Subset-specific regulation of the lymphatic exit of recirculating lymphocytes in vivo.

The blood-to-lymph recirculation of lymphocytes is required for the maintenance of immune surveillance and the dissemination of memory. Although the ability of lymph-borne cells to recirculate has been well documented, relatively less is known about the migration capacity of PBLs. We have found a clear preference for PBLs to recirculate through s.c. rather than intestinal lymph nodes. This preference could be directly attributed to the migratory characteristics of gammadelta-T cells. gammadelta-T cells were found to express significantly higher levels of L-selectin than other subsets, suggesting that at least some of this preferential migration could be attributed to their interaction with ligands on vascular endothelium. More detailed experiments showed that gammadelta-T cells migrated through lymph nodes with greater efficiency than alphabeta T cells or B cells, which clearly indicated an enhanced ability of gammadelta-T cells to exit lymph nodes in the efferent lymph independent of entry from the blood. This hypothesis was supported by histological examination, where gammadelta-T cells were found almost exclusively in the interfollicular traffic areas within lymph nodes. These data indicate that gammadelta-T cells are the most active recirculating lymphocyte subset in ruminants and suggest new mechanisms to regulate the traffic of lymphocyte subsets through normal lymph nodes.

Splenectomy selectively affects the distribution and mobility of the recirculating lymphocyte pool.

The spleen plays a major role in immune surveillance, but the impact that splenectomy exerts on the immune competence of an individual is not fully resolved. Here we show that neonatal splenectomy in sheep does not abrogate the development of a large, nonrecirculating pool of lymphocytes and that it has no effect on the acquisition of a normal blood lymphocyte profile. Splenectomy did, however, result in a significant decrease in blood residency time of recirculating lymphocytes and in an enhanced accumulation of recirculating lymphocytes in lymph nodes. Furthermore, nonrecirculating peripheral blood lymphocytes were less likely to migrate to the lung, possibly because of saturation of the marginal pool by recirculating lymphocytes. Although splenectomy has little effect on the development or distribution of lymphocyte subsets in blood and lymph, it has marked effects on the rate of recirculation of lymphocytes, which may have significant implications for peripheral immune surveillance in patients who undergo splenectomy.

An enhanced role for the recirculating lymphocyte in the neonatal immune system.

Lymphocytes continually recirculate between the blood and the tissues via the lymph independent of antigen. A great deal is known regarding both the physiology and the molecular mechanisms responsible for the process in adults. However, relatively little is known regarding the development of the recirculating lymphocyte pool in very young animals or fetuses. We have directly measured the recirculation of lymphocyte subsets in antigen-inexperienced newborn animals, and found extensive recirculation of T cells through both intestinal and subcutaneous lymph nodes. Apparent selective migration of recirculating lymphocytes could be attributed to subset-specific migration of gammadelta-T cells through subcutaneous lymph nodes. This clearly demonstrates that the preference for gammadelta-T cells to recirculate through SCLN is lineage specific, and independent of the presence of antigen. Most surprising was the observation that the recirculating lymphocyte pool was proportionately larger in neonatal animals than in adults, which correlated with the histological appearance of newborn lymph nodes. This data strongly suggests that development of the recirculating lymphocyte pool is inversely correlated with antigen exposure, and decreases in size with age and the acquisition of immunological memory.

A role for lymphatic endothelium in the sequestration of recirculating gamma delta T cells in TNF-alpha-stimulated lymph nodes.

TNF-alpha is one of the most potent immunoregulatory molecules in vivo. In addition to important regulatory effects, it is also a potent inducer of extravascular lymphocyte infiltration. To examine the dynamic changes that are induced in local lymphocyte migration through regional lymph nodes following TNF-alpha injection, we used a protocol of direct lymphatic cannulation to quantitatively and qualitatively examine the traffic of lymphocytes through regional lymph nodes. We observed that local TNF-alpha injection reduced the output of lymphocytes from lymph nodes up to 90% within 6-10 h following stimulation. TNF-alpha also altered the specificity of migration of lymphocyte traffic through subcutaneous lymph nodes. In addition to the decreased output, phenotypic analysis demonstrated decreases in the concentration of gamma delta T cells by up to 30% following TNF-alpha injection. Histological examination showed that the gamma delta T cells were found in close association with VCAM-1-expressing cells in TNF-stimulated lymph nodes, at least some of which appeared to be lymphatic endothelium. These data indicate that TNF-alpha is capable of altering the number and specificity of lymphocytes recirculating through stimulated lymph nodes by selectively altering the entry of lymphocytes into the efferent lymphatics of inflamed lymph nodes in vivo.

Diversification of sheep immunoglobulins.

The sheep antibody repertoire was assessed by sequence analysis of Vlambda Vkappa and V(H) cDNA clones. In fetuses, all immunoglobulin chains were essentially in germline configuration, although limited diversity occurred at the junctional regions of Vkappa chains. Fetal B cells expressed 12 distinct Vlambda and six Vkappa gene segments. V(H) gene segments were also in germline configuration in fetuses but became extensively mutated with advancing post natal age. Two distinct types of mutations, point mutations and block mutations, were detected in V(H) transcripts and these localised differently along the gene. This raises a question as to whether a novel mutational mechanism may be involved in V(H) diversification in sheep.

Structure and expression of ovine complement receptor type 2.

The structure of sheep complement receptor type 2 (CR2) was characterised by cDNA cloning, protein sequencing and immunoprecipitation. The primary structure of sheep CR2 is similar to known mammalian homologues but the higher-order structure is unusual. Two distinct CR2 isoforms occur, one of which is ubiquitinated in the cytoplasmic domain, and the two molecular forms are expressed at the cell surface as non-covalently associated dimers. The percentage of sheep B-cells that express CR2 changes during development and varies between different body compartments. CR2+ and CR2 B-cell subsets also differ in the expression of other surface markers and in functional properties. Differential expression of CR2 may, therefore, delineate B-cells that arose by alternative developmental pathways, or it could be a marker for B-cells at different phases of antigen exposure.

Diversity of Ig light chain variable region gene expression in fetal lambs.

The usage of lambda and kappa light chain variable region genes in peripheral fetal lamb B cells was assessed at intervals from 61 to 146 days of gestation (term = 150 days). Transcripts of 12 distinct Vlambda genes were identified, eight of which belonged to the previously described families I or II, while four segments defined a new Vlambda family, provisionally called family VI. A total of six different Vkappa gene segments were identified and grouped into four families, while two Jkappa gene segments were expressed. Southern blot analysis indicated that the sheep Igkappa locus probably contains approximately 10 Vkappa genes and suggests that the V gene cluster has been duplicated. Throughout fetal ontogeny, Vkappa transcripts contained more diverse CDR3 sequences than did Vlambda transcripts, where the V-J junctions were nearly always invariant and canonical. There was also a shift in usage of Vkappa genes and in the pattern of Vkappa-Jkappa gene recombination during fetal ontogeny.

Ubiquitination and dimerization of complement receptor type 2 on sheep B cells.

Complement receptor type 2 (CR2) is a membrane-anchored glycoprotein that specifically binds C3d, as well as other ligands, and plays diverse roles in regulating immunity. Here we show that two distinct isoforms of CR2 are expressed on the surface of sheep B lymphocytes. One (CR2no 150 kDa) is structurally similar to known mammalian homologues while the other (CR2ub 190 kDa) has been modified by the covalent attachment of ubiquitin to the cytoplasmic domain and is identified for the first time. CR2no and CR2ub are expressed on the surface of sheep B cells as noncovalently associated dimers and the external topography of the two isoforms differs in some respect. The basis for these unusual higher-order structural properties may lie in the primary sequence of sheep CR2, since the transmembrane domain contains a region resembling a rare 7-amino acid dimerization motif, and two lysine residues in the cytoplasmic domain provide potential sites for posttranslational ubiquitination. The primary structures of sheep ubiquitin and C3d ligand are extensively conserved. In conjunction with the results of separate in vivo studies, these findings suggest that selective ubiquitination plays a role in modulating the higher-order structure and/or expression of CR2 during B cell development.

An immune system switch in T cell lifespan at birth results in extensive loss of naive fetal T cells during the first week of postnatal life.

Lymphocyte recirculation is critical to maximize the efficiency of immunological surveillance and is an absolute requirement for the development of systemic memory. The consensus view of the lifespan of peripheral T cells holds that naive T cells are long-lived cells and most memory T cells are short-lived cells, although the question of the lifespan of peripheral T cells is not yet fully resolved. We have studied the lifespan of T cells circulating in efferent lymph draining lymph nodes (LN) in the immunologically naive sheep fetus and in postnatal lambs immediately following birth by examining the in vivo incorporation of [3H]thymidine by newly formed T cells during continuous administration of [3H]thymidine. We report that authentically naive fetal T cells are long-lived cells which continue to recirculate between blood and lymph during fetal life. At birth, however, a process is triggered whereby fetal T cells circulating through LN are rapidly lost from the peripheral T cell pool and are replaced by freshly arriving T cells which have been formed since birth. Our results indicate that by the end of the first week of postnatal life, around three-quarters of the T cells circulating through peripheral LN have been formed since birth.

