In this report, we have addressed two questions concerning immunological memory: the way in which naive and memory T cells recirculate through the body, and the intrinsic rate of division within the naive and memory populations. We identified naive and memory T cells in sheep by their cell surface phenotype and their ability to respond to recall antigen. Memory T cells were CD2hi, CD58hi, CD44hi, CD11ahi, and CD45R-, as pertains in man. T cells that crossed from blood to the tissues of the hind leg and accumulated in the popliteal afferent lymph were all of memory phenotype. Conversely, T cells in efferent lymph, 90% of which entered the lymph node (LN) via high endothelial venules (HEV), were mostly of the naive phenotype (CD2lo, CD58lo, CD44lo, CD11alo, and CD45R+). The marked enrichment of these two phenotypes in different recirculatory compartments indicated that memory T cells selectively traffic from blood to peripheral tissues to LN (via afferent lymph), whereas naive T cells selectively traffic from blood to LN (via HEV). We argue that the differential use of these two recirculation pathways probably optimizes lymphocyte interactions with antigen. The nonrandom distribution of T cell subsets in various recirculatory compartments may be related to the relative proportion of memory cells in each subset. In particular, gamma/delta T cells in blood were almost exclusively of memory phenotype, and accumulated preferentially in afferent, but not in efferent, lymph. Finally, using the bromo-deoxyuridine labeling technique, we found that at least a sizeable proportion of memory T cells, whether in blood or afferent lymph, were a dividing population of cells, whereas naive T cells were a nondividing population. This result supports an alternative model of lymphocyte memory that assumes that maintenance of memory requires persistent antigenic stimulation.

A proportion of T cells recirculate in a tissue-selective manner. Recent studies which showed that the skin-tropic subset of T cells was of memory/activated type, led us to examine whether the preferential homing of T cells to the gut also involved memory T cells, and if so whether these memory T cells were phenotypically distinct from other memory T cells. Lymphocytes migrating through the gut and the skin of sheep was collected by cannulating the lymphatic ducts draining these tissues. Both naive and memory T cells were found to recirculate through the gