STUDIES ON LYMPHOCYTES

Tritiated thymidine was administered to calves continually for 2 to 8 days via the thymic artery in an attempt to label intensively thymic lymphocytes. Heavily labeled cells which had migrated from the thymus were observed in the spleen, lymph nodes and Peyer's patches. Cell maps were made for the various lymphoid tissues and in all cases the majority of labeled thymic cells were found in the ‘thymus dependent areas’of the spleen and lymph nodes. The number of labeled thymic cells per thousand lymphocytes was highest in the ‘thymus dependent areas’. A few labeled thymic cells were seen in or near the post capillary venules. The labeling pattern in the Peyer's patches was different from that in the spleen and lymph nodes. Labeled thymic cells were not observed in the bone marrow. Heavily labeled cells were not detected in any of the lymphoid tissues of those calves which received continuous intravenous infusion of comparable amounts of tritiated thymidine.     

Molecular mechanisms of lymphocyte extravasation. III. The loss of lymphocyte extravasation potential induced by pertussis toxin is not mediated via the activation of protein kinase C

The present study evaluated whether protein kinase C (PKC) activation was involved in the lymphocytosis promoting properties of pertussis toxin (Ptx). The exposure of mouse lymphocytes to phorbol esters (as a means to selectively activate PKC) caused a depression in their subsequent capacity to localize into lymph nodes and Peyer's patches in vivo. This pattern of inhibition was quite similar to that observed with lymphocytes treated with Ptx. The mechanisms responsible for the observed decreases in localization to lymphoid organs caused by these two agents, however, appeared to be distinct. Exposure of lymphocytes to PMA was followed by a time and dosage-dependent decrease in the surface density of MEL-14 defined homing receptors. Ptx-treated lymphocytes retained normal density of this homing receptor. Consequently, PMA-treated lymphocytes lost their capacity to bind to high-endothelial venules in in vitro lymph node binding assays while Ptx-treated cells retained normal high-endothelial venule binding potential. We conclude from this study that: 1) the activation of PKC in lymphocytes by PMA can alter their recirculation properties via mechanisms that diminish their expression of surface receptors which support extravasation into lymph node and mucosal lymphoid tissues, and 2) even though Ptx has been reported to elevate the rate of inositol phosphate turnover in lymphocytes, the loss of extravasation potential of Ptx-treated lymphocytes is not mediated via the modification of surface homing receptors as observed in cells exposed to the known PKC activator, PMA.     

Plasmacytoid dendritic cells do not migrate in intestinal or hepatic lymph

Plasmacytoid dendritic cells (pDCs) recognize pathogen-associated molecules, particularly viral, and represent an important mechanism in innate defense. They may however, also have roles in steady-state tolerogenic responses at mucosal sites. pDCs can be isolated from blood, mucosa, and lymph nodes (LNs). Although pDCs can express peripherally derived Ags in LNs and at mucosal sites, it is not clear whether pDCs actually migrate from the periphery in lymph or whether LN pDCs acquire Ags by other mechanisms. To determine whether pDCs migrate in lymph, intestine or liver-draining LNs were removed and thoracic duct leukocytes (TDLs) were collected. TDLs expressing MHC-II and CD45R, but not TCRalphabeta or CD45RA, were then analyzed. These enriched TDLs neither transcribe type I IFNs nor secrete inflammatory cytokines in response to viral stimuli in vitro or after a TLR7/8 stimulus in vivo. In addition, these TDLs do not express CD5, CD90, CD200, or Siglec-H, but do express Ig, and therefore represent B cells, despite their lack of CD45RA expression. Intestinal and hepatic lymph are hence devoid of bona fide pDCs under both steady-state conditions and after TLR7/8 stimulation. This shows that any role for pDCs in Ag-specific T cell activation or tolerance must differ from the roles of classical dendritic cells, because it cannot result from peripheral Ag capture, followed by migration of pDCs via lymph to the LN.     