TCR gamma delta+ cells are prominent in normal bovine skin and express a diverse repertoire of antigen receptors.

More than 80% of T cells in bovine skin localized in the superficial 0.5 mm of the dermis. Only 3% occurred within the epidermis or made contact with the stratum basale while the remainder occupied deeper dermal sites. The gamma delta-T-cell receptor (TCR) was expressed by 44% of T cells in skin and 39% and 35% expressed, respectively, the CD4 and CD8 markers. Some cells co-expressed CD8 and the gamma delta TCR. A highly diverse repertoire of gamma delta TCR was expressed in skin due mainly to the usage of multiple V delta segments and to extensive sequence variation at the junctions of both TCR gamma and TCR delta chains. However, a single receptor isotype was used. Transcripts encoding several new components of the bovine gamma delta TCR were identified, including three new V gamma segments, the C gamma 5 region and 13 new functional V delta segments. Taken together with earlier findings, these results emphasize that ruminant gamma delta T cells express exceptionally diverse antigen receptors and suggest they may have a more elaborate recognitive capacity than do their counterparts in other species.

Distinct recirculating and non-recirculating B-lymphocyte pools in the peripheral blood are defined by coordinated expression of CD21 and L-selectin.

The continual recirculation of lymphocytes between the blood, tissues, and lymph is essential for the coordination and dissemination of immune responses. We have compared the functional and phenotypic properties of lymphocytes isolated from blood and lymph, the two major migratory populations. Lymph-borne lymphocytes migrated readily into the lymphatic recirculation pathway, but greater than one third of all peripheral blood lymphocytes (PBLs) were excluded from the lymphatic circuit and showed an enhanced migration to the spleen. Phenotypic analysis showed that most non-recirculating PBLs were B cells. The migration competence of B cells correlated with the surface expression of CD21 and L-selectin; recirculating B cells expressed both of these molecules, whereas non-recirculating B cells lacked both antigens. These results establish that blood contains distinct pools of lymphocytes that differ in their recirculation competence. Clearly, blood sampling is not an efficient method to directly measure the status of the recirculating immune system, and implies important constraints and restrictions in the interpretation of experimental or clinical data that include phenotypic and quantitative analyses of blood lymphocytes.

Late-term fetal thymectomy does not prevent the development of gut-homing T cells after birth.

Tissue-specific circulation of T cells is a critical element in the integration of systemic immune responses. Current models of T-cell migration suggest that homing specificities of T cells for tissues such as gut and skin are generated outside the thymus as a result of activation of virgin T cells by antigen in lymph nodes. We have used the sheep fetus (which is immunologically virgin and contains no memory or effector T-cell subsets) to examine the migration of 51Cr-labelled T cells in vivo. We report that gut-homing T cells are not present in the fetus and that gut-homing T cells from postnatal lambs home normally to fetal gut. Fetal thymectomy performed immediately prior to birth failed to prevent the development of gut-homing T cells in postnatal life. Gut-homing specificities on T cells are thus acquired extrathymically.

Phenotype and function of stromal cells cloned from the ileal Peyer's patch of sheep.

The ileal Peyer's patch (PP) is the major site of B cell production and immunoglobulin diversification in lambs, but the factors which regulate these processes are poorly understood. As a first step toward identifying possible regulatory mechanisms, stable long-term cultures of ileal PP stromal cells were established at the clonal level. Four distinct cell types were identified by their phenotype and growth requirements. Immunohistochemical staining confirmed that all clones were mesenchymal (vimentin+; cytokeratin-) in origin and were negative for T cell, B cell, and macrophage markers. Three cell lines were negative for major histocompatibility complex (MHC) I and II molecules, but one cell line, SCN, expressed MHC I, MHC II and CD44 molecules, and a subpopulation of SCN cells expressed BAQ44A, a B cell differentiation molecule. The four cell lines produced different types and amounts of extracellular matrix proteins, and their growth was not influenced by exogenous human interleukin 1 (IL-1), IL-2, transforming growth factor beta 1 (TGF beta 1), or bovine fibroblast growth factor (FGF) but was influenced by serum. When tested for their capacity to support lymphocyte growth, all clones produced a soluble factor(s) that was mitogenic for ileal and jejunal PP cells and thymocytes. Similar growth promoting activity was observed with culture supernatants of murine, human and bovine fibroblasts but could not be reproduced using recombinant human cytokines. Furthermore, coculture of stromal cells with ileal PP follicular B cells elicited a proliferative response unique to each stromal cell line. Coculture with increasing numbers of SCN cells inhibited B cell proliferative responses, whereas coculture with SCG2 and SCF32 cells enhanced B cell proliferative response at both low and high stromal cell densities. Ileal PP follicular B cells rapidly bound to the surface of all stromal cell clones, and this interaction was specific when compared with thymocytes or peripheral blood lymphocytes. These results suggest that ileal PP stromal cells are a phenotypically and functionally heterogeneous population that may enhance or inhibit B lymphopoiesis in the ileal PP.

