cellular therapy that will improve both expansion and cytotoxic effect., Competing Interests: Declarations of Competing Interest SAG reports grants and personal fees from Novartis; reports grants from Kite and Servier; reports personal fees from and is on advisory boards for Cellectis, Adaptimmune, Eureka, TCR2, Juno, GlaxoSmithKline, Vertex, Cure Genetics, Humanigen, Roche and Cellular Biomedicine Group; and has a patent of toxicity management for anti-tumor activity of CARs (WO 2014011984 A1) issued. The other authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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.

Mechanical loading conditions are likely to play a key role in passive and active (contractile) behaviour of lymphatic vessels. The development of a microstructurally motivated model of lymphatic tissue is necessary for quantification of mechanically mediated maladaptive remodelling in the lymphatic vasculature. Towards this end, we performed cylindrical biaxial testing of Sprague-Dawley rat thoracic ducts (n = 6) and constitutive modelling to characterize their mechanical behaviour. Spontaneous contraction was quantified at transmural pressures of 3, 6 and 9 cmH2O. Cyclic inflation in calcium-free saline was performed at fixed axial stretches between 1.30 and 1.60, while recording pressure, outer diameter and axial force. A microstructurally motivated four-fibre family constitutive model originally proposed by Holzapfel et al. (Holzapfel et al. 2000 J. Elast. 61, 1-48. (doi:10.1023/A:1010835316564)) was used to quantify the passive mechanical response, and the model of Rachev and Hayashi was used to quantify the active (contractile) mechanical response. The average error between data and theory was 8.9 ± 0.8% for passive data and 6.6 ± 2.6% and 6.8 ± 3.4% for the systolic and basal conditions, respectively, for active data. Multi-photon microscopy was performed to quantify vessel wall thickness (32.2 ± 1.60 µm) and elastin and collagen organization for three loading conditions. Elastin exhibited structural 'fibre families' oriented nearly circumferentially and axially. Sample-to-sample variation was observed in collagen fibre distributions, which were often non-axisymmetric, suggesting material asymmetry. In closure, this paper presents a microstructurally motivated model that accurately captures the biaxial active and passive mechanical behaviour in lymphatics and offers potential for future research to identify parameters contributing to mechanically mediated disease development., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

Calcium channel blockers (CCB) are widely prescribed anti-hypertensive agents. The commonest side-effect, peripheral oedema, is attributed to a larger arterial than venous dilatation causing increased fluid filtration. Whether CCB treatment is detrimental to human lymphatic vessel function and thereby exacerbates oedema formation is unknown. We observed that spontaneous lymphatic contractions in isolated human vessels (thoracic duct and mesenteric lymphatics) maintained under isometric conditions were inhibited by therapeutic concentrations (nanomolar) of the CCB nifedipine while higher than therapeutic concentrations of verapamil (micromolar) were necessary to inhibit activity. Nifedipine also inhibited spontaneous action potentials measured by sharp microelectrodes. Furthermore, noradrenaline did not elicit normal increases in lymphatic vessel tone when maximal constriction was reduced to 29.4 ± 4.9% of control in the presence of 20 nmol l(-1) nifedipine. Transcripts for the L-type calcium channel gene CACNA1C were consistently detected from human thoracic duct samples examined and the CaV1.2 protein was localized by immunoreactivity to lymphatic smooth muscle cells. While human lymphatics ex vivo were highly sensitive to nifedipine, this was not apparent in vivo when nifedipine was compared to placebo in a randomized, double-blinded clinical trial: conversely, lymphatic vessel contraction frequency was increased and refill time was faster despite all subjects achieving target nifedipine plasma concentrations. We conclude that human lymphatic vessels are highly sensitive to nifedipine in vitro but that care must be taken when extrapolating in vitro observations of lymphatic vessel function to the clinical situation, as similar changes in lymphatic function were not evident in our clinical trial comparing nifedipine treatment to placebo., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

The small intestine epithelium (SI-Ep) harbors millions of unconventional (γδ and CD4(-) CD8(-) NK1.1(-) TCRαβ) and conventional (CD8αβ and CD4) T cells, designated intraepithelial lymphocytes (IELs). Here, we identified the circulating pool of SI-Ep-tropic T cells and studied their capacity to colonize the SI-Ep under steady-state conditions in SPF mice. Developmentally regulated levels of α4β7 endowed recent thymic emigrants (RTEs) of unconventional types with higher SI-Ep tropism than their conventional homologues. SI-Ep-tropic RTEs, which in all lineages emerged naive, homed to the SI-Ep, but this environment was inadequate to stimulate them to cycle. In contrast, conventional and, unexpectedly, unconventional T cells, particularly Vγ7(+) (hallmark of γδ IELs), previously stimulated to cycle in the gut-associated lymphoid tissue (GALT), proliferated in the SI-Ep. Cycling unconventional SI-Ep immigrants divided far more efficiently than their conventional homologues, thereby becoming predominant. This difference impacted on acquisition of high Granzyme B content, which required extensive proliferation. In conclusion, SI-Ep-tropic T cells follow a thymus-SI-Ep or a GALT-SI-Ep pathway, the latter generating highly competitive immigrants that are the sole precursors of cytotoxic IELs. These events occur continuously as part of the normal IEL dynamics.

Lung cancer is so strategically situated as regards the heart and aorta that it ought to scatter its metastasizing cells far and wide. However, at careful autopsy, instead of giant opportunities, only dwarf deposition may be detected. Indeed, up to seven patterns of its metastases demonstrate surprises. What explains these surprises? Consider the thoracic duct. When this 45 cm long duct was obtained in its entirety, coiled in the Swiss-roll manner, processed in the usual way, and examined on a single microscope slide, necrosis of some transported lung cancer cells was found to be very intimately associated with the erythrocytes. Therefore, let this underlying natural mechanism be named as the "erythrocyte associated necrosis factor", i.e., EANF. It is argued that this Factor operates differently from the suspected roles of both anoikis and stem cells. Accordingly, it is hypothesized that, if intravital video microscopy is used to obtain subsets of both necrotic and lively cancer cells from the thoracic duct of consenting lung cancer patients, the underlying EANF will definitely materialize. It is predicted that the manipulative replication of this Factor in leading centers will ensure progress. In sum, EANF would not only aid in our understanding of the outlined highly inefficient metastatic processes but also effect a breakthrough in the realms of target therapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Interstitial Cajal-like cells (ICLCs) are speculated to be pacemakers in smooth muscle tissues. While the human thoracic duct (TD) is spontaneously active, the origin of this activity is unknown. We hypothesized that ICLCs could be present in the TD and using histological techniques, immunohistochemistry and immunofluorescence we have investigated the presence of ICLCs, protein markers for ICLCs and the cellular morphology of the human TD. Transmission electron microscopy was employed to investigate ultrastructure. Methylene blue staining, calcium-dependent fluorophores and confocal microscopy were used to identify ICLCs in live tissue. Methylene blue stained cells with morphology suggestive of ICLCs in the TD. Immunoreactivity localized the ICLC protein markers c-kit, CD34 and vimentin to many cells and processes associated with smooth muscle cells (SMCs): coexpression of c-kit with vimentin or CD34 was observed in some cells. Electron microscopy analysis confirmed ICLCs as a major cell type of the human TD. Lymphatic ICLCs possess caveolae, dense bands, a patchy basal lamina, intermediate filaments and specific junctions to SMCs. ICLCs were ultrastructurally differentiable from other interstitial cells observed: fibroblasts, mast cells, macrophages and pericytes. Lymphatic ICLCs were localized to the subendothelial region of the wall as well as in intimate association with smooth muscle bundles throughout the media. ICLCs were morphologically distinct with multiple processes and also spindle shapes. Confocal imaging with calcium-dependent fluorophores corroborated cell morphology and localization observed in fixed tissues. Lymphatic ICLCs thus constitute a significant cell type of the human TD and physically interact with lymphatic SMCs., (Copyright © 2012 S. Karger AG, Basel.)

Background: Lymphatic pump techniques (LPT) are used by osteopathic practitioners for the treatment of edema and infection; however, the mechanisms by which LPT enhances the lymphatic and immune systems are poorly understood., Methods and Results: To measure the effect of LPT on the rat, the cisterna chyli (CC) of 10 rats were cannulated and lymph was collected during 4 min of 1) pre-LPT baseline, 2) 4 min LPT, and 3) 10 min post-LPT recovery. LPT increased significantly (p < 0.05) lymph flow from a baseline of 24 ± 5 μl/min to 89 ± 30 μl/min. The baseline CC lymphocyte flux was 0.65 ± 0.21 × 10⁶ lymphocytes/min, and LPT increased CC lymphocyte flux to 6.10 ± 0.99 × 10⁶ lymphocytes/min (p < 0.01). LPT had no preferential effect on any lymphocyte population, since total lymphocytes, CD4+ T cells, CD8+ T cells, and B cell numbers were similarly increased. To determine if LPT mobilized gut-associated lymphocytes into the CC lymph, gut-associated lymphocytes in the CC lymph were identified by staining CC lymphocytes for the gut homing receptor integrin α4β7. LPT significantly increased (p < 0.01) the flux of α4β7 positive CC lymphocytes from a baseline of 0.70 ± 0.03 × 10⁵ lymphocytes/min to 6.50 ± 0.10 × 10⁵ lymphocytes/min during LPT. Finally, lymphocyte flux during recovery was similar to baseline, indicating the effects of LPT are transient., Conclusions: Collectively, these results suggest that LPT may enhance immune surveillance by increasing the numbers of lymphocytes released in to lymphatic circulation, especially from the gut associated lymphoid tissue. The rat provides a useful model to further investigate the effect of LPT on the lymphatic and immune systems.

Spleen tyrosine kinase (Syk) plays critical roles in B-cell and T-cell development, the maintenance of vascular integrity, and proper partitioning of the blood vascular and lymphatic vascular system. Here, we utilize the zebrafish as an in vivo system to demonstrate novel roles for Syk and the related kinase Zeta associated protein (Zap-70) in promoting angioblast migration. Partial knockdown of either gene results in early angiogenic delay of the intersegmental vessels, dorsal intersegmental vessel patterning defects, and partial loss of the thoracic duct. Higher dose knockdown of both genes results in little to no angiogenic sprouting of the intersegmental vessels, a phenotype which resembles knockdown of vegfa. Di-phosphorylated ERK, an effector of the vegfa pathway, is also downregulated in the aorta of syk:zap double morphants. Over-expression of syk under the control of a blood-specific or vascular-specific promoter rescues sprouting defects after loss of vegfa. Together these results suggest that syk and zap-70 function redundantly in an early progenitor to promote the migration of intersegmental vessel angioblasts and lymphangioblasts that contribute to the thoracic duct, either downstream of, or in parallel to vegfa., (Copyright 2010 Elsevier Inc. All rights reserved.)

Objective: To develop the techniques needed for the specific gene/protein targeting transfection experiments in isolated lymphatic vessels, we completed two major tasks: 1) optimize the experimental conditions to maintain the viability of isolated rat lymphatic vessels in culture for sufficiently long periods of time to permit knockdown or overexpression of selected proteins/genes and 2) develop effective transfection protocols for lymphatic muscle and endothelial cells in intact lymphatic vessels without nonspecific impairment of lymphatic contractile function due to the transfection protocol itself., Methods: Experimental protocols were developed for the maintenance of isolated lymphatic vessels under nonpressurized and pressurized conditions for 3-12 days in culture and for adenoviral gene transfection of the lymphatic muscle and endothelial cells., Results: The data demonstrate the effectiveness of the newly developed experimental protocols for the maintenance of isolated rat mesenteric lymphatic vessels and thoracic duct in culture up to 3-12 days without significant impairment of the parameters of their pumping and effective adenoviral/GFP transfection of lymphatic endothelial and muscle cells in isolated rat mesenteric lymphatic vessels., Conclusions: These experimental techniques will extend the set of the modern experimental tools available to researchers investigating the physiology of lymphatic function.

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom and modulates immune and inflammatory responses, interfering with the activity of leukocytes. In the present work, the effects of crotoxin on the number of blood and lymphatic leukocytes and on lymph nodes and spleen lymphocytes population were investigated. The toxin s.c. administered to male Wistar rats, decreases the number of lymphocytes in blood and lymph circulation and increases the content of B and T-lymphocytes in lymph nodes. These effects were detected 1-2h after treatment. The crotoxin molecule is composed of two subunits, an acidic non-toxic polypeptide, named crotapotin and a toxic basic phospholipase A(2) (PLA(2)). PLA(2), but not crotapotin, decreased the number of circulating blood and lymph lymphocytes. Crotoxin promotes leukocyte adherence to endothelial cells of blood microcirculation and to lymph node high endothelial venules, which might contribute to the drop in the number of circulating lymphocytes. Crotoxin increases expression of the adhesion molecule LFA-1 in lymphocytes. The changes in the expression of the adhesion molecule might contribute, at least in part, for the increased leukocyte adhesion to endothelium. Zileuton, a 5-lipoxygenase inhibitor, blocked the decrease in the number of circulating leukocytes induced by crotoxin and also abolished the changes observed in leukocyte-endothelial interactions, suggesting the involvement of lipoxygenase-derived mediators in the effects of the toxin.

This study was designed to examine the effects of angiogenesis inhibitors IFN-alpha and TIMP-1 on lymphangiogenesis. We cultured lymphatic endothelial (LE) cells from pig thoracic ducts and performed morphological observations using light microscopy, TEM, and confocal microscopy to confirm their lymphatic origin. We tested these cells for growth inhibition by angiogenesis inhibitors IFN-alpha and TIMP-1 using both the scraping line and MTT methods. In addition, we analyzed apoptosis using the Hoechst and Caspase staining methods. Finally, we tested IFN-alpha and TIMP-1 using in vivo inhibitory assays. By morphological observations, all LE cells in vivo and in vitro were found to be of very similar morphology. Both in vitro inhibitory assays of scraping line and MTT showed significant differences for the IFN-alpha treatment (p < 0.01) and no significant difference for TIMP-1. Hoechst and Caspase apoptosis assays demonstrated that IFN-alpha could induce apoptosis of LE cells, and TIMP-1 had little effect. IFN-alpha and TIMP-1 inhibitory in vivo assays showed a lack of healing following IFN-alpha treatment compared to control and TIMP-1 treatment. In summary, these different angiogenesis inhibitors have different effects on lymphangiogenesis. IFN-alpha inhibits proliferation and migration of LE cells in a dose-dependent fashion and induces apoptosis of LE cells while TIMP-1 has no significant inhibitory effects on proliferation, migration, or inducing apoptosis.

