An in vitro homologous antibody-dependent cell-mediated cytolytic (ADCC) assay system was developed using human peripheral blood mononuclear cells, blood group 0 Rhesus (D) positive red blood cells and anti-11 antibodies. Extensive studies were carried out using monocyte-depleted cultures, where the lymphoid K-cell is the putative cyglytic effector cell, and the degree of specific lysis estimated from Cr release from damaged RBC. Enhanced lysis was obtained when RBC were treated with the proteolytic enzyme papain. The coefficients of variation of replicates processed in an identical manner were less than 10%. The degree of specific lysis of anti-D sensitised D-positive RBC depended upon the number of effector cells present, the number of RBC, and the amount of antibody. Under appropriate conditions, lysis was observed with only 3 ng anti-D per culture. The specificity of lysis resided with the appropriate combination of antibody and antigen (is D-anti-D) and not with the effector cells. Donors of all ABO Rh groups were equally effective donors of K-cells and under appropriate conditions autologous RBC were lysed. The degree of specific lysis was influenced by the D-antigen dosage of RBC and the potential for determining the D-zygosity of RBC was explored. Time-course experiments showed that RBC lysis was measurable by 30 minutes incubation, and was approximately linear over 18 hrs, at which time maximum lysis was seen. Cell-to-cell contact was essential for lysis and the cytolytic process was extremely short-range with no "innocent bystander" effect being seen. Phagocytosis was not necessary for cytolysis to be observed, but effector cell mobility and intact microfilament function was required (inhibition by cytochalasin B). The divalent cations Ca and Mg were required for efficient itBC lysis. Studies with metabolic inhibitors showed that a metabolically active cell is required for K-cell activity, that both RNA and protein synthesis are necessary for maximum lysis, and that an intact microtubule system is also required. The trigger to lysis appears to be the activated Fe component of red cell-bound anti-D which interacts with the K-cell Fc receptor. Lysis was mediated by IgG antibodies and was significantly inhibited immbulins IgG. This suggests that the K-cell Fe receptor by specific for IIgG but that IgG1 and IgG3 cross-react. Application of the K-cell assay to normal individuals reveals that there was inherent biological variation between individuals and that there appeared to be 'good' and 'poor' K-cell donors in terms of cytolytic activity. K-cells were present in the cord blood of neonates and were slightly less active than adult cells. Adrenaline infusion produced a transient rise in K-cell activity at 15-30 minutes and infusion of D positive RHC into a D negative individual resulted in a transient fall at 12-14 days followed by enhanced lytic potential some weeks later. Preliminary studies into the biological activities of different sources of anti-D revealed that there was poor correlation between agglutination titre and specific RBC lysis. The potential applications of the K-cell assay system are discussed. The K-cell has the morphologic appearance of a small lymphoid cell, does not appear to have surface immunoglobulin or sheep RHC (R) receptors but does have Fe and C3 receptors. The cell is non-adherent, non-phagocytic and negative for the non-specific esterase stain. It therefore does not have any of the characteristics of a mature monocyte, but does have monocyte-specific surface antigens as demonstrated by anti-monocyte serum. It may therefore be a monocyte precursor.

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The object of this thesis was to study the role of thymic humoral factors in the maturation of rat lymphoid cells. The response, in vitro, of lymphoid cells to PHA, measured by the incorporation of tritiated thymidine, was used as a measure of thymic function. Neonatal thymectomy led to a persistent lymphopenia and a drastically reduced response of rat whole blood and spleen cells to PHA. Partial restoration of the lymphocyte count and PHA response occurred in thymectomized rats following the transplantation of two syngeneic thymuses. Analysis of the PHA response and lymphocyte count in individual whole blood samples suggested that the response to PHA was a good measure of the peripheral blood T cell population. Addition of serum to the medium was not necessary in order to obtain a satisfactory response to PHA provided that erythrocytes were present. No difference was found between the ability of pooled intact rat and pooled thymectomized rat serum to depress, or increase, tritiated thymidine incorporation in cultures of rat blood or spleen cells responding to PHA. Incubation of unfractionated lymphoid cells with crude cell-free saline extracts of rat thymus was not found to increase the responsiveness of the cells to PHA. When the extract was present throughout the culture decreased isotope incorporation occurred. When the extract was removed from the cells after a preculture and before the addition of PHA the isotope incorporated in response to PHA was either slightly increased or decreased. Incubation of unfractionated spleen, bone marrow and thymus cells with cell-free medium obtained from three-day and six-day thymus organ cultures did not load to increased responsiveness to PHA. Also, 40 injections of thymus organ culture medium given to each of 12 neonatally thymectomized rats over an eight week period did not increase the whole blood response to PHA.

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Until two decades ago the fate and function of lymphocytes were largely unknown. The discovery that these cells were long-lived, showed unique mobility in migrating from the blood into the specialized lymphatic tissue of lymph nodes and spleen and repeatedly returning to the blood, and were immunologically competent, provided the basic answers to these questions. However, many aspects of the mechanisms involved in the initiation and propagation of immune reactions remained unclear. In particular it was not known to what extent lymphocytes entered other tissues and how, if at all, this behaviour was altered in sites of inflammation. It might be expected that immunologically active cells would enter non-lymphoid tissues as many immune reactions are implemented at least in part in these peripheral sites; also direct examination of afferent lymph draining nonlymphoid tissue has detected some lymphocyte traffic. The studies reported here were designed to provide quantitative estimates of lymphocyte migration into non-lymphoid tissues under normal and pathological conditions and attempts were made to define the kinetics of this flow of cells and to investigate some aspects of the control mechanisms that may regulate it. To this end the basic techniques of marking rat thoracic duct lymphocytes and tracing their distribution in syngeneic recipients, that allowed Gowans and his colleagues to establish the concept of the recirculating pool of small lymphocytes, were used. Critical evaluation of different labelling methods was essential, as great sensitivity was required in order to detect accurately the small concentrations of labelled cells expected in non-lymphoid tissues. Radioisotopic labelling of lymphocytes would allow estimation of their tissue distribution after iv transfer by assay of total tissue radioactivity provided the label remained attached to the cells and these cells were extravascular. The contamination duct to intravascular labelled cells was minimized by perfusing a large volume of saline through the whole circulatory system before sampling the tissues. An indirect measure of the extent of loss of isotope from the injected cells was made by assaying the activity in many compartments particularly those free from cells such as plasma, urine and the perfusion washout fluid. Examination of the latter also provided information about widespread contamination of tissues by freely diffusible isotopes and was important in assessing the usefulness of particular labelling compounds for the purpose of tracing lymphocytes to areas of low cell concentration. A comparison of the distribution of activity after labelling lymphocytes with many isotopes showed serious limitations in the use of some 'labels' but suggested that the results obtained using $1Cr were most likely to provide realistic estimates of labelled cell content of tissues. Ranges for lymphocyte cell content and flux (104-105 lymphocytes/gm/hr) in nonlymphoid tissues were derived from kinetic distribution studies using multiple recipients of the same labelled cell population and examining groups of recipients at intervals after cell transfer. The estimates were in the same range as those derived from data on afferent lymph cellular flow obtained by direct measurements in other species such as sheep and pigs. The time taken for labelled cells to traverse nonlymphoid tissues while not measurable precisely was probably shorter than that taken to enter and leave either spleen (5-6 hrs) or lymph nodes (18 hrs) migration of lymphocytes into inflammatory lesions in the skin was found to be increased and usually the transit time remained rapid. Exposure of lymphocytes to a low dose of trypsin in vitro drastically reduces their efficient migration into lymph nodes: it was found that such treated cells also failed to migrate in increased numbers into lesions in the skin but entry into normal tissues was unaffected. It was suggested that in both lymph nodes and cell-mediated immune lesions the enzyme treatment in some way interfered with the lymphocyte-endothelial cell interaction; this implied there was a change in the inflamed venular endothelium that allowed a more efficient clearance of lymphocytes from the blood. It was further suggested that the highly specialized function of lymph node post-capillary venular endothelium that is associated with a morphological specialization may be an adaptation of a temporary functional change that occurs in venular endothelium in cell-mediated immune lesions because this latter type of lesion is widespread even in invertebrate animals but lymph nodes are very recent developments in phylogenetic terms. Using the same techniques of following labelled cells supplemented by autoradiography of recipients' tissues the migration of thoracic duct lymphocytes to the 'primary' lymphoid organs was studied. The thymus and bone marrow are both sites of very active lymphocytopoiesis which is mainly independent of antigenic stimulation. Both have been regarded as insignificant compartments of the recirculating lymphocyte pool. However, the thymus was found to contain a greater concentration of radioactivity than all other non-lymphoid tissues except liver and kidney and contained approximately 0.1 % of the total recirculating pool at equilibrium. The kinetic distribution curve was similar to that for lymph nodes suggesting a modal transit time of at least eighteen hours. Autoradiography showed that the migrating cells were limited to the thymic medulla. The bone marrow was found to accept up to one quarter of an injected 'bolus' of labelled lymphocytes. These cells were extravascular and later returned to the blood somewhat more rapidly than the lymphocytes that entered the spleen. The existence and size of this migration pathway was confirmed using a functional indicator of lymphocytes and the capability of the bone marrow to act as a 'secondary' lymphoid organ was examined. It was shown that this tissue could concentrate particulate antigen from the blood, and support antigen-induced transformation and proliferation of lymphocytes and these findings taken with the high flux of immune competent lymphocytes suggested that this organ could function in the initiation and propagation of immune responses.

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