Sheep Red Blood Cell Instillation at Palatine Tonsil Effectively Induces Specific IgA Class Antibody in Saliva in Rabbits

We have shown that the palatine tonsil effectively incorporates exogenous foreign substances instilled at its surface. It is not clear whether antigen-specific IgA can be induced by the instillation. Sheep red blood cells (SRBC) were instilled at the palatine tonsil every three days as the antigen, and the agglutination titer of specific IgA in saliva was examined. Nasal or intragastric administration, which have been shown to induce specific antibody in saliva, were done as control experiments. Anti-SRBC antibody in saliva from the tonsillar instillation group was detected in the second week, and the agglutination titer reached a maximum in the 6th week after the instillation. The maximum titers in the tonsillar instillation group and nasal administration group were 16 (P<0.01, n=7) and 4 times (P<0.01, n = 7) higher, respectively, than that in the intragastric administration group. In the tonsillar instillation group, the number of specific antibody-producing cells per 10⁵ lymphocytes was the highest in the parotid glands compared with the lymphoid tissues such as the retropharyngeal lymph nodes, nasal mucosa, mesenteric lymph nodes, Peyer's patches, cervical lymph nodes, palatine tonsil and spleen. In the nasal administration group, the number of lymphocytes was the highest in the nasal mucosa. The results indicate that tonsillar instillation was more effective than nasal administration in inducing specific IgA in saliva.     

Nonrandom migration of CD4+, CD8+, and gamma delta+T19+ lymphocyte subsets following in vivo stimulation with antigen

We report here the results of experiments in which the migration of three T cell subsets (CD4+, CD8+, and gamma delta+T19+ cells) through antigen-stimulated lymph nodes and subcutaneous granulomas has been compared with that through normal skin and resting lymph nodes. The percentage of gamma delta+T19+ lymphocytes was halved and the percentage of CD8+ lymphocytes was doubled in lymph draining stimulated compared with control tissues, and all lymphocyte subsets except gamma delta+T19+ lymphocytes had higher hourly outputs in lymph draining antigen-stimulated compared with control tissues. Antigen also resulted in a higher percentage of CD8+ lymphoblasts and a lower percentage of gamma delta+T19+ lymphoblasts in efferent lymph draining antigen-stimulated lymph nodes. The data indicate that lymphocyte subsets leave the blood with differing efficiencies in different vascular beds and raise the possibility that antigen can influence the rate at which tissues extract individual T cell subsets from the blood.     

Expression of low levels of peripheral lymph node-associated vascular addressin in mucosal lymphoid tissues: possible relevance to the dissemination of passaged AKR lymphomas

Lymphoid tumors display a wide variety of growth patterns in vivo, from that of a solitary extralymphoid tumor, to a general involvement of all lymphoid organs. Normal lymphocytes are uniquely mobile cells continuously recirculating between blood and lymph throughout much of their life cycle. Therefore, it is reasonable to propose that disseminating malignant lymphocytes may express recirculation characteristics or homing properties consistent with that of their normal lymphoid counterparts. Trafficking of lymphocytes involves the expression and recognition of both lymphocyte homing receptors and their opposing receptors on endothelium, the vascular addressins. These cell surface elements direct the tissue-selective localization of lymphocyte subsets in vivo into organized lymphoid organs and sites of chronic inflammation where specific binding events occur between lymphocytes and the endothelium of specialized high endothelial venules (HEV). In a recent murine study of 13 lymphoma lines, we found that lymphomas that bind well to high endothelial venules, in the Stamper-Woodruff in vitro assay (an assay of lymphocyte binding to venules in frozen sections of peripheral lymph nodes or Peyer's patches), spread hematogenously to all high endothelial venule bearing lymphoid organs, whereas non-binding lymphomas did not. In some cases lymphomas that bound with a high degree of selectivity to peripheral lymph node (PLN) high endothelial venules exhibited only limited organ preference of metastasis, involving the mucosal lymphoid organs Peyer's patches (PP) in addition to the peripheral lymph nodes of adoptive recipients. Here we demonstrate that Peyer's patch high endothelial venules express a low but functional level of peripheral lymph node addressin (MECA-79) that can be recognized by lymphomas expressing the peripheral lymph node homing receptor (MEL-14 antigen).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes

Dendritic cells (DCs) emigrate to regional lymph nodes (LNs) during immune responses via afferent lymphatic channels. Secondary lymphoid-tissue chemokine (SLC), a CC chemokine, is expressed in secondary lymphoid organs and mediates the chemotaxis of lymphocytes and DCs via its receptor, CC chemokine receptor 7 (CCR7). By dual-label fluorescence confocal microscopy, we showed MHC class II-positive cells within SLC-staining lymphatic channels in the mouse dermis. SLC was a potent in vitro chemoattractant for cultured, migratory skin DCs, and it enhanced the emigration of MHC class II-positive DCs from mouse skin explants by an average of 2.5-fold. Mature or cytokine-activated, but not resting, Langerhans cells expressed CCR7 mRNA by RT-PCR. Anti-SLC Abs, but not control or anti-eotaxin Abs, blocked the in vivo migration of 51Cr-labeled, skin-derived DCs from footpads to draining LNs by 50% (n = 9, p < 0. 005). Thus, we provide direct evidence that SLC and CCR7 participate in the emigration of DCs from peripheral tissue to LNs via lymphatics.     

