Activated T cells enter rat lymph nodes and Peyer's patches via high endothelial venules: survival by tissue-specific proliferation and preferential exit of CD8+ T cell progeny.

Activated T cells reach the lymph nodes via afferent lymphatics but it is unknown to what extent they also enter them directly via high endothelial venules (HEV). Little is known about the mechanism mediating the proliferation of activated T cells within lymphoid tissues in vivo or the subsequent fate of the progeny. Therefore, we stimulated rat T cells via TCR and CD28 in vitro and after injection identified them in the blood and the HEV of lymphoid organs at several time points. In addition, the proliferation of these cells was studied after entering different lymphoid organs. Our results show that, firstly, activated T cells continuously enter lymph nodes and Peyer's patches directly via HEV. Second, they proliferate within lymphoid organs, the rate significantly depending on the microenvironment. Third, mainly CD8+ progeny are able to leave the tissues and re-enter the blood. Thus, the distribution of activated T cells circulating through the body can be regulated during entry, but also within the tissue by influencing their proliferation and subsequent release.     

B and T lymphocyte subsets enter peripheral lymph nodes and Peyer's patches without preference in vivo: no correlation occurs between their localization in different types of high endothelial venules and the expression of CD44, VLA-4, LFA-1, ICAM-1, CD2 or L-selectin

Many lymphocytes enter tissues such as peripheral lymph nodes and Peyer's patches through high endothelial venules (HEV). It is known that HEV differ in the expression of adhesion molecules as lymphocyte subsets do. Through the interaction of these molecules B and T lymphocyte subsets are thought to be preferentially directed into lymphoid organs. However, it is unclear which role these mechanisms play in vivo, since there are no studies demonstrating that blood lymphocyte subsets preferentially interact with different types of HEV in vivo. Therefore, in the present study the frequency of B, T, CD4⁺ and CD8⁺ lymphocytes in the wall of the HEV of rat peripheral lymph nodes and Peyer's patches was analyzed by immunohistology. In addition, the expression of CD44, VLA-4, LFA-1, ICAM-1, CD2 and L-selectin on B and T lymphocyte subsets of the blood was determined by flow cytometry. Although B and T lymphocytes showed significantly different levels of expression for each adhesion molecule investigated, the relation of B and T lymphocytes within the HEV of peripheral lymph nodes and Peyer's patches was strikingly comparable (38.0 ± 5.2% vs. 40.6 ± 5.7% and 62.0 ± 5.2% vs. 59.4 ± 5.7%, respectively). The same was true for CD4⁺ and CD8⁺ cells. Thus, although HEV and the blood lymphocyte subsets differ markedly in their expression pattern of adhesion molecules, the existing levels are sufficient to mediate comparable entrance of B and T lymphocyte subsets into both types of HEV.     

The interaction of high endothelial venules with T and B cells in peripheral lymph nodes after antigenic stimulation

The influence of an ongoing immune response on the specific interaction of high endothelial venules (HEV) in peripheral lymph nodes with T and B cells was determined using short-term homing experiments and an in vitro HEV adherence assay. It was demonstrated that stimulation with various antigens did not change the receptor specificity of the HEV. However, the retention and/or the efflux of immigrant cells in the stimulated lymph nodes had changed.     

Activation of high endothelial venules in peripheral lymph nodes. The involvement of interferon-gamma

The introduction of Freund's complete adjuvant into footpadsof mice leads to functional changes of the high endothelialvenules (HEV) in the draining lymph nodes. Using an in vitroadhesion assay, increased binding of monocytes and dendriticcells could be seen. These effects are also seen after injectionof high doses of IFN-. No changes in the expression of vascularaddressins, VCAM-1, E selectin or ICAM-1 could be observed butthe binding of monocytes could be blocked with an antibody againstMac-1. Depletion studies have made it clear that macrophagesin the lymph node are not directly involved in the activationof the HEV. The induction of additional adhesion mechanismson HEV could be an efficient way to recruit inflammatory cellsinto the lymph node, thereby regulating the local immune response.     