Divergent evolution of T cell repertoires: extensive diversity and developmentally regulated expression of the sheep gamma delta T cell receptor.

Sheep gamma delta T cells express an unprecedented repertoire of antigen receptors contributed by increased diversity in both variable and constant region gene segments. Variable region diversity results mainly from the utilization of a large family of duplicated V delta genes that have retained two distinct hypervariable segments comparable with the complementarity determining regions present in other antigen receptor V genes. This implies that sheep V delta chains have been intensely selected during evolution, probably at sites involved in ligand recognition. The sheep gamma delta heterodimer occurs in at least five isotypic variants formed by the association of a single C delta segment with one of five functional C gamma segments, each with distinctive hinge regions. Our analysis also shows that the establishment of a normal peripheral repertoire is both developmentally regulated and dependent on the continual presence of a functional thymus during ontogeny. The existence of an expanded V gene repertoire and multiple receptor isotypes together with the prominence of gamma delta T cells in the sheep immune system argues that this lineage of T cells has a more elaborate functional role in this evolutionary pathway.

Altered patterns of T cell migration through lymph nodes and skin following antigen challenge.

Antigen challenge has profound effects on a regional lymph node (LN); it leads to an increase in blood flow to the node, and a marked increase in lymphocyte output through the efferent lymphatics. We used the isolated LN model developed in the sheep to see if antigen challenge in a LN resembled inflammation in peripheral tissues. Following stimulation with an antigen (purified protein derivative of tuberculin), lymphocyte output from the LN showed the typical periods of "lymphocyte shutdown" and "recruitment". The shutdown phase, when cell numbers in efferent lymph dropped by approximately 80%, affected almost exclusively the naive-type (adhesionlo, L-selectin+) T cell population. The large increase in T cell traffic through the node during the recruitment phase was mostly due to CD4+ memory-type T cells and, moreover, the majority of these T cells were L-selectin-, indicating that these cells were crossing from the blood by a molecular mechanism other than L-selectin interaction with its ligand, the "lymph node vascular addressin" (MECA-79). Examination of LN high endothelial venules revealed the presence of vascular cell adhesion molecule-1 (VCAM-1), an endothelial adhesion molecule which has been reported to bind preferentially memory-type T cells in inflammatory lesions. Within the skin, antigen challenge also induced the rapid expression of VCAM-1 on vascular endothelium. It was purely memory-type T cells (beta 1+, L-selectin+/-) that collected in lymph draining from this tissue. However within chronically inflamed skin, the MECA-79 determinant appeared on vascular endothelium, and a small proportion of T cells draining from chronically inflamed skin were of naive-type. The present results illustrate that there are similarities in the cellular and molecular events that characterize antigen stimulation of a LN and inflammation in a peripheral tissue.

Tissue-specific migration pathways by phenotypically distinct subpopulations of memory T cells.

A proportion of T cells recirculate in a tissue-selective manner. Recent studies which showed that the skin-tropic subset of T cells was of memory/activated type, led us to examine whether the preferential homing of T cells to the gut also involved memory T cells, and if so whether these memory T cells were phenotypically distinct from other memory T cells. Lymphocytes migrating through the gut and the skin of sheep was collected by cannulating the lymphatic ducts draining these tissues. Both naive and memory T cells were found to recirculate through the gut, although only memory T cells migrated through the skin. However, when T cells from the gut were labeled with fluorescein isothiocyanate and assessed for their migration back to the gut, it was the memory population which showed a tropism for the gut. Gut-tropic memory T cells migrated poorly through the skin, indicating that these cells were distinct from skin-tropic memory T cells. This was confirmed by phenotypic analysis. Gut memory T cells expressed very low levels of the alpha 6 and beta 1 integrins, in contrast to skin memory T cells which expressed high levels. There was no evidence for heterogeneity within the naive T cell population, which migrated preferentially to lymph nodes. This migration pattern could be explained in part by the high expression of the L-selectin (lymph node homing receptor, LAM-1) on naive T cells, in contrast to memory T cells from gut or skin which were mostly L-selectin negative. These results in sheep indicate that subsets of alpha/beta memory T cells show tissue-selective migration patterns, which probably develop in a particular environment following encounter with antigen.