Independent Bottlenecks Characterize Colonization of Systemic Compartments and Gut Lymphoid Tissue by Salmonella

Vaccination represents an important instrument to control typhoid fever in humans and protects mice from lethal infection with mouse pathogenic serovars of Salmonella species. Mixed infections with tagged Salmonella can be used in combination with probabilistic models to describe the dynamics of the infection process. Here we used mixed oral infections with tagged Salmonella strains to identify bottlenecks in the infection process in naïve and vaccinated mice. We established a next generation sequencing based method to characterize the composition of tagged Salmonella strains which offers a fast and reliable method to characterise the composition of genome-tagged Salmonella strains. We show that initial colonization of Salmonella was distinguished by a non-Darwinian selection of few bacteria setting up the infection independently in gut associated lymphoid tissue and systemic compartments. Colonization of Peyer''s patches fuels the sustained spread of bacteria into mesenteric lymph nodes via dendritic cells. In contrast, infection of liver and spleen originated from an independent pool of bacteria. Vaccination only moderately reduced invasion of Peyer''s patches but potently uncoupled bacterial populations present in different systemic compartments. Our data indicate that vaccination differentially skews the capacity of Salmonella to colonize systemic and gut immune compartments and provide a framework for the further dissection of infection dynamics.     

Trafficking and Replication Patterns Reveal Splenic Macrophages as Major Targets of Dengue Virus in Mice

Human postmortem studies of natural dengue virus (DENV) infection have reported systemically distributed viral antigen. Although it is widely accepted that DENV infects mononuclear phagocytes, the sequence in which specific tissues and cell types are targeted remains uncharacterized. We previously reported that mice lacking alpha/beta and gamma interferon receptors permit high levels of DENV replication and show signs of systemic disease (T. R. Prestwood et al., J. Virol. 82:8411–8421, 2008). Here we demonstrate that within 6 h, DENV traffics to and replicates in both CD169⁺ and SIGN-R1⁺ macrophages of the splenic marginal zone or draining lymph node, respectively, following intravenous or intrafootpad inoculation. Subsequently, high levels of replication are detected in F4/80⁺ splenic red pulp macrophages and in the bone marrow, lymph nodes, and Peyer''s patches. Intravenously inoculated mice begin to succumb to dengue disease 72 h after infection, at which time viral replication occurs systemically, except in lymphoid tissues. In particular, high levels of replication occur in CD68⁺ macrophages of the kidneys, heart, thymus, and gastrointestinal tract. Over the course of infection, proportionately large quantities of DENV traffic to the liver and spleen. However, late during infection, viral trafficking to the spleen decreases, while trafficking to the liver, thymus, and kidneys increases. The present study demonstrates that macrophage populations, initially in the spleen and other lymphoid tissues and later in nonlymphoid tissues, are major targets of DENV infection in vivo.     

Quantitative evaluation of the total number and distribution of lymphocytes in young pigs.

In young pigs, the spleen, thymus and all lymph nodes were dissected out and weighed. The relative content of lymphoid cells was determined from histological sections. The number of nucleated cells was evaluated by two different methods: firstly, by measuring the DNA content of samples of lymphoid tissue and dividing by the DNA content of a single nucleus; and, secondly, by counting all lymphoid cells in histological sections of defined volumes of these organs. The number of lymphoid cells in tonsils, gut, bone marrow and lung were determined using histological evaluations and the volumes or weights of these organs. The resulting average number of lymphocytes was 321 times 10 (9) for a pig of 26 kg body weight. The lymphocytes showed the following distribution in lymphoid and non-lymphoid organs: thymus 44%, spleen 9%, mesenteric lymph nodes 17%, cervical lymph nodes 9%, other peripheral lymph nodes 3%, gut-associated lymphocytes 5%, tonsils 2%, bone marrow 5%, blood 3%, lung 0.2% and an estimated figure of 3% for all other tissues.     