CCR7 mediates the migration of Foxp3+ regulatory T cells to the paracortical areas of peripheral lymph nodes through high endothelial venules

Thymus-derived forkhead box p3(+) naturally occurring regulatory T cells (nTreg) are thought to circulate throughout the body to maintain peripheral immunological self-tolerance through interactions with dendritic cells (DCs), resulting in regulation of conventional T cells. However, the chemokine receptors, which are putatively involved in the in vivo migration of nTreg, have not been fully established. Here, we demonstrated that lymph node nTreg preferentially migrated to the paracortical area of lymph nodes after adoptive transfer, where they were observed to make contact frequently with CD8alpha(+) DCs and CD8alpha(-) CD11b(-) DCs. This migration of nTreg to the paracortical areas was impaired severely when cells were prepared from CCR7-deficient mice. However, to some extent, CCR7-independent migration of nTreg in such CCR7-deficient mice was also observed, but this occurred mainly in the medullary high endothelial venules. Taken together, these data provide the evidence that CCR7 mediates nTreg migration to the paracortical areas of lymph nodes under steady-state conditions; however, CCR7-independent migration also takes place in the medulla.     

Interaction of B and T lymphocyte subsets with high endothelial venules in the rat: binding in vitro does not reflect homing in vivo

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.     

P-selectin glycoprotein ligand-1 mediates L-selectin-independent leukocyte rolling in high endothelial venules of peripheral lymph nodes

Lymphocyte homing to peripheral lymph nodes (LNs) requires L-selectin. Previous studies, however, suggest that there are L-selectin-independent mechanisms of lymphocyte homing. P-selectin glycoprotein ligand-1 (PSGL-1) is a major ligand for P-selectin expressed in a selectin-binding form on myeloid cells and subsets of lymphoid cells. To discover whether PSGL-1 plays a role in lymphocyte homing, we examined leukocyte rolling and adhesion in the high endothelial venules (HEVs) of the subiliac LNs of wild-type and PSGL-1-deficient mice by intravital microscopy. There were no significant differences in blood velocity or wall shear stress between wild-type and PSGL-1-deficient mice. Although the leukocyte rolling fraction was not altered in PSGL-1-deficient mice, infusion of an anti-L-selectin mAb into these mice completely abolished leukocyte rolling, while the same treatment in wild-type mice inhibited 90% of the leukocyte rolling. This residual rolling in wild-type mice appears to depend on the PSGL-1-P-selectin interaction, since infusion of an anti-L-selectin mAb together with an anti-PSGL-1 mAb or anti-P-selectin mAb almost completely abolished the rolling. PSGL-1 deficiency also led to a higher rolling velocity, suggesting that PSGL-1 mediates leukocyte rolling at low velocities. P-selectin was found to be expressed on the HEVs of subiliac LNs under the conditions of intravital microscopy. Taken together, these results indicate that the interaction of PSGL-1 with P-selectin constitutes a second mechanism of leukocyte rolling in the HEVs of peripheral LNs.     

Rapid decrease in lymphocyte adherence to high endothelial venules in lymph nodes deprived of afferent lymphatic vessels

Occlusion of the afferent lymph flow to the lymph node (LN) results in both flattening of the endothelium of high endothelial venules (HEV) and a severe decrease in numbers of lymphocytes in transit across the walls of the flattened HEV. In the present study we have used the in vitro lymphocyte-binding assay to investigate the ability of HEV in rat LN to bind lymphocytes at various time points after occlusion of the afferent lymph flow. In addition the specificity of T and B lymphocyte adherence to HEV of such operated LN was studied. In normal LN, lymphocytes adhered to virtually all HEV using the in vitro binding assay. However, 1 and 2 weeks after operation lymphocytes bound to only 50-60% of the HEV and by 3-6 weeks 20-30%. The total numbers of lymphocytes bound to these HEV had also diminished to 10% of the control value 3-6 weeks after operation. Morphometric analysis showed that this was not only due to a reduction in the area of HEV endothelium available for lymphocyte adherence by flattening of the high endothelial cells, but also to a strong decrease in the numbers of bound lymphocytes per unit area high endothelium. In spite of the reduction in numbers of adhering lymphocytes the T/B cell ratio did not change. The results show that the reduction in lymphocyte binding of HEV in operated LN is a rapid event, probably due to loss of high endothelial cell determinants involved in binding of lymphocytes. The decrease in lymphocyte binding clearly precedes flattening of HEV endothelium suggesting that the height of high endothelial cells is of secondary importance to lymphocyte adherence.     

