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We have studied the appearance and phenotype of recent thymic emigrants in blood, spleen and lymph nodes (LN) of neonatal lambs. Using in situ labelling of thymocytes with fluoroscein isothiocyanate (FITC), we examined the expression of the LN homing receptor L-selectin on alphabeta and gammadelta subsets of recent thymic emigrants 24 hr after labelling. There were marked differences in the proportions of CD4+, CD8+ and gammadelta T-cell receptor (TCR+) cells exported from the thymus to spleen compared to lymph nodes. Spleen was enriched in CD8+ and gammadelta TCR+ emigrants while LN were enriched in CD4+ emigrants. There were also marked differences in the expression of L-selectin by emigrants homing to spleen compared with those homing to lymph nodes. While the majority of thymic emigrants in LN expressed L-selectin, considerably fewer emigrants in spleen were L-selectin+. The presence of large numbers of CD8+ L-selectin- and gammadelta TCR+ L-selectin- thymic emigrants homing to spleen raises the possibility that unique homing receptor specificities underpin the migration of T cells to spleen as distinct from lymph nodes.

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.

Tissue-specific and lymphocyte subset-specific lymphocyte recirculation patterns have been analysed simultaneously. Lymphocytes obtained from one lymph compartment were directly labelled with fluorochrome in vitro and returned to the blood of the same animal. Over the next 48-72 h, the recirculation of these cells into both the same lymph compartment and at least one different lymph compartment was monitored. The cells in all of these lymph collections, as well as an aliquot of the cells used for direct fluorescent labelling, were then phenotyped with monoclonal antibodies (mAb) which define the mutually exclusive small CD4+ and CD8+ T-lymphocyte subsets in sheep. All cell samples were analysed by flow cytometry and CD4/CD8 ratios were determined for the recirculated, fluorochrome-labelled population in each lymph collection. The mean CD4/CD8 ratio calculated for each lymph compartment was then compared with the CD4/CD8 ratio calculated for each lymph compartment was then compared with the CD4/CD8 ratio of the transfused, starting population. In one experiment employing efferent prescapular lymph cells, three experiments employing efferent intestinal lymph cells, and two experiments employing afferent intestinal lymph cells, tissue-specific recirculation was observed. In all of these experiments, the pattern of recirculation of small CD4+ and CD8+ T lymphocytes was non-random. Moreover, in each experiment, this non-randomness was completely unrelated to tissue-specific phenomena, since the mean CD4/CD8 ratio of the recirculated population was higher than the CD4/CD8 ratio of the transfused, starting population regardless of the lymph compartment examined. These data are therefore consistent with the hypothesis that tissue-specific and lymphocyte subset-specific lymphocyte-endothelial cell recognition mechanisms independently direct the recirculation of small lymphocytes from blood to lymph.

The experiments described in this paper have examined the migration of three fluorochrome-labelled T-lymphocyte subsets (CD4+, CD8+ and gamma delta+T19+) on a single passage from blood to lymph, through prescapular lymph nodes. Lymphocytes obtained from prescapular efferent lymph were labelled in vitro with fluorochrome and returned to the blood of the same animal. Over the next 2 days, lymph was continuously monitored and the cells in all collections, including the one used for intravenous infusion, were phenotyped and analysed by flow cytometry. Significant differences in the subset ratios between the infused, starting population and the recirculated population indicated that CD4+ and gamma delta+T19+ lymphocytes are extracted by a resting lymph node at the same rate and that both are extracted at a faster rate than CD8+ lymphocytes. The results presented here also suggest that a unique subset of gamma delta+T19+ lymphocytes may be present in blood that does not recirculate through peripheral lymph nodes.

The surface phenotypes (CD5+, CD4+, CD8+, SBU-T19+, MHC class II+, T80+ and sIg+) of lymphocytes in blood, and in prescapular and ileocaecal efferent lymph of sheep, have been determined. The similarity in the distribution of lymphocyte subsets in both lymph compartments indicated that the non-random migration of lymphocyte populations to peripheral lymph nodes and gut could not be due to preferential migration of a particular lymphocyte subset such as CD4+ or CD8+ cells to one tissue. Marked differences in the percentage of lymphocyte subsets between blood and efferent lymph suggested that some lymphocyte subsets leave the blood with differing efficiencies and that differential extraction of lymphocyte subsets from blood by a lymph node may be due to subset-specific lymphocyte-endothelial interactions.

The recruitment of radiolabelled blood-borne lymphocytes into rat popliteal lymph nodes (PLN) was investigated following the injection of varying doses of chicken (CRBC) or sheep red blood cells (SRBC) into the hind foot pad. Within the dose range tested (10(5)-10(8) RBC) an increase in the dose of injected antigen resulted in elevated levels of lymphocyte recruitment into the draining popliteal lymph node. The kinetics of lymphocyte recruitment with the same dose of CRBC or SRBC was similar even though these red cells differ in size and are antigenically non-cross-reactive. While little is known of the mechanisms which control the rate of entry of blood-borne lymphocytes into antigen-stimulated lymph nodes, the extent of lymphocyte recruitment was shown to be directly related to the quantity of antigen injected.

The experiments reported in this paper examine the cell-surface phenotype (CD5, CD4, CD8, T19, MHC class II and sIg) and cell output of lymphocyte subsets circulating through a subcutaneous lymph node in the sheep fetus, in an environment unaffected by foreign antigen and circulating immunoglobulins. CD4+ lymphocytes were the major T-cell subset in fetal lymph and were clearly enriched in lymph compared with blood, whereas T19+, CD8+ and B lymphocytes were not. It seems likely that in the fetus CD4+ lymphocytes are extracted from the blood at a faster rate than are other T-cell subsets and B cells. There was a much higher percentage of CD8+ and T null cells and a lower percentage of MHC class II+ and B cells circulating in the fetal lymph than in adult lymph, while the percentage of T19+ lymphocytes in fetal blood was twice that in the adult. Although the hourly cell output from an adult prescapular lymph node was far higher than that from a fetal lymph node, the circulation of lymphocytes through fetal lymph nodes was much greater per gram lymph node weight than that through adult lymph nodes. The wholesale recirculation in the fetus of all the major T-cell subsets found in the adult is paradoxical because it is not known what function they serve in the fetus in the absence of antigen and ongoing immune responses, although clearly they are not memory cells.