Search

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.

The in vitro induction of specific primary and secondary immune responses in sheep lymph node cell suspensions is described and suitable culture conditions determined. The induction of primary immune responses required supplementation of the culture medium with antigen-absorbed homologous serum or lymph, whereas the requirements for the induction of a secondary response were less stringent. The addition of 2-mercaptoethanol to the medium was required. The amounts of heterologous erythrocytes used for immunization were critical and optimal responses were obtained when 50 microleters of a 1% suspension were added to 1 ml cultures. Lymphocyte densities of about 5 X 10(6)/ml were found optimal in primary immune responses in vitro. Less than 2 X 10(6) cells/ml rarely gave rise to plaque-forming cell (PFC) generation, whereas densities of 10 x 10(6) and above reduced the number of PFC obtained per number of cultured cells. Lymphocytes obtained from the efferent lymph draining lymph nodes previously immunized with heterologous erythrocytes were found to generate PFC in vitro when specific antigen was added to the cultures, but attempts to generate PFC in vitro with cells from efferent lymph draining non-immunized nodes failed.

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.

When an "isolated" single lymph node is challenged with irradiated allogeneic lymphocytes, there is a change in the reactivity of the lymphocytes flowing out of the node when they are cultured in vitro in unidirectional mixed lymphocyte cultures (MLC) against the immunizing lymphocytes. These changes in the reactivity of the recipient lymphocytes are shortlived, follow a set time sequence in relation to the cell traffic changes accompanying the immune response, are the property of small lymphocytes and not blast cells, are exhibited by surface Ig-negative cells, and they are specific for the donor lymphocytes. It is suggested that antigen causes the selective retention of antigen-specific lymphocytes within the stimulated node followed by the proliferation and differentiation of large numbers of antigen-specific cells, which then leave the lymph node as small lymphocytes.

The technique of separating lymphocytes on nylon wool columns has been applied to sheep lymphocytes obtained from efferent lymph. The non-immunoglobulin-bearing (sIg-) lymphocytes which pass through the column were MLR-reactive, responsive only to T cell mitogens, and migrated in vivo like T lymphocytes described in rodents. Immunoglobulin-bearing (sIg+) lymphocytes were MLR-non-reactive, responsive to lipopolysaccharide and migrated in vivo like B lymphocytes in rodents. It is considered that the majority of sIg- lymphocytes obtained in this way are T lymphocytes and the majority of sIg+ lymphocytes are B lymphocytes.

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.

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.

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 oft cells. This technique led to confirmation of the earlier reports in sheep ora 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 ceils 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. [ABSTRACT FROM AUTHOR]