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.

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.

A novel glycoprotein expressed on sheep T and B lymphocytes is involved in a T cell activation pathway.

We have produced a new mouse mAb that identifies a sheep T cell activation Ag. The mAb B5-5 is specific for low m.w. components on nearly all sheep thymocytes and peripheral T and B lymphocytes but does not label immature B cells in Peyer's patches or germinal centers. After cross-linking of target structures either directly by plastic-bound mAb or indirectly using anti-Ig reagents, peripheral T cells, but not thymocytes or peripheral B cells, were activated. IL-2 was secreted by T cells after cross-linking and activation was strongly augmented in the presence of PMA. The addition of soluble B5-5 mAb to mitogen-stimulated cultures of sheep lymphocytes resulted in a suppression of PHA responses and augmentation of PWM responses and had a variable effect on Con A responses but had no effect on LPS- or protein A-induced proliferation. When added to alloantigen-stimulated cultures, B5-5 augmented the proliferative response. The B5-5 membrane component consists of 14- to 19-kDa glycoproteins but the banding patterns obtained during SDS-PAGE analysis of 125I-labeled Ag differed between thymocytes, peripheral T cells, and peripheral B cells. On the basis of its range of expression on lymphoid cells and known biochemical and functional properties, we conclude that the B5-5 component on sheep lymphocytes is different from T cell activation Ag in other species.

A murine anti-sheep T8 monoclonal antibody, ST-8, that defines the cytotoxic T lymphocyte population.

A mouse monoclonal antibody (mAb), ST-8, reactive with a subpopulation of sheep T lymphocytes was investigated by tissue distribution analysis and functional studies of the antigen-bearing (ST-8+) cells. Two other mouse mAb, ST-1 and T-80 that recognize all sheep T cells and a T-cell subset, respectively, were also examined for comparison. The ST-8+ cells represented 61-69% of thymocytes and about 10-30% of peripheral T lymphocytes. Histologically, ST-8+ cells were found mainly in the thymic cortex, T-dependent areas of the peripheral lymphoid tissues and the splenic red pulp and marginal zone, but not in B-dependent areas such as germinal centers of lymph follicles of the Peyer's patches. ST-8 mAb plus complement treatment completely abolished both the induction phase and the effector phase of alloreactive cytotoxic T lymphocytes but did not affect proliferative responses to T-dependent mitogens and allogeneic antigens. ST-8 mAb also blocked the cytotoxic T lymphocyte function of lysing specific targets in the absence of complement. ST-8 mAb immunoprecipitated an antigen from the surface of sheep T lymphocytes under reducing conditions with an apparent molecular weight of 33-35 kilodaltons. Therefore the antigen recognized by the ST-8 mAb showed striking similarities to human T8/Leu-2a and mouse Lyt-2 antigens not only in the tissue distribution and function of antigen-bearing cells but also the molecular weight. We conclude that the ST-8 antigen is the ovine homologue of the human T8 and mouse Lyt-2 antigens.

Lymphocyte migration and differentiation in a large-animal model: the sheep.

The size and docility of the sheep permit various surgical interventions and repeated collections of biological samples. Development of lymphatic cannulation techniques in this species enabled the investigation of the kinetics of lymphocyte migration in single lymph nodes of not only postnatal animals but also of fetuses at various stages of gestation. It was first demonstrated in the sheep that lymphocyte recirculation commences in the fetus without any exogenous antigenic stimulus. Using these cannulation techniques, it is also possible to investigate humoral events such as the secretion of lymphokines taking place in single lymph nodes with regard to the regulation of lymphopoiesis and the immune response. An extracorporeal perfusion system has been used successfully to investigate the emigration of cells from various lymphoid organs in the sheep. This apparatus enables cells to be labelled in their normal microenvironment with radioisotopes and/or fluorescent probes without destroying the normal tissue architecture. In studies with outbred animals such as the sheep, an investigation in which an individual animal is studied as a case history over a long time often provides much more information than studies based on single-point examinations of many animals and is much closer to the clinical study of immunological problems in individual humans. The recent development of an array of monoclonal antibodies against lymphocyte surface antigens in sheep will help to further dissect the complexity of immunological phenomena. Therefore, the sheep is a useful animal model to study physiological events taking place in the lymphoid system, and in vivo studies in this species will continue to offer a great potential for research of biological relevance and supplement the research done on the in vitro manipulation of cells and biological products related to the immune system.