Disappearance and reappearance of high endothelial venules and immigrating lymphocytes in lymph nodes deprived of afferent lymphatic vessels: a possible regulatory role of macrophages in lymphocyte migration

Interruption of the afferent lymphatic vessels of the popliteal lymph node resulted in the disappearance of high endothelial venules (HEV) and immigrating lymphocytes within 3 weeks. HEV showed several characteristic morphological changes: the endothelial cells became flattened and less pyroninophilic, the chromatine became condensed and protein synthetizing and secretory cell organelles became scarce. At the same time the number of macrophages in the lymph node was severely reduced. Injection of sheep red blood cells into such lymph nodes, 6 weeks after operation, resulted in reappearance of HEV and immigrating lymphocytes, and development of many plasma cells and some germinal centres. Injection of lipopolysaccharide into the operated lymph nodes resulted in the appearance of many plasma cells and a few poorly developed germinal centres; HEV and immigrating lymphocytes, however, remained almost absent. The results show a relationship between the immigration of lymphocytes and the activity of the endothelial cells in the HEV. The activation of the latter may occur by mediators released by antigen-stimulated macrophages and T cells. Moreover, the morphological features of the HEV are independent of the presence of recirculating lymphocytes.     

CD4+ T cells of both the naive and the memory phenotype enter rat lymph nodes and Peyer's patches via high endothelial venules: Within the tissue their migratory behavior differs

It is thought that naive T cells predominantly enter lymphoid organs such as lymph nodes (LN) and Peyer's patches (PP) via high endothelial venules (HEV), whereas memory T cells migrate mainly into non-lymphoid organs. However, direct evidence for the existence of these distinct migration pathways in vivo is incomplete, and nothing is known about their migration through the different compartments of lymphoid organs. Such knowledge would be of considerable interest for understanding T cell memory in vivo. In the present study we separated naive and memory CD4⁺ T cells from the rat thoracic duct according to the expression of the high and low molecular weight isoforms of CD45R, respectively. At various time points after injection into congenic animals, these cells were identified by quantitative immunohistology in HEV, and T and B cell areas of different LN and PP. Three major findings emerged. First, both naive and memory CD4⁺ T cells enter lymphoid organs via the HEV in comparable numbers. Second, naive and memory CD4⁺ T cells migrate into the B cell area, although in small numbers and continuously enter established germinal centers (GC) with a bias for memory CD4⁺ T cells. Third, memory CD4⁺ T cells migrate faster through the T cell area of lymphoid organs than naive CD4⁺ T cells. Thus, our study shows that memory CD4⁺ T cells are not excluded from the HEV route. In addition, “memory” might depend in part on the ability of T cells to specifically enter the B cell area and GC and to screen large quantities of lymphoid tissues in a short time.     

CD4+ lymphocytes are extracted from blood by peripheral lymph nodes at different rates from other T cell subsets and B cells

The circulation of lymphocyte subsets through prescapular lymph nodes in sheep has been quantified using a panel of monoclonal antibodies against sheep lymphocyte surface antigens. Differences in the extraction of lymphocyte subsets from blood by the lymph node were found with CD4+ lymphocytes being extracted at a faster rate (1/2) than CD8+, SBU-T19+, major histocompatibility complex class II+ and B cells (1/4 to 1/5). In order to accommodate existing data on organ-specific adhesion molecules, one subset specific and one tissue specific, expressed on vascular endothelium could act jointly to regulate the migration of recirculating lymphocytes.     

Isolation, culture, and characterization of endothelial cells derived from the post-capillary venules of sheep mesenteric lymph nodes, Peyer's patches, and associated small bowel

Lymphocyte recirculation is mediated principally by specialized endothelial cells which line the post-capillary venules of lymph nodes and other secondary lymphoid tissues. The ontogeny and physiology of this process have been characterized in sheep in considerable detail. To further enhance the analytical potential of this experimental system we have isolated endothelial cells from the post-capillary venules of ovine mesenteric lymph nodes by perfusion with small (37-74 microns diameter), sulfonated microcarrier beads. Cells isolated in this manner have been maintained in vitro for greater than 12 months through greater than 30 passages. The endothelial nature of these cells has been conclusively established on the basis of morphologic, metabolic, and immunologic criteria. Virtually all (greater than 99%) cells in primary and passaged cultures metabolized Dil-AC-LDL, a known marker for endothelial cells. Furthermore, nearly all (greater than 95%) cells expressed cell-surface von Willebrand factor and antithrombin III, which are known endothelial antigens. All cells expressed major histocompatibility class I antigens but no cells expressed class II antigens. In vitro lymphocyte-binding studies revealed that these cells bound lymphocytes in a dose-dependent fashion. The microcarrier perfusion technique was also used to isolate endothelial cells from the post-capillary venules of ileal Peyer's patches and associated small bowel in sheep. The majority (70%) of cells isolated in this manner resembled the cells isolated from mesenteric lymph nodes both morphologically and metabolically.     

Development of apoptosis and changes in lymphocyte subsets in thymus, mesenteric lymph nodes and Peyer's patches of mice orally inoculated with T-2 toxin.

Development of apoptosis and changes in lymphocyte subsets were examined mainly by flow cytometer in thymus, mesenteric lymph nodes and Peyer's patches of mice up to 24 hours after oral inoculation with T-2 toxin (10 mg/kg). T-2 toxin attacked Peyer's patches first, then mesenteric lymph nodes, and finally thymus in relation to the course of enteric absorption of orally inoculated T-2 toxin. The degree of lymphocyte apoptosis was prominent in the thymus, moderate in the Peyer's patches, and somewhat mild in the mesenteric lymph nodes, suggesting the difference in lymphocyte population susceptible to T-2 toxin. As to the changes in lymphocyte subsets, CD4+ CD8+ T cells were most sensitive to T-2 toxin, and CD4+ CD8- T cells were more severely depressed than CD4- CD8+ T cells in the thymus. In the mesenteric lymph nodes, CD3+ cells was more clearly affected than CD19+ cells, and the numbers of CD4+ and CD8+ cells were similarly decreased. In the Peyer's patches, the numbers of CD3+, CD 19+, CD4+ and CD8+ cells were unexceptionally decreased. In addition, among IgM+, IgG+ and IgA+ B cells, the number of IA+ B cells which are more important in the mucosal immunity was most severely affected.     

慢性乙肝病毒携带者外周血皮质醇水平与T细胞亚群状态的研究

目的 探讨慢性乙肝病毒(HBV)携带者血浆皮质醇水平与细胞免疫状态的变化.方法 选取符合诊断标准的慢性HBV携带者60例,正常对照10例,放射免疫法测定血浆皮质醇水平,流式细胞仪检测外周血T细胞亚群CD3+、CD4+、CD8+、CD4+/CD8+比例.结果 与正常对照组相比,慢性HBV携带者血浆皮质醇水平相对升高(P＜0.05),CD4+细胞比例无明显变化(P＞0.05),CD8+细胞比例明显增加(P＜0.05),CD4+/CD8+明显下降(P＜0.05).HBeAg阳性组与HBeAg阴性组比较,HBeAg阳性组血浆皮质醇升高较为明显(P＜0.05),CD4+细胞比例无明显变化(P＞0.05).CD8+细胞比例明显增加(P＜0.05),CD4+/CD8+明显下降(P＜0.05).结论 慢性乙肝病毒携带者存在着血浆皮质醇水平的增高及细胞免疫功能的失衡,慢性HBV感染过程中存在内分泌-免疫系统紊乱.     

CD44 is not directly involved in the binding of lymphocytes to cultured high endothelial cells from peripheral lymph nodes.

The Ager assay was adapted to a porcine lymphocyte-rat high endothelial cell (HEC) system. Using this in vitro assay, the role of porcine CD44 in lymphocyte binding to HEC was examined. The results show that the presence of soluble CD44 molecules did not inhibit the binding of porcine lymphocytes to the cultured rat HEC. Treatment of lymphocytes with anti-CD44 monoclonal antibodies (mAb), or with papain, which removes a 45,000 MW peptide from the intact CD44 molecule, did not inhibit the binding. Binding to the rat HEC did not induce modulation of CD44 molecules on the cell surface. Furthermore, modulation of the CD44 molecule by biotinylated anti-CD44 antibody followed by streptavidin-phycoerythrin, which had caused the molecule to cap on the cell surface, did not prevent the cells binding to the HEC. Similarly, cells denuded of CD44 by anti-CD44 antibody retained the capacity to bind to HEC. Moreover, the binding cells were mainly those which had been stripped of CD44 by the antigenic modulation. It is concluded that CD44 is not directly involved in the binding of lymphocytes to the cultured HEC from peripheral lymph nodes (PLN).     

Non-random migration of CD4+, CD8+ and gamma delta+T19+ lymphocytes through peripheral lymph nodes.

The experiments described in this paper have examined the migration of three fluorochrome-labelled T-lymphocyte subsets (CD4+, CD8+ and gamma delta+T19+) on a single passage from blood to lymph, through prescapular lymph nodes. Lymphocytes obtained from prescapular efferent lymph were labelled in vitro with fluorochrome and returned to the blood of the same animal. Over the next 2 days, lymph was continuously monitored and the cells in all collections, including the one used for intravenous infusion, were phenotyped and analysed by flow cytometry. Significant differences in the subset ratios between the infused, starting population and the recirculated population indicated that CD4+ and gamma delta+T19+ lymphocytes are extracted by a resting lymph node at the same rate and that both are extracted at a faster rate than CD8+ lymphocytes. The results presented here also suggest that a unique subset of gamma delta+T19+ lymphocytes may be present in blood that does not recirculate through peripheral lymph nodes.     

T lymphocyte rolling and recruitment into peripheral lymph nodes is regulated by a saturable density of L-selectin (CD62L)

L-selectin mediates tethering and rolling of lymphocytes in high endothelial venules (HEV) of lymph nodes (LN) and of leukocytes at inflammatory sites. We used transgenic mice expressing varying levels of wild-type or a non-cleavable mutant form of L-selectin on T cells to determine the relationship between L-selectin density, tethering and rolling, and migration into LN. T cells expressing supraphysiological levels of either wild-type or non-cleavable L-selectin showed rolling parameters similar to C57BL/6 T cells in hydrodynamic flow assays and during rolling in Peyer's patch HEV. In contrast, PMA- or antigen-activated T cells and L-selectin(+/-) T cells expressing subphysiological levels of L-selectin showed reduced numbers of rolling cells with increased rolling velocity. Short-term homing studies showed that elevated expression of L-selectin above physiological levels had no effect on T cell migration to LN; however, low L-selectin expression resulted in reduced T cell homing to LN. Thus, T lymphocyte migration into LN is regulated by the density of cell surface L-selectin. In addition, there is a saturable density of L-selectin required for optimal homing to PLN in C57BL/6 mice, the L-selectin level on circulating naive T cells promotes optimal homing, and increased expression above saturating levels promotes no further increase in T cell recruitment.     

Recirculation of lymphocyte subsets (CD5+, CD4+, CD8+, T19+ and B cells) through fetal lymph nodes

The experiments reported in this paper examine the cell-surface phenotype (CD5, CD4, CD8, T19, MHC class II and sIg) and cell output of lymphocyte subsets circulating through a subcutaneous lymph node in the sheep fetus, in an environment unaffected by foreign antigen and circulating immunoglobulins. CD4+ lymphocytes were the major T-cell subset in fetal lymph and were clearly enriched in lymph compared with blood, whereas T19+, CD8+ and B lymphocytes were not. It seems likely that in the fetus CD4+ lymphocytes are extracted from the blood at a faster rate than are other T-cell subsets and B cells. There was a much higher percentage of CD8+ and T null cells and a lower percentage of MHC class II+ and B cells circulating in the fetal lymph than in adult lymph, while the percentage of T19+ lymphocytes in fetal blood was twice that in the adult. Although the hourly cell output from an adult prescapular lymph node was far higher than that from a fetal lymph node, the circulation of lymphocytes through fetal lymph nodes was much greater per gram lymph node weight than that through adult lymph nodes. The wholesale recirculation in the fetus of all the major T-cell subsets found in the adult is paradoxical because it is not known what function they serve in the fetus in the absence of antigen and ongoing immune responses, although clearly they are not memory cells.     

Recirculation of lymphocyte subsets (CD5+, CD4+, CD8+, SBU-T19+ and B cells) through gut and peripheral lymph nodes

The surface phenotypes (CD5+, CD4+, CD8+, SBU-T19+, MHC class II+, T80+ and sIg+) of lymphocytes in blood, and in prescapular and ileocaecal efferent lymph of sheep, have been determined. The similarity in the distribution of lymphocyte subsets in both lymph compartments indicated that the non-random migration of lymphocyte populations to peripheral lymph nodes and gut could not be due to preferential migration of a particular lymphocyte subset such as CD4+ or CD8+ cells to one tissue. Marked differences in the percentage of lymphocyte subsets between blood and efferent lymph suggested that some lymphocyte subsets leave the blood with differing efficiencies and that differential extraction of lymphocyte subsets from blood by a lymph node may be due to subset-specific lymphocyte-endothelial interactions.     

Activation of T lymphocytes to M locus determinants in vivo. I. Quantitation of T cell proliferation and migration into thoracic duct lymph.

Proliferative responses to M locus (Mls) determinants were investigated in vivo by injecting T cell-containing populations intravenously into irradiated H2-identical, Mls-incompatible mice. Responses were studied 1-6 days later by measuring levels of radioactivity in the spleen and lymph nodes of the recipient 45 min after intravenous injection of tritiated thymidine. High responses were observed against Mlsa and Mlsd determinants, i.e. determinants which give strong proliferative responses in vitro. With injection of thoracic duct lymphocytes (TDL), lymph node or thymus cells, the responses, both in the spleen and the lymph nodes, reached a peak at day 3-4 and then fell sharply. This was in marked contrast to the stimulation observed with H-2-incompatible mice where, with TDL or lymph node cells (though not with thymus cells), responses were low at day 3-4 but reached high levels at day 6. Significant responses were also observed against Mlsb and Mlsc determinants, i.e. determinants which stimulate poorly in vitro. The kinetics of this response differed from that against Mlsa or Mlsd determinants in in that proliferation was very low at day 4 but high at day 6. No evidence was found that the responses observed reflected "back-stimulation" by the host. Since congenic resistant strains were not used, the possibility that the stimulation observed was in fact due to minor transplantation antigens rather than Mls determinants could not be excluded. Large numbers of blast cells appeared in thoracic duct lymph of mice sustaining responses to Mlsa or Mlsd determinants. Nearly all of these cells had recently synthesized DNA and entered the lymph after day 3.