Unlabelled: Donor dendritic cell (DC) migration and allosensitization in host secondary lymphoid organs after liver transplantation are ill defined. We used rat models to investigate graft-derived cells and intrahost allosensitization. Liver transplantation induced diffuse blood-borne migration of donor major histocompatibility class II antigen-positive (MHCII(+)) cells and MHCI(+) cells from the graft to host secondary lymphoid organs, not only the spleen, but also lymph nodes and Peyer's patches. The migrated MHCII(+) cells included DCs and some T cells and B cells. The DCs formed clusters with host BrdU(+) cells where they up-regulated CD86(+), and a CD8(+) T cell proliferative response originated within 24 hours after liver transplantation, demonstrating that these DCs can quickly mature and trigger direct allosensitization in host lymphoid organs. Transfer of allogeneic bone marrow cells also induced DC transmigration and a similar host response. In contrast, allogeneic thoracic duct lymph cells contained many fewer transmigrating DCs, and their transfer induced a comparable T cell response but significantly weaker CD8(+) T cell proliferation. Thus, there is a different outcome via the indirect pathway by host DCs that have captured donor alloantigens., Conclusion: The rat liver as well as bone marrow contains an immature DC population that can systemically transmigrate through blood vessel walls of the host secondary lymphoid organs, quickly mature, and induce diffuse intrahost CD8(+) T cell responses, which may promote graft rejection.

Initial lymphatic vessel endothelial cells are connected to the surrounding elastic fibers by fibrillin anchoring filaments that have been hypothesized to favor interstitial fluid drainage in edema pulling apart interendothelial junctions. We hypothesized a biochemical mechanism involving mechanotransduction. This study was designed to verify whether a relation exists between focal adhesion molecules and anchoring filaments and whether they may transduce extracellular forces to the nucleus. We first performed an immunohistochemical study on human skin cryostat sections to evaluate whether fibrillin and alphav-beta3 integrins, FAK and fibrillin, or alphav-beta3 integrins and FAK co-localize in lymphatic endothelium. We observed that integrins and FAK co-localize and that fibrillin filament attachment sites to endothelial cells merge with these molecules. These data may suggest that fibrillin anchoring filaments are connected to endothelial cells through focal adhesions. Mechanotransduction was investigated applying static stretching to bovine thoracic duct segments and lymphatic endothelial cells cultured on elastic membranes and immunohistochemically evaluating the expression of ERK1/2. Under stretching conditions, ERK1/2 labels the nucleus. Western blotting on cultured cells confirmed the presence of ERK1/2 in stretched cells. Based on our data we speculate that anchoring filaments may trigger a focal adhesion-mediated cascade of mechanotransduction toward the nucleus for genetic modulation and thus contribute to endothelial adaptation to interstitial requirements.

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33 degrees C. A temperature shift to 37 degrees C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.

This paper reviews the results of the studies performed mainly by the Russian anatomists on the functional anatomy of lymphangion as a structural and functional unit of lymphatic vessel. One of the peculiar features of functional anatomy of lymphangion is the heterogeneity of its structures (myocytes, endothelium, blood supply and innervation). The functional heterogeneity of different lymphangions, which depends on the local differences in the combination of lymph flow factors, was demonstrated. The role of lymphatic vessels is discussed on the basis of significance of lymphangion in active lymph transport. This is demonstrated by the multiple correlations between lymphangion structure, rate and type of its contractions, length of thoracic duct lymphangions and contraction type (peristaltic, rhythmic), number of lymphangions of thoracic duct and its type (presence of collaterals), form of individual variability of thoracic duct and its myoarchitecture, body mass and lymphangion volume in various animals and at different stages of ontogenesis.

Objective: The objective of the present study was to establish a rat lymphatic endothelial cell line and then to investigate the morphological and immunohistochemical properties of the cells., Methods: The lymphatic endothelial cells of rat thoracic ducts were isolated enzymatically by trypsin digestion and were cultured in endothelium growth medium (EGM)-2 in an atmosphere of low oxygen (5% O(2), 5% CO(2), and 90% N(2)) or high oxygen (21% O(2), 5% CO(2), and 74% N(2))., Results: The number of the cells cultured in the low-oxygen atmosphere was significantly larger than that obtained in the high-oxygen atmosphere. The cultured cells in the low-oxygen atmosphere showed a monolayer with uniform cobblestone appearance, suggesting the morphological properties of endothelial cells. Factor VIII-related antigen and cell surface carbohydrates (i.e., D-galactose alpha and D-N-acetylgalactosamine alpha) were found on the lymphatic cultured cells. The phagocytosis of 1,1-diocadecyl1-3,3,3',3'-tetramethylindo-carbocyanine perchlorate-labeled acetylated low-density lipoprotein also was observed in the cultured cells. The cytoskeleton protein F-actin was located on the plasma membrane of the cultured cells as circumferential thin bundles and in the cytoplasma as filamentous bundles., Conclusions: The present study indicates that the choice of EGM-2 as a culture medium and the hypoxic atmosphere ( approximately 5%) enabled us to establish rat lymphatic endothelial cell line.

The microfibrils of anchoring filaments, a typical ultrastructural feature of initial lymphatic vessels, consist mainly of fibrillin and are similar to the microfibrils of elastic fibers. As we previously demonstrated, they radiate from focal adhesions of lymphatic endothelium to the perivascular elastic network. Although present in large blood vessels, fibrillin microfibrils have never been detected in blood capillaries. Here we report immunohistochemical evidence that cultured bovine aortic and lymphatic endothelial cells express fibrillin microfibrils. These microfibrils form an irregular web in lymphatic endothelial cells, whereas in blood vessel endothelial cells they are arranged in a honeycomb pattern. Cultured lymphatic and blood vessel endothelial cells also produce focal adhesion molecules: focal adhesion kinase, vinculin, talin, and cytoskeletal beta-actin. Our data suggest that anchoring filaments of initial lymphatic vessels in vivo may be produced by endothelium. Through their connection with focal adhesions, they may form a mechanical anchorage for the thin wall of initial lymphatic vessels and a transduction device for mechanical signals from the extracellular matrix into biochemical signals in endothelial cells. The complex anchoring filaments-focal adhesions may control the permeability of lymphatic endothelium and finely adjust lymph formation to the physiological conditions of the extracellular matrix. The different deposition of fibrillin microfibrils in blood vessel endothelial cells may be related to the necessity of withstanding shear forces. Thus, in our opinion, differences in fibrillin deposition imply a different role of fibrillin in blood vessel and lymphatic endothelium.

The distribution of lymphoblasts(lymphocytes in cell cycle) obtained from the central lymph of donor rats and transferred adoptively to syngeneic recipients has been shown previously to be influenced by the presence of arthritis in either donor or recipient rats. The intent of the present study was to examine patterns of distribution of lymphoblasts in the early period after transfer, when extravasation of donor lymphoblasts was expected to occur. Thoracic duct lymphoblasts labelled in vitro with [125I]-iododeoxyuridine were detected in recipient rats by external radiometry and autoradiography. Irrespective of donor status, fewer donor lymphoblasts accumulated in the feet of normal recipients when compared to arthritic recipients at 15 min, 2 h and 24 h after cell transfer.When recipients of similar disease status were compared, the percentages of injected lymphoblasts from normal and arthritic donors recovered in the feet were similar at 15 min and 2 h after transfer. The proportions of lymphoblasts recovered in the feetat 24 h after injection declined in normal recipients and arthritic recipients of cells from normal donor rats. Importantly,this decline did not occur when both the donor and the recipient were arthritic. In the hindpaws, donor lymphoblasts were located predominantly in the bone marrow, except in transfers between arthriticrats, when at 24 h they were predominantly in the synovium. At 15 min, lymphoblasts were detected within the lumen of vessels within synovium, whereas by 2 h extravasation of these cells was evident. In conclusion, lymphoblasts accumulate more readily in hindfeet that are inflamed. In the early hours after injection, lymphoblasts from normal and arthritic donors are recruited equally, but these early levels are only maintained for 24 hin the combination of arthritic donor and arthritic recipient. Adramatic change in the proportion of lymphoblasts located in synoviumat this later time suggests that a dynamic process of relocation,retention and/or local cell division maintains the numbers of arthritic donor cells in the latter combination.

CD8alphabeta(+) and CD4(+) intraepithelial lymphocytes, the progeny of double-positive thymocytes, are oligoclonal T-cell populations that have accumulated in the gut wall as the result of repeated antigenic stimulations, which lead to rounds of traffic through the lymph/blood circuit ending in an alpha4beta7-integrin-driven homing all along the gut mucosa. In contrast, CD8alphaalpha(+) intraepithelial lymphocytes, which may be TCRgammadelta(+) or alphabeta(+), result in part from local differentiation in the gut, but studies comparing euthymic and athymic mice suggest a thymic double-negative origin for many of them.

Previously we have shown that autoimmune diabetes, induced in rats by a protocol of adult thymectomy and split-dose gamma irradiation, can be prevented by the transfer of a subset of CD4+ T cells with a memory phenotype (CD45RC-), as well as by CD4+CD8- thymocytes, from syngeneic donors. Further studies now reveal that in the thymus the regulatory cells are observed in the CD25+ subset of CD4+CD8- cells, whereas transfer of the corresponding CD25- thymocyte subset leads to acceleration of disease onset in prediabetic recipients. However, in the periphery, not all regulatory T cells were found to be CD25+. In thoracic duct lymph, cells that could prevent diabetes were found in both CD25- and CD25+ subsets of CD4+CD45RC- cells. Further, CD25- regulatory T cells were also present within the CD4+CD45RC- cell subset from spleen and lymph nodes, but were effective in preventing diabetes only after the removal of CD25- recent thymic emigrants. Phenotypic analysis of human thymocytes showed the presence of CD25+ cells in the same proportions as in rat thymus. The possible developmental relationship between CD25+ and CD25- regulatory T cells is discussed.

We investigated the effects of vascular endothelial growth factor (VEGF(165)) on [Ca(2+)](i)-transient in cultured lymphatic endothelial cells (LEC) and mechanical activity of isolated dog thoracic ducts. VEGF (0.1-10 ng/ml) caused a dose-dependent increase of the [Ca(2+)](i) in LEC. Pretreatment with 10(-5) M genistein or 5x10(-6) M herbimycin A produced a significant reduction of the VEGF-induced [Ca(2+)](i)-transient. In the presence of 10(-6) M thapsigargin, VEGF caused no significant effect on the [Ca(2+)](i)-transient. Pretreatment with Ca(2+)-free solution containing 0.1 mM EGTA produced no significant effect on the peak increase of [Ca(2+)](i) induced by 0.1 or 10 ng/ml VEGF, but significantly depressed the sustained part of [Ca(2+)](i) observed at the higher concentration of VEGF. The VEGF (0.1-10 ng/ml) caused a significant dilation of the isolated lymph vessels with intact endothelium, which were precontracted with U46,619. The 10 ng/ml VEGF-induced dilation was significantly reduced by 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME). The action of L-NAME was inhibited by the simultaneous application of 10(-3) M L-arginine. Mechanical rubbing of the endothelium also caused significant inhibition of the VEGF-induced dilation. The findings suggest that VEGF(165) may activate the receptor-related tyrosine kinase and cause the release of Ca(2+) from the inositol 1,4, 5-triphosphate-sensitive intracellular Ca(2+) stores in LEC. VEGF(165) also produces endothelium-dependent nitric oxide-mediated dilation of the precontracted isolated lymph vessels.

Murine gut intraepithelial (IEL) T cell receptor (TCR)-alpha/beta lymphocytes bearing CD8alpha/13 or CD8alpha/alpha coreceptors have been shown previously to express different oligoclonal TCR beta chain repertoires in the same mouse, in agreement with other evidence indicating that these two populations belong to different ontogenic lineages, with only CD8alpha/beta+ IELs being fully thymus dependent. CD8alpha/beta+, but not CD8alpha/alpha+, T lymphocytes are also present in the lamina propria. Here, we show that CD8alpha/beta+ lymphocytes from the lamina propria and the epithelium are both oligoclonal, and that they share the same TCR-beta clonotypes in the same mouse, as is also the case for CD4alpha T cells. Furthermore, identical T cell clones were detected among CD8alpha/beta IELs and CD8alpha/beta+ blasts circulating into the thoracic duct (TD) lymph of the same mouse, whereas TD small lymphocytes are polyclonal. These findings must be considered in light of previous observations showing that T blasts, but not small T lymphocytes, circulating in the TD lymph have the capacity of homing into the gut epithelium and lamina propria. These combined observations have interesting implications for our understanding of the recirculation of gut thymus-dependent lymphocytes and their precursors, and of the events leading up to the selection of their restricted TCR repertoire.

Lymphocytes continuously circulate between the bloodstream and lymphoid organs, and their migration into lymphatic tissues presumably occurs through selective mechanisms. Although bronchus-associated lymphoid tissue (BALT) is known as an inductive tissue of the common mucosal immune system, little is known about how effectively the lymphocytes in the blood vessels migrate into the BALT, thereby enabling the BALT to act as an effector tissue in the immunologic responses of the lungs. To analyze whether or not thoracic duct lymphocytes (TDL) from immunized and nonimmunized rats possess different migratory patterns to the BALT, 5-(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled TDL were injected into rats with BALT hyperplasia that was produced by intratracheal administration of TNP-KLH, and then the number of labeled cells in the BALT were examined by immunohistochemical methods. We studied the following three groups at 12 h after the injection: group A, intraintestinally immunized donors and intratracheally immunized recipients; group B, nonimmunized donors and intratracheally immunized recipients; group C (control group), nonimmunized donors and nonimmunized recipients. Time course studies 0.5, 4, 12, and 24 h after the injection were done in groups A and C. In a cytokinetic study, larger numbers of CFSE-labeled lymphocytes were found at 12 h and 24 h in group A than in group C. At 12 h after the injection, the absolute number of CFSE-labeled lymphocytes per BALT was significantly higher in group A than in group B (p < 0.05), and was lowest in group C. Histologically, there was a marked proliferation of high endothelial venules (HEV), with findings of adhesion and influx of lymphocytes inside the HEV in group A. These observations indicate that the immunized BALT actively recruits immunized TDL through a specific mechanism of lymphocyte-endothelium recognition in HEV, which partially explains the process of BALT development as an effector tissue for local immunity.

Graft-vs-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. Experimentally, lethal GVHD can be induced in MHC-matched strain combinations differing in expression of multiple minor histocompatibility Ags (miHA). Recently, the GVHD potential of C57BL/6By (B6) T cells in irradiated BALB.B (both H2b) and related CXB recombinant inbred strains of mice has been studied to determine the scope of the response to miHA in vivo and how it compared with CTL responses to immunodominant miHA in vitro. The GVHD response in these strain combinations appeared to be limited to a few Ags, yet there was no correlation of these miHA with that of in vitro CTL responses. To further investigate the role of CD8+ T cells in GVHD, we analyzed positively selected miHA-specific donor CD8+ thoracic duct lymphocytes (TDL) collected from irradiated BALB.B and CXBE mice, 5 to 6 days after transplantation of B6 T cells. Flow cytometric analysis of B6-->BALB.B TDL did not indicate expansion of any particular TCR Vbeta family, whereas Vbeta10 and Vbeta14 families were significantly expanded in the B6-->CXBE TDL. However, PCR-based complementarity-determining region 3 size spectratyping revealed overlapping involvement of donor Vbeta1, 6, 8, 9, 10, and 14 families in both BALB.B and CXBE recipients and unique utilization of the Vbeta4 family in BALB.B mice, suggesting oligoclonal T cell responses to a limited number of miHA. In addition, the injection of CD8+ Vbeta14+ B6 T cells into irradiated BALB.B and CXBE mice induced lethal GVHD, confirming the involvement of miHA-specific T cells within an individual Vbeta family.

We have attempted to evaluate whether, similar to the angiogenesis of blood vessels, cultures of lymphatic endothelial cells (LEC) isolated from dog thoracic ducts have an ability to induce lymphangiogenesis in response to basic fibroblast growth factor (bFGF), then to examine the effects of heparin on the bFGF-mediated morphogenesis. The effects of bFGF and/or heparin on the proliferation and migration of the LEC were evaluated by changing the number of the subconfluent cells and by wound migration assay, respectively. The effects of the agents on invasion and tube formation of the LEC into a three-dimensional collagen gel and on collagen gel induced tube formation of the LEC were also investigated by a phase-contrast microscope and an electron microscope. The bFGF (10 ng/ml) caused a significant induction of proliferation and migration of the LEC, the induction of which was augmented dose-dependently by an additional treatment with heparin ranging from 1 to 100 microg/ml. The bFGF produced invasion and tube formation of the LEC into a three-dimensional collagen gel. The bFGF also facilitated to form capillary-like tubes of the LEC between two layers of collagen gels. Heparin (10 microg/ml) accelerated both processes of bFGF-mediated lymphangiogenesis of the LEC. These findings suggest that the cultured LEC isolated from dog thoracic ducts have an ability to form lymphatic capillary-like tubes in response to bFGF and that heparin accelerates dose-dependently the process of the bFGF-mediated neovascularization of lymph vessels.

Initiation of an Ab response requires interaction between dendritic cells (DC), T cells, and B cells in a T cell area. We demonstrate that rat DC and B cells form T cell-independent clusters in vitro and in vivo. In vitro clusters form within 1 h and dissociate within 24 to 48 h. Clustering is restricted to resting B cells, is energy, cytoskeleton, and protein kinase C dependent, and is inhibited by anti-LFA-1 but not anti-ICAM-1 mAbs. Spleen and lymph node B cells cluster more strongly than those from lymph or blood, suggesting up-regulation of adhesiveness during transendothelial migration. Bone marrow B cells do not form clusters. DC from spleen and lymph nodes show the most clustering, lymph-borne DC are intermediate, and DC from lamina propria, Peyer's patches, and those grown from bone marrow form the fewest clusters. Clustering is stimulated by cross-linking MHC class II (whole mAb or F(ab')2) on DC or B cells or Thy-1 on DC, but not MHC class I, CD45, or CD44. Stimulation by mAb is energy, cytoskeletal, and protein kinase C dependent, but is not inhibited by anti-LFA-1 mAbs, suggesting involvement of other, unidentified adhesion molecules. We suggest that interactions between DC and B cells will occur regularly during B cell recirculation. Cross-linking of MHC class II-peptide molecules on DC by specific T cells would increase binding avidity, causing retention of Ag-specific B cells on DC long enough for the B cells to process Ag, thereby facilitating cognate interactions between T and B cells.

The subset composition and recirculation properties of the migrating lymphocyte pool in humans is largely unknown. The present study was conducted in order to phenotypically characterize cells in human thoracic duct lymph of patients under non-inflammatory and inflammatory conditions. These data were compared with data from peripheral blood, with special emphasis on those cells homing to the gut. Thoracic duct lymph and peripheral blood contained comparable proportions of B and T lymphocytes and CD8+ cells. Thoracic duct lymph contained proportionally more CD4+ cells, more CD4+CD45RO+ that express alpha 4 beta 7 cells and more CD8+CD45RO+ that express alpha 4 beta 7, as compared to peripheral blood. These data suggest an equal recirculation rate of B and T lymphocytes; a more active recirculation of CD4+ cells compared to CD8+ cells; and a more active recirculation of memory cells to the gut as compared to other extra-lymphoid sites in patients under non-inflammatory conditions. Data were also obtained in patients with the system inflammatory response syndrome and multiple organ failure. Although it is generally assumed that granulocytes and monocytes do not recirculate, lymph of multiple organ failure patients contained significantly more granulocytes than monocytes, indicating that in severe generalized inflammatory states these cells re-enter the circulation through the thoracic duct. Furthermore, no increased activation of cells homing to the gut was found in these patients.

This study was undertaken to distinguish between several mechanisms responsible for graft-vs-host disease (GVHD) protection in anti-CD3epsilonF(ab')2 fragment (Fr)-treated recipients: TCR down-modulation, deletion, failure of expansion, or anergy induction. To quantify alloreactive T cell expansion and function, thoracic duct lymphocytes (TDL) were analyzed. Sixfold fewer donor TDL T cells were recoverable from anti-CD3epsilonF(ab')2 Fr as compared with irrelevant F(ab')2 Fr-treated recipients at the time of peak T cell expansion in vivo. Kinetic analysis revealed that donor T cell expansion was inhibited and not simply delayed by anti-CD3epsilonF(ab')2 Fr. Similar proportions of TDL T cells in irrelevant and anti-CD3epsilonF(ab')2 Fr were undergoing apoptosis. Although TCR modulation was observed, donor TDL T cells had intact anti-host alloresponses as compared with irrelevant F(ab')2 Fr-treated recipients. Because donor CD4+ T cells are primarily responsible for GVHD in this model, an adoptive transfer system was used in which the function and kinetics of expansion of OVA-specific CD4+ TCR transgenic cells could be physically tracked. Relevant Fr severely blunted CD4+ TCR transgenic T cell clonal expansion after OVA administration. Nonviable transgenic and nontransgenic T cells were proportionally similar in OVA-pulsed recipients, regardless of whether relevant or irrelevant F(ab')2 Fr were given. After discontinuing Fr, transgenic T cells were found to have intact in vitro OVA-specific responses. Our current and previous results suggest that reduced donor T cell expansion and T cell depletion both contribute to GVHD protection by anti-CD3epsilonF(ab')2 Fr. These data have implications for designing therapeutic approaches directed toward TCR targeting in humans.

A method has been devised for labeling whole blood with the fluorescent dye fluorescein isothiocyanate (FITC) so the migration of blood lymphocytes can be studied in the sheep. Although lymphocytes can be purified from blood using density gradient media or elutriation it is difficult to obtain a large number of cells, because many cells are usually lost during the purification steps. It is desirable to label at least 10(8)-10(9) lymphocytes for lymphocyte tracking studies, because a smaller number is difficult to subsequently detect and quantitate in blood and lymph even using flow cytometry. Also, it is desirable to minimize the in vitro manipulation of lymphocytes, because dead or damaged lymphocytes will not recirculate. By labeling all the cellular components of a sample of whole blood rather than first purifying the lymphocytes we have been able to satisfy both of these criteria. Although labeled blood cells of all types are reinjected into the animal, the lymphocytes are easily distinguishable from other cells using a flow cytometer. In these studies between 2.4-12.4 x 10(8) lymphocytes were injected intravenously, and they were detectable in the blood and lymph for at least 10 days. The recovery of FITC-labeled (FITC+) lymphocytes in efferent lymph is comparable to that of lymphocytes labeled with other fluorescent or radioactive markers. The presence of labeled non-lymphoid cells in the animal makes this technique impractical for studies of lymphocyte localization within histologic sections. However, it is useful for studies in animals in which lymphatic vessels can be cannulated and the blood-to-lymph recirculation of labeled lymphocytes monitored, and it also may be applicable for studies in which lymphoid organ suspensions are analyzed using flow cytometry.

Pancreatic islets obtained from the congenic LEW.1BB/OK rat strain (MHC identical, different in genetic background from BB/OK rats) were grated into diabetic BB/OK rats. The recipients were treated for 10 d with 1.0 mg/kg bw anti-IL2 receptor monoclonal antibody (ART-18) in combination with 1.5 mg/kg bw cyclosporin A, which resulted in indefinite graft survival in the majority of animals. The successfully treated recipients (normoglycaemia for > 120 days) relapsed immediately into hyperglycaemia after graft removal. A second donor-identical graft was accepted without any further immunotherapy, whereas MHC-different islet grafts (obtained from LEW.1A or DA rats) were rejected, demonstrating the induction of donor-specific tolerance. Splenocytes or thoracic duct lymphocytes (TDL) obtained from successfully treated recipients were transfused into naive diabetic BB/OK rats grafted with LEW.1BB/OK islets. Independent of the origin of the transfused cells, 64% of recipients maintained normoglycaemia for more than 120 days. To characterize the cell population(s) responsible for transfer of tolerance, B-lymphocytes were removed from the TDLs using the monoclonal antibody OX33 and magnetic beads. When OX33-depleted TDLs were transfused into naive diabetic BB/OK with a LEW.1BB/OK islet graft, all recipients maintained normoglycaemia. The OX33-depleted TDLs consisted only of CD4+ T-lymphocytes, which were either negative for CD45RC or coexpressed the CD45RC at low levels. We conclude that the cell-dependent tolerance transfer is mediated by a TH2-like suppressor cell.

The adhesion molecules integrin alpha 4 beta 7 and L-selectin have been hypothesized to help direct selective migration (homing) of lymphocytes to the gut and peripheral lymph nodes, respectively. An important prediction of current models is that lymphocytes selectively recirculating through an organ will preferentially express adhesion receptors for organ-specific endothelial ligands. To test this prediction, we directly examined the expression of cell adhesion molecules on lymphocytes recirculating through the gut, the periphery, and the lung. Integrin beta 7 was highly expressed on CD4+CD45R- "memory/effector" T cells recirculating through the gut (mesenteric efferent and lower thoracic duct lymph). In contrast, cells recirculating through the periphery (prescapular efferent lymph) and the lung (caudal mediastinal efferent lymph) had much less beta 7 expression. A similar pattern of organ-specific beta 7 expression was seen on B cells. Beta 7 expression corresponded with adhesion to the gut mucosal addressin, MAdCAM-1, in vitro. The peripheral lymph node homing receptor, L-selectin, was expressed at higher levels on CD4+CD45R- T cells from peripheral lymph than on cells from gut or lung lymph. These results provide additional strong support for alpha 4 beta 7 and L-selectin involvement in lymphocyte homing to the gut and to peripheral lymph nodes, respectively. Lymphocytes emigrating from the lung expressed low levels of both homing receptors and likely utilize molecules other than alpha 4 beta 7 and L-selectin for migration to the lung and associated lymphoid tissue.

Salivary immune responses depend on localization of immunocytes in salivary glands. We tested effects of anti-adhesion molecule antibodies and several ligand analogs on in vitro adherence of rat thoracic duct lymphocytes (TDL) to parotid and submandibular gland sections. While TDL adherence to both tissues was markedly decreased by anti-L-selectin mAbs, binding ability after removal of L-selectin by chymotrypsin or PMA suggested that other adhesion systems were involved. Integrin involvement in parotid interactions was indicated by inhibitory effects of anti-HEBF(PP), LFA-1, ICAM-1, and alpha4 integrin antibodies as well as by the PMA-enhanced adherence. Anti-Thy-1 partially inhibited TDL binding to parotid gland, and anti-CD44 partially inhibited submandibular binding. The majority of salivary gland-bound TDL were sIg+ B cells. FACS analysis showed differences in parotid and submandibular endogenous lymphocyte adhesion molecule expression with greater percentages of L-selectin, HEBF(PP), alpha4 integrin, LFA-1, ICAM-1, CD44, and Thy-1-positive cells present in parotid gland. While precise roles of known or novel adhesion molecules in salivary gland lymphocyte retention are not clear, these data suggest that selectins (parotid, submandibular), integrins (parotid), Thy-1 (parotid), and CD-44 (submandibular), as well as other unidentified molecules, are involved.

Purpose: In lacrimal glands, cell-cell interactions control the localization of lymphocyte populations that play a role in immune defense at the ocular surface. This study describes lymphocyte adhesive interactions with cultured lacrimal gland acinar epithelial cells., Methods: Primary cultures of lacrimal gland epithelial cells were used as targets for in vitro lymphocyte binding assays. The relative adherence of lymphocyte populations was determined. Various physiologically active agents and putative ligand analogs were tested for their effect in the binding assay., Results: Thoracic duct lymphocytes (TDL) bound to cultured lacrimal acinar epithelial cells in greater numbers than did thymocytes (54 cells/mm2 versus 8 cells/mm2). B cells showed preferential adherence compared with T cells (75% sIg+, 14% W3/13+). Thoracic duct lymphocyte binding required intact metabolic and membrane-cytoskeletal function and was inhibited by treating the lymphocytes with sodium azide, formaldehyde, or cytochalasin B (23%, 12%, and 10% of control binding, respectively). Further, adherence was dependent on divalent cations. Ethylenediaminetetraacetic acid-mediated inhibition (42% of untreated) was restored by replacing calcium (89%) but not magnesium (41%). Lymphocyte adherence was inhibited in the presence of fucoidin or phosphomannan polysaccharides (36% and 48% of control binding, respectively). Fibronectin peptides, which are involved in certain types of integrin-mediated adherence, had no effect in this system. Lacrimal culture supernatants contained a factor that was inhibitory for TDL adherence (more than 50% inhibition when concentrated 5 or 10 times)., Conclusions: Thoracic duct lymphocyte adherence to cultured lacrimal gland acinar epithelial cells shows good correlation with previously reported adherence to lacrimal gland frozen sections. Further, lacrimal cell culture supernatants contain soluble factors that inhibit TDL adherence to epithelial cells. These findings suggest that the lacrimal molecules involved in lymphocyte localization are shed and that lacrimal epithelial cell cultures will be useful for ligand isolation and characterization.