Studies on the differentiation of T lymphocytes in sheep. II. Two monoclonal antibodies that recognize all ovine T lymphocytes.

Two mouse monoclonal cytotoxic antibodies (ST-1a and ST-1b) recognize an antigen present on the large majority of thymocytes and all T cells in the periphery, but not B cells or other haemopoietic cells in sheep. Examination of frozen sections of various fetal tissues revealed that the cells expressing this antigen first appeared in the thymus, and these cells markedly increase in numbers in the peripheral lymphoid tissues after mid-gestation. Large accumulations of positive cells were located in the paracortex of lymph nodes, the periarteriolar lymphoid sheath of the spleen, and interfollicular areas of jejunal Peyer's patches, all of which are known to be T-dependent areas. Treatment of lymphocytes with ST-1a and complement resulted in the abrogation of T-proliferative responses, but the response to a B-cell mitogen, lipopolysaccharide, was not reduced. Neither ST-1a nor ST-1b cross-reacted to lymphocytes obtained from other species of animals (man, monkey, mouse, rat, guinea-pig, chicken, frog, pig, horse, goat and cattle). Based on these findings, it was concluded that the expression of the antigen recognized by ST-1a and ST-1b is restricted to the T-cell lineage of sheep, and that all ovine T cells express this antigen. Furthermore, ST-1a and ST-1b were determined to recognize the same antigen by reciprocal blocking experiments.

Differentiation of B lymphocytes in sheep. I. Phenotypic analysis of ileal Peyer's patch cells and the demonstration of a precursor population for sIg+ cells in the ileal Peyer's patches.

The ileal Peyer's patches (IPP) of sheep may be a primary lymphoid organ for b cells since they have a number of important features in common with the bursa of Fabricius of chickens. We have examined the surface phenotype of IPP cells. Approximately 90% to 95% of IPP cells are 'low sIgM+'; that is, they have surface IgM, but in much smaller amounts than peripheral B cells, which are 'high sIgM+'. IPP cells with sIgG or sIgA are very rare. Upon exposure to a tumour promotor, phorbol myristate acetate (PMA), in vitro, low sIgM+ cells differentiated into high sIgM+ cells. The amount of Ia-like antigens on the surface also increased after PMA treatment. Approximately 5% of IPP cells bore no identifiable markers. However, these cells could also be induced into high sIgM+ cells upon exposure to PMA; this may indicate the presence of precursors of sIgM+ cells within the IPP. While PNA (peanut agglutinin) binds strongly to the vast majority of IPP cells, it binds very little, if at all, to B cells obtained from the periphery, unless they have been treated with neuraminidase; this suggests that cells in the B lineage retain their PNA receptors, but that these become masked by sialic acid on mature B cells.

Actin activity is high in the immunocompetent fraction of thymocytes.

We have shown previously that actin activity of lymphocytes correlates with their migratory abilities. To determine whether any subpopulation of thymocytes would possess the potential for peripheral migration, sheep thymocytes were separated into four fractions by Percoll discontinuous gradient centrifugation and actin activity of each fraction was determined. The fraction enriched for immunocompetent medullary-type cells showed the highest actin activity among thymocytes, but the activity was significantly lower than that of peripheral lymphocytes. The implications of the observation in relation to thymocyte migration is discussed.

Studies on the differentiation of T lymphocytes in sheep. I. Recognition of a sheep T-lymphocyte differentiation antigen by a monoclonal antibody T-80.

The results presented in this paper demonstrate that a mouse IgM monoclonal antibody (T-80) recognizes an antigen on cells of the T-lymphocyte lineage of sheep. However, this antibody does not identify all T cells, as 10-20% of thymocytes and some peripheral-blood T cells are negative. T-80- thymocytes reside in the medulla. The majority of cortical thymocytes are T-80+ and classified as dull cells on the basis of antigen density per cell as measured by flow microfluorometry. In contrast, T-80+ cells in the periphery can be categorized into two populations, i.e., dull cells and bright cells. Suggestive evidence was obtained that bright T-80+ cells are fast recirculating T cells, whereas dull cells are sessile or less easily mobilizable T cells in the periphery. In foetal environment, over 90% of thymocytes and approximately 5% of spleen cells are T-80+ at 54 days of gestation (gestation period = 150 days), which may indicate that T-cell emigration from the thymus commences well before mid-gestation in sheep.

T-cell recirculation in the sheep: migratory properties of cells from lymph nodes.

T lymphocytes derived from different sources in sheep were compared for their ability to recirculate from blood to lymph. Nylon wool columns were used to prepare T-cell-enriched populations from efferent intestinal lymph, efferent prescapular lymph and from cell suspensions of mesenteric lymph nodes and prescapular lymph nodes. With each animal, T cells from two of the above sources were labelled in vitro, one population with fluorescein isothiocyanate the other with rhodamine isothiocyanate; both populations were returned to the animal at the same time by intravenous injection. The intestinal lymph and prescapular lymph were continuously monitored to compare the recirculating properties of the two populations of T cells. This technique led to confirmation of the earlier reports in sheep of a preferential recovery of intestinal lymph T cells and of prescapular lymph T cells in the lymph from which the cells were originally collected. This phenomenon was much less evident with T cells from mesenteric nodes and prescapular nodes and in a number of experiments a random pattern of recirculation occurred. It is concluded that there are differences in the composition of the T-cell population in a node compared with that of the lymph draining the node. The advantages of using fluorescently-labelled cells to study lymphocyte migration are discussed.

Lymphocyte recirculation in the sheep fetus.

The numbers of circulating thymus-derived and surface Ig-bearing lymphocytes in the fetal lamb increase exponentially over the last third of gestation. Experiments in which [3H]thymidine was continuously infused into fetal lambs have established that these cells are long-lived in the fetus. The migration of 51Cr-labelled autologous lymphocytes from intestinal or prescapular lymph was compared in fetal lambs and adult sheep. A subpopulation of thymus-derived lymphocytes present in intestinal lymph of adults which migrated preferentially to the small intestine was not found in fetal intestinal lymph. There were marked differences in the migration of fetal and adult lymphocytes to the lungs and liver. In spite of the absence of circulating antibodies or immunoglobulins and of extrinsic antigen in the immunologically virgin sheep fetus, the circulation of lymphocytes through the spleen and lymph nodes of fetal lambs was more intense than in the adult, indicating that the pathways of recirculation and the capacity of cells to recirculate arise as a physiological process independently of antigenic stimulation.

Changing patterns of lymphocyte circulation during development.

Despite the absence of antibody, immunoglobulin, and extrinsic antigen, the output of T- and B-lymphocytes from single lymph nodes and the intestines in the sheeep fetus increases exponentially over the last third of gestation. The fetus possesses a huge pool of recirculating lymphocytes which have the same blood to lymph transit time through lymph nodes as adult sheep. A subpopulation of intestinal lymphocytes which migrates preferentially through the small intestines of adult sheep was not found in the fetus. The sheep fetus is immunologically virgin, so fetal recirculating lymphocytes cannot be memory cells nor can they be dependent on antigen for their development.

The migration of lymphocytes in the fetal lamb.

The migration of 51Cr-labeled autologous lymphocytes from intestinal or prescapular lymph was compared in fetal lambs and adult sheep. A subpopulation of lymphocytes present in intestinal lymph of adults which migrated to the small intestine was not found in fetal intestinal lymph. There were marked differences in the migration of fetal and adult lymphocytes to the lungs and liver. In spite of the absence of circulating antibodies or immunoglobulins and of extrinsic antigen in the immunologically virgin sheep fetus, the circulation of lymphocytes through the spleen and lymph nodes of fetal lambs was more intense than in the adult.