Z. Trnka

The kinetics of antigen-reactive cells during lymphocyte recruitment

Abstract Lymphocyte recruitment, the increased traffic of lymphocytes from blood to lymph which occurs within antigenically stimulated lymph nodes, was monitored in the efferent lymph of single lymph nodes in sheep after immunization with allogeneic lymphocytes or purified protein derivative. Specific antigen-reactive cells were assayed by their ability to proliferate in vitro in the presence of the priming antigen. During lymphocyte recruitment such cells were no longer detected in the efferent lymph draining either the immunized node or a nonstimulated node remote from the region of antigen administration. These results probably reflect the selective removal of specific lymphocytes from the recirculating pool. Alternatively, the findings could involve a state of specific unresponsiveness of the cells.

24 – Sheep as an Experimental Model for Immunology: Immunological Techniques in Vitro and in Vivo

Publisher Summary This chapter presents sheep as an experimental model for immunology in discussing the immunological techniques both in vitro and in vivo. Surgical procedures that insert vinyl and plastic cannula into the afferent and efferent lymphatic ducts of lymph nodes and the intestinal and thoracic lymphatic ducts have enabled the study of cell kinetics in the immune response to percutaneous or intralymphatic infusions of immunogens and in the responses to grafted tissues and organs. By the use of such techniques, the kinetics of entry of blood-borne lymphocytes into lymphoid and other tissues may be monitored, and the cellular and humoral products of immune reactions taking place within lymphoid tissues may be quantitatively and qualitatively assessed in the efferent lymph issuing from the lymph node. In this way, an immune response induced by the infusion of antigen via an afferent lymphatic vessel can be confined to a single lymph node provided the efferent lymphatic vessel has been cannulated and exteriorized.

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.

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 Peyers 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.

Behaviour of Sheep-Immunoglobulin-Bearing and Non-Immunoglobulin-Bearing Lymphocytes Isolated by Nylon Wool Columns

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.

PEYER'S PATCHES AS A BURSAL EQUIVALENT: A NEW LOOK AT SOME OLD ARGUMENTS

ABSTRACT Peyers patches in sheep and the bursa of Fabricius in chickens have important features in common: 1. Histological appearance. 2. Prenatal maturation. 3. Involution before adulthood. 4. Intense lymphopoiesis occurs prenatally and continues after isolation from the digestive tract. (This shows that cell proliferation occurs without antigen.) 5. Antigen crosses the intestinal epithelium and enters the follicles. (It is not clear if antigen also causes lymphopoiesis). 6. Lymphocytes are produced at an enormous rate, some die within the follicles, but others leave and make a substantial contribution to the B lymphocyte pool. (Evidence of this in sheep was seen in the marked deficiency of B lymphocytes after surgical removal of a large proportion of PP at or before birth.

Differentiation of B lymphocytes in sheep. II. Surface phenotype of B cells leaving the 'bursa-equivalent' lymphoid tissue of sheep, ileal Peyer's patches.

It has been shown that there are two different types of Peyer’s patches, jejunal Peyer’s patches (JPP) and ileocecal or ileal Peyer’s patches (IPP) in sheep1. IPP have been proposed to be ‘bursa-equivalent’ lymphoid tissue of sheep and bear some characteristics of the primary lymphoid organ2. Like the bursa of Fabricius of chickens, IPP consist of numerous follicles of B cells whereas T cells account for only less than 1% of total cells. The majority of IPP cells express small amounts of surface IgM (low sIgM+ or slg10)3,4,5.

Collection of Lymph from Single Lymph Nodes and the Intestines of Fetal Lambs in utero

Techniques for establishing chronic lymphatic fistulae in the intestinal and prescapular lymphatic ducts of fetal lambs during the test third of gestation are described. Despite the absence of antibody, immunoglobulin and extrinsic antigen, the number of recirculating lymphocytes increases considerably through gestation. Both thymus-derived and surface Ig-bearing B lymphocytes are present in the fetal recirculating pool and they appear to increase in number at the same rate.

Changes in mixed lymphocyte culture-reactive lymphocytes following alloimmunization of single lymph nodes in sheep.

SUMMARY 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.

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.