Lymphocytes continuously migrate through the body, and their efficient extravasation from the blood via high endothelial venules (HEV) is essential for initiating an appropriate immune response. Most investigations have focused on the lymphocyte/HEV interaction in vitro. However, to what extent such systems reflect the situation in vivo is not known. It is also unclear whether lymphocyte subsets immigrate into the HEV in proportion to their presence in the blood, and whether import capacity is limited by the HEV. When rat mesenteric lymph node lymphocytes were incubated in vitro on cryostat sections, the well-known preferential binding of B lymphocytes to HEV of Peyer's patches (PP) and T cells to HEV of axillary lymph nodes (axLN) was observed (axLN vs. PP: B lymphocytes 21.2 +/- 5.0% vs. 40.6 +/- 11.0%, T lymphocytes 84.6 +/- 6.3% vs. 56.5 +/- 12.9%). However, when labeled mesenteric lymph node lymphocytes were injected and their location within the HEV was analyzed 15 min later, no preferential interaction was seen. After injection of labeled thoracic duct lymphocytes, the percentage of labeled cells among B and T lymphocytes in the blood was significantly different (4.4 +/- 0.9% vs. 8.9 +/- 3.6%), whereas that in HEV of axLN (19.0 +/- 6.4% vs. 16.6 +/- 6.0%) and PP (30.6 +/- 6.1% vs. 33.9 +/- 4.4%) was comparable. Although the number of injected lymphocytes was similar in magnitude to the total blood lymphocyte pool, after injection there was no increase in lymphocyte numbers in the HEV. Thus, the adhesion assay in vitro does not completely reflect immigration into HEV in vivo. In addition, our data suggest that both the availability of lymphocyte subsets in small venules and the immigration rate into HEV are actively regulated in vivo.

We analysed the cytokine profile of a T cell subset (CD4+ CD45 RC-) that confers protection against Trichinella spiralis infection in rats. These CD4+ cells are generated in the gut and appear in the thoracic duct lymph within 72 h after infection. Cytokine mRNA levels for IL-2, IL-3, IL-4, IL-5, IL-10 and IFN-gamma and functional cytokine secretion for IL-4, IL-5, IFN-gamma, TNF-alpha and mast cell differentiation activity were tested in vitro following stimulation with T. spiralis antigens. Compared to a non-protective T cell population (CD4+ CD45 RC+ or CD8+), also isolated from the same thoracic lymph, no significant differences were observed in the levels of mRNA for IL-2, IL-3, IL-4, IL-5, IL-10 or IFN-gamma in the protective CD4+ CD45 RC- cells. However, analysis of the cytokine activities in culture supernatant of these T cell subsets following 24 h stimulation in vitro with T. spiralis antigens showed that significant IL-4 and IL-5 activity but little IFN-gamma or TNF-alpha was secreted by the protective CD4+ CD45- RC- cells. Whereas the non-protective CD4+ CD45 RC+ cells secreted significant levels of IL-4, IFN-gamma, mast cell differentiating activity and TNF-alpha but little IL-5 activity. Non-protective CD8+ cells were found to secrete IL-4 but not IL-5. Production of IL-4 was essentially equal for both protective and non-protective T cell subsets. These findings suggest that the presence or absence of IFN-gamma secretion, rather than IL-4 alone, determines whether a T cell subset has protective activity against T. spiralis infection in rats.

Glandular mucosal tissues contain lymphocyte populations that contribute to expression of IgA antibodies in external secretions. Interaction of circulating lymphocytes with glandular structures may regulate lymphocyte accumulation. An in vitro assay was used to investigate adhesive interactions between lymphocytes and salivary gland tissues. Thoracic duct lymphocytes (TDL) bound to the serous acinar epithelia of parotid salivary glands and to the mucous tubulo-acinar epithelium of submandibular salivary glands. Lymph node cells and splenocytes adhered to these tissues in lesser numbers and thymocytes bound in negligible numbers. TDL adherence was an active process, being time- and cell dose-dependent and requiring intact membrane as well as cytoskeletal and metabolic function. Calcium was required in each case and binding was mediated by a trypsin-sensitive lymphocyte surface determinant. These findings suggest that the lymphocyte composition of salivary gland tissues is regulated by active lymphocyte interaction with the glandular epithelium.

Thoracic duct lymphocytes (TDL) continuously patrol through the body, facilitating immune responses at most sites. IFN-gamma might regulate immune responses by influencing the migration of TDL. Therefore, it was investigated in vivo whether IFN-gamma affects the migration of thoracic duct B, T, CD4+, and CD8+ lymphocytes from blood to lymph. Labeled TDL were injected i.v. into rats continuously receiving IFN-gamma via a central venous catheter. The numbers of B, T, CD4+, and CD8+ lymphocytes were determined in blood and thoracic duct lymph for 120 h. IFN-gamma increased the disappearance of TDL from the blood to a similar extent in all subsets. In contrast, the reappearance of B and T lymphocyte subsets in the lymph was decreased: B lymphocytes were affected significantly more than T lymphocytes, whereas CD4+ and CD8+ lymphocytes were affected to a similar extent. Our study suggests that differential retention within the tissue rather than preferential immigration into the tissue creates a microenvironment with a distinct composition of lymphocyte subsets.

Genetically marked thoracic duct B cell subpopulations rich in either IgD+ or IgD- B cells were transferred to non-irradiated, congenic rats in order to compare the capacities of IgD+ versus IgD- B cells to form germinal centers (GCs). This comparison was made quantitatively based on flow cytometric analyses of lymph node cells prepared from chimeric rats 7 days after s.c. immunization. Donor-origin and host-origin B cells were distinguished using anti-Igk antibodies, and GC B cells were distinguished from other B cells in suspension by their lack of labeling with the mAb HIS22. IgK+ HIS22- lymph node cells corresponded well to GC B cells: they contained many large cells, were IgM+ but mostly IgD-, expressed relatively lower levels of IgM than HIS22+ B cells, and increased in number and frequency in response to antigen. Results from flow cytometric analyses, corroborated by immunofluorescence histochemical studies, showed that cell-for-cell, IgD- B cells from GCs much more efficiently than IgD+ cells. B cell populations enriched for IgD- cells became relatively more distributed to GCs than to other lymph node B cell areas and gave rise to many more GC B cells of donor origin per transferred B cell than whole, unseparated thoracic duct B cells (for which greater than 97% were IgD+). IgD- B cells from rats primed deliberately with antigen also became relatively more distributed to GCs and gave rise to more GC B cells of donor origin than either IgD+ B cells from primed donors or IgD- B cells from unprimed donors.(ABSTRACT TRUNCATED AT 250 WORDS)

Monolayer cultures of rat lymphatic endothelial cell were obtained from explants of rat thoracic ducts. The cells displayed a typical cobblestone morphology, expressed von Willebrand factor (factor VIII-associated antigen) and angiotensin converting enzyme, and took up acetylated-low density lipoprotein, being indistinguishable by these criteria from blood vessel endothelial cells. The availability of these cells will facilitate the use of rat experimental models for functional studies of lymphatic endothelium.

The possibility of obtaining useful scintigrams of secondary lymphoid organs after infusion of syngeneic lymphocytes labelled with technetium-99m (99mTc) was explored in a rat model. Thoracic duct lymphocyte (TDL) accumulation in various organs was measured with both 99mTc and 51Cr labelled cells, the latter processed with a method that has been shown not to damage lymphocytes. 99mTc labelled TDL did not localize properly in the lymph nodes and spleen. We could not visualize lymph nodes in scintigrams, neither could we demonstrate any difference between normal and hyperplastic spleens. Our conclusion is that radiation from the 99mTc label readily influences lymphocyte migration so that useful scintigraphy in rats and other small experimental animals becomes impossible. This was supported by results from culture experiments with 99mTc labelled, radiosensitive mouse haemopoietic progenitor cells. Theoretical considerations, including the calculations of lymphocyte self-irradiation and signal/noise ratios during scintigraphy of rat tissues, supported our conclusion that scintigraphy in small animals, to disclose the physiological migration of lymphocytes, may be impossible with the present sensitivity of gamma cameras.

Canalization of the jugular lymphatic sac is accompanied with disengagement from the blood stream of the thoracic subcardinal and the thoracic parts of the superior mesocardinal veins, forming the paired thoracic anlage of the thoracic duct (ThD). Canalization of the retroperitoneal lymphatic sac (RLS) is combined with differentiation of the antevertebral plexus of the abdominal veins, formation of the ThD cistern. Lateroaortal, retrocaval and retroaortal lumbar trunks (LT) become its roots. Together with RLS and the ThD cistern they form the lymphatic "muff" around the abdominal aorta and inferior vena cava. The degree of its parts separation and its substitution by collaterals of LT and by lymph nodes determines the variants of the definitive organization of the ThD initial part. Already at the stage of anlage the intestinal trunk is not included in the ThD root system, but serves as the RLS anterior tributary, or its lumbar, preaortic tributary.

We studied the effect of antiantidonor major histocompatibility complex antibodies (antiidiotypic antibodies) in vivo on ACI cardiac allograft survival in Lewis rats and correlated the results with in vitro mixed lymphocyte culture. Lewis anti-ACI hyperimmune sera (Ab1) were obtained from animals that have rejected successive ACI skin grafts. Purified immunoglobulin (Ig) G and IgM fractions were obtained from the sera. These putative "idiotypic antibodies" (IgG fractions) were used to immunize groups of five naive Lewis rats. Purified Ig (0.5 mg) was mixed with 0.5 ml incomplete Freund's adjuvant and injected intraperitoneally -15, -7, and -3 days before and at the time of ACI cardiac allografting. The median allograft survival time was 11.2 +/- 0.7 days in animals treated with Ab1 compared with 6.4 +/- 0.5 days in untreated control rats (p less than 0.001). Use of IgM with adjuvant did not prolong graft survival. Purified IgG obtained from sera collected before transplantation was tested for antiidiotypic antibodies with the complement-mediated cytotoxicity assay. For this, serial dilutions of the hyperimmune serum were tested for cytotoxicity against ACI lymphocyte in the presence of Ig from sera collected after immunization with Ab1. Blocking was demonstrated by sera and IgG obtained from Lewis rats that received anti-ACI IgG (Ab1) with adjuvant. The blocking activity of purified IgG (Ab2) was strain specific because it did not block the reaction of Lewis hyperimmune sera against third-party Wistar-Furth rats. Thoracic duct lymphocytes from immunized recipients showed no blastogenic responses when tested in in mixed lymphocyte culture for reactivities against splenocytes from ACI rats. The finding that Lewis rats treated with Ig from sera containing anti-ACI antibodies exhibit impaired anti-ACI T- and B-cell reactivity and prolong allograft survival suggests that pretreatment of recipients with idiotypic antibodies leads to development of antiidiotypic antibodies that modulate alloreactivity suppressing allograft rejection.

Rapid expulsion of Trichinella spiralis could be transferred to naive adult rats with thoracic duct lymphocytes and immune serum. Thoracic duct cells collected from Days 3-5 and immune serum collected on Day 28, respectively, after infection were effective. Both cells and serum were unable to transfer rapid expulsion when given alone, even in large volumes. Recipients of immune serum and cells eliminated a significantly higher number of larvae than control rats by 1 hr after challenge with muscle larvae. Rapid expulsion produced 30-80% larval worm rejection but could not be increased by the transfer of more cells or immune serum. Mucus trappings did not appear to play a role in the rejection process. After transfer of 2 x 10(8) cells and 4.0 ml immune serum, rapid expulsion persisted for less than 1 week. However, after adoptive transfer of cells alone, the gut remained functionally receptive to the passive transfer of immune serum for 7 weeks. Therefore, the changes effected by transfer of cells were long lived in contrast to the 1 week, or less, of functional persistence by transferred immune serum. The data indicate that two separate processes, one cell mediated and the other immune serum mediated, interact synergistically in the intestine and lead to the expression of rapid expulsion.

Migration pathways of B cell and CD4+ and CD8+ T cell subsets of murine thoracic duct lymphocytes (TDL) were mapped. Per weight, the spleen accumulated more TDL than any other organ, regardless of lymphocyte subset. Spleen autoradiographs showed early accumulations of TDL in marginal zone and red pulp. Many TDL exited the red pulp within 1 hr via splenic veins. The remaining TDL entered the white pulp, not directly from the adjacent marginal zone but via distal periarterial lymphatic sheaths (dPALS). From dPALS, T cells migrated proximally along the central artery into proximal sheaths (pPALS) and exited the white pulp via deep lymphatic vessels. B cells left dPALS to enter lymphatic nodules (NOD), then also exited via deep lymphatics. T cells homed to lymph nodes more efficiently than B cells. Lymphocytes entered nodes via high-endothelial venules (HEV). CD4+ TDL reached higher absolute concentrations in diffuse cortex than did CD8+ T cells. However, CD8+ TDL moved more quickly through diffuse cortex than did CD4+ TDL. B cells migrated from HEV into NOD. Both T and B TDL exited via cortical and medullary sinuses and efferent lymphatics. A migration pathway across medullary cords is described. All TDL subsets homed equally well to Peyer's patches. T TDL migrated from HEV into paranodular zones while B cells moved from HEV into NOD. All TDL exited via lymphatics. Few TDL entered zones beneath dome epithelium. All subsets were observed within indentations in presumptive M cells of the dome epithelium.

Immigration of B lymphocytes into established germinal centers in the rat was studied by transferring genetically marked thoracic duct B cells to non-irradiated congenic hosts at various times between 3 days before and 6 days after host immunization. Seven days after host immunization, the distribution of donor B cells to lymph node germinal centers (relative to their distribution to non-germinal center lymph node areas) was measured by two-color flow cytometry in which (a) donor and host B cells were distinguished by their Ig kappa chain allotypes, and (b) germinal center B cells were distinguished by their lack of labeling with the monoclonal antibody HIS22. Thoracic duct B cells from long-term antigen-primed rats were found to immigrate into host germinal centers much better than B cells from unprimed donors. This effect was antigen specific: primed B cells only immigrated well into host germinal centers induced by the priming antigen. Although B cells localized in germinal centers most efficiently when injected before immunization, specifically primed donor B cells injected after immunization were still found to be at least as evenly distributed to germinal centers as to other lymph node areas, whereas unprimed B cells transferred after immunization localized poorly in host germinal centers. These findings are discussed in light of recent suggestions that memory B cell clones are maintained by continued antigenic stimulation within secondary lymphoid follicles.