Lymphocyte traffic through lymph nodes and Peyer's patches of the rat: B- and T-cell-specific migration patterns within the tissue,and their dependence on splenic tissue

The migration routes of lymphocyte subsets through organ compartments are of importance when trying to understand the local events taking place during immune responses. We have therefore studied the traffic of B, T, CD4⁺, and CD8⁺ lymphocytes through lymph nodes and Peyer's patches. At various time points after injection into the rat, labeled lymphocytes were localized, and their phenotype characterized in cryostat sections using immunohistochemistry. Morphometry was also performed, and the recovery of ⁵¹Cr-labeled lymphocytes in these organs was determined. B and T lymphocytes entered the lymph nodes via the high endothelial venules in similar numbers. Most B lymphocytes migrated via the paracortex (T cell area) into the cortex (B cell area), and then back in substantial numbers into the paracortex. In contrast, T lymphocytes predominantly migrated into the paracortex and were rarely seen in the cortex. No obvious differences were seen between various lymph nodes and Peyer's patches and the routes of CD4⁺ and CD8⁺ lymphocytes. After injection of lymphocytes into animals with autotransplanted splenic tissue, the number of B lymphocytes that had migrated into the B cell area of lymph nodes and of Peyer's patches was significantly decreased, whereas CD4⁺ lymphocytes migrated in larger numbers into the T cell area of both organs.This study was supported by the Deutsche Forschungsgemein-schaft (SFB 244, A7).     

Cutting edge: the B cell chemokine CXC chemokine ligand 13/B lymphocyte chemoattractant is expressed in the high endothelial venules of lymph nodes and Peyer's patches and affects B cell trafficking across high endothelial venules

While CCR7 ligands direct T cell trafficking into lymph nodes (LNs) and Peyer's patches (PPs), chemokines that regulate B cell trafficking across high endothelial venules (HEVs) remain to be fully elucidated. Here we report that CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant) is detected immunohistologically in the majority of HEVs in LNs and PPs of nonimmunized mice. Systemically administered anti-CXCL13 Ab bound to the surface of approximately 50% of HEVs in LNs and PPs, but not to other types of blood vessels, indicating that CXCL13 is expressed in the HEV lumen. In CXCL13-null mice, B cells rarely adhered to PP HEVs, whereas T cells did efficiently. Superfusion of CXCL13-null PPs with CXCL13 restored the luminal presentation of CXCL13 and also B cell arrest in PP HEVs at least partially. Collectively, these results indicate that CXCL13 expressed in the HEV lumen plays a crucial role in B cell trafficking into secondary lymphoid tissues such as PPs.     

Role of Palatine Tonsils as a Prion Entry Site in Classical and Atypical Experimental Sheep Scrapie

Atypical and classical scrapie-infected sheep brain tissue was monolaterally injected into the tonsils of lambs to investigate their role as a prion entry point. We first detected classical PrP^Sc within the inoculated tonsil and in the ipsilateral retropharyngeal lymph node at 3 months postinoculation (p.i.). At 7 months p.i., PrP^Sc colonized other lymphoid tissues bilaterally, including ileal Peyer''s patches. The earliest PrP^Sc deposition within the brain was ipsilaterally observed at 9 months p.i. in the substantia reticularis of the medulla oblongata. At 12 months p.i., PrP^Sc deposition was present bilaterally in the nucleus parasympathicus nervi vagi, as well as in the intermediolateral cell column of the thoracolumbar spinal cord. No PrP^Sc was detected in the lambs inoculated with atypical scrapie. These findings suggest that neuroinvasion may naturally occur from the tonsil after a widespread prion replication within the lymphoid tissues during classical scrapie only, thus mimicking the pathogenesis after oral ingestion.     

Colony-stimulating factor (CSF-1): its enhancement of plasminogen activator production and inhibition of cell growth in a mouse macrophage cell line

The cell-mediated immune response by the gut-associated lymphoid tissues to antigens within the intestinal tract is poorly understood. Our objective was to investigate the antigen-specific T cell proliferative response and cytotoxic T lymphocyte (CTL) response of cells from the GALT after enteric immunization with vaccinia virus. Lymphocytes able to proliferate in the presence of vaccinia virus in vitro were found in large numbers in mesenteric lymph nodes (MLN) 6 days after the injection of vaccinia virus into the lumen of the small bowel. The MLN at this time also contained vaccinia-specific CTL, but unlike the proliferating cells, which were found for several weeks after immunization, the CTL were demonstrable in the MLN for only a few days. Peyer's patches were found to contain neither antigen-stimulated proliferating cells nor CTL. The viral-specific proliferating lymphocytes from the MLN 10 days after immunization were sIg-, monoclonal antibody W3/25+, MRC OX-8- large lymphoblasts. The vaccinia-specific CTL were also large lymphoblasts, but they belonged to the W3/25-, OX-8+ subset. Thus, a strong T helper and cytotoxic T lymphoblast response is generated within the MLN after viral challenge of the gut.     

MAdCAM-1 Dependent Colonization of Developing Lymph Nodes Involves a Unique Subset of CD4+CD3- Hematolymphoid Cells

During fetal lymph node organogenesis in mice, lymph node postcapillary high endothelial venules briefly express the Peyer's patch addressin MAdCAM-1. This allows initial seeding by two unusual lymphocyte populations selectively expressing the Peyer's patch homing receptor integrin alpha4beta7: CD4 + CD3- oligolineage progenitors and TCR gammadelta + T cells. It was found that the CD4 + CD3- cells are lineage-restricted progenitors that express surface lymphotoxin-beta (LTbeta) and the chemokine receptor BLR1. They can differentiate into natural killer cells, dendritic antigen-presenting cells, and follicular cells of unknown outcome, but these cells do not become T or B lymphocytes.

In addition to LN, CD4 + CD3- cells can also be found in fetal spleen starting at 13.5 dpc, while absent from fetal liver. In view of the necessity of lymphotoxin in lymphoid organ development, it is thought that the novel subset of CD4 + CD3-LTbeta + fetal cells is instrumental in the development of lymphoid tissue architecture.     

Evolutionary conservation of tissue-specific lymphocyte-endothelial cell recognition mechanisms involved in lymphocyte homing

Tissue-specific interactions with specialized high endothelial venules (HEV) direct the homing of lymphocytes from the blood into peripheral lymph nodes, mucosal lymphoid organs, and tissue sites of chronic inflammation. These interactions have been demonstrated in all mammalian species examined and thus appear highly conserved. To assess the degree of evolutionary divergence in lymphocyte-HEV recognition mechanisms, we have studied the ability of lymphocytes to interact with HEV across species barriers. By using an in vitro assay of lymphocyte binding to HEV in frozen sections of lymphoid tissues, we confirm that mouse, guinea pig, and human lymphocytes bind to xenogeneic as well as homologous HEV. In addition, we show that mouse and human lymphoid cell lines that bind selectively to either peripheral lymph node or mucosal vessels (Peyer's patches, appendix) in homologous lymphoid tissues exhibit the same organ specificity in binding to xenogeneic HEV. Furthermore, monoclonal antibodies that recognize lymphocyte "homing receptors" and block homologous lymphocyte binding to peripheral lymph node or to mucosal HEV, also inhibit lymphocyte interactions with xenogeneic HEV in a tissue-specific fashion. Similarly, anti-HEV antibodies against organ-specific mouse high endothelial cell "addressins" involved in lymphocyte homing to peripheral lymph node or mucosal lymphoid organs, not only block the adhesion of mouse lymphocytes but also of human lymphocytes to target mouse HEV. The results illustrate a remarkable degree of functional conservation of elements mediating these cell-cell recognition events involved in organ-specific lymphocyte homing.     

Migration patterns of dendritic cells in the rat: comparison of the effects of gamma and UV-B irradiation on the migration of dendritic cells and Lymphocytes

To further define the underlying mechanisms of immune suppression induced by UV-B irradiation, we have examined the kinetics of homing patterns of in vitro UV-B-irradiated and gamma-irradiated-thoracic duct lymphocytes (TDL) compared to dendritic cells (DC). Our findings show that 111In-oxine-labeled TDL specifically home to the spleen, liver, lymph nodes, and bone marrow with subsequent recirculation of a large number of cells from the spleen to lymph nodes. In contrast, DC preferentially migrate to the spleen and liver with a relatively insignificant distribution to lymph nodes and an absence of subsequent recirculation. Splenectomy prior to cell injection significantly diverts the spleen-seeking DC to the liver but not to the lymph nodes, while the homing of TDL to lymph nodes is significantly increased. In vitro exposure of 111In-oxine labeled TDL to gamma irradiation does not significantly impair immediate homing to lymphoid tissues but inhibits cell recirculation between 3 and 24 hr. In contrast, gamma irradiation does not affect the tissue distribution of labeled DC, suggesting that DC are more radioresistant to gamma irradiation than TDL. Unlike the findings in animals injected with gamma-irradiated cells, UV-B irradiation virtually abolished the homing of TDL to lymph nodes and significantly reduced the homing of the spleen-seeking DC to the splenic compartment while a large number of cells were sequestered in the liver. The results of in vitro cell binding assay show that TDL, unlike DC, have the capacity to bind to high endothelial venules (HEV) within lymph node frozen sections while gamma and UV-B irradiation significantly inhibit the binding of TDL to lymph node HEV. These findings suggest that: (i) DC, unlike TDL, are unable to recirculate from blood to lymph nodes through HEV; (ii) although gamma irradiation impairs TDL recirculation, it does not affect DC tissue distribution; and (iii) UV-B irradiation impairs both TDL and DC migration patterns. We conclude that the lack of capacity of irradiated TDL to home to lymph nodes is due to damage to cell surface homing receptors and that the failure of DC to home to the lymph node microenvironment is related to the absence of HEV homing receptors on their cell surface.     

Expression of Wiskott-Aldrich syndrome protein in dendritic cells regulates synapse formation and activation of naive CD8+ T cells

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin polimerization in hematopoietic cells. Mutations in WASp cause a severe immunodeficiency characterized by defective initiation of primary immune response and autoimmunity. The contribution of altered dendritic cells (DCs) functions to the disease pathogenesis has not been fully elucidated. In this study, we show that conventional DCs develop normally in WASp-deficient mice. However, Ag targeting to lymphoid organ-resident DCs via anti-DEC205 results in impaired naive CD8(+) T cell activation, especially at low Ag doses. Altered trafficking of Ag-bearing DCs to lymph nodes (LNs) accounts only partially for defective priming because correction of DCs migration does not rescue T cell activation. In vitro and in vivo imaging of DC-T cell interactions in LNs showed that cytoskeletal alterations in WASp null DCs causes a reduction in the ability to form and stabilize conjugates with naive CD8(+) T lymphocytes both in vitro and in vivo. These data indicate that WASp expression in DCs regulates both the ability to traffic to secondary lymphoid organs and to activate naive T cells in LNs.     

Homing of germinal-center cells into germinal centers of lymph node via afferent lymphatics

Summary Affinity of lymphoid cells for the microenvironment of germinal centers (GC), as detectable in transfer experiments by rapid homing in spleen GC from the blood, is a capacity expressed by only a subset of lymphoid cells, in particular by those constituting a GC. However, when introduced into the blood stream, these cells do not home into GC of lymph nodes and gut-associated lymphoid tissues. To investigate further this homing inability for high endothelial venule (HEV)-containing lymphoid tissues, GC cells isolated from donor rabbit appendix were labeled in vitro with ³H-leucine and injected into an afferent lymph vessel of recipient popliteal lymph nodes. Draining lymph nodes were removed 15 min to 24 h after cell administration and prepared for radioautography. For reference, the migration of cells isolated from Peyer's patches and thoracic duct lymph was also studied. By use of appendix GC cells, large numbers of labeled cells were found to migrate into GCs of the outer cortex centripetally, i.e., from the subcapsular sinus through the lymphocyte corona into the GC proper. The same was observed for cells from Peyer's patches, although in smaller numbers. Thoracic duct lymphocytes were only localized in the lymphocyte corona and the deep cortex. Thus, appendix GC cells and a subpopulation of cells from Peyer's patches can reach lymph node GC, but only when administered intralymphatically. We conclude that cells expressing affinity for the GC microenvironment do so for both spleen and lymph node GC, but do not have the capacity to interact with the wall of HEV; its implication for the understanding of the dynamics of a GC reaction is discussed.Abbreviations GC germinal center - GCC germinal-center cells - AGCC appendix germinal-center cells - GCPC germinal-center precursor cells - GCSC germinal-center seeking cells - HEV high endothelial venules - SRBC sheep red blood cells - PP Peyer's patch - TDL thoracic duct lymphocytes - NCS newborn calf serum - PBS phosphate-buffered saline - PNA peanut agglutinin - LN lymph node - LC lymphocyte corona - DC deep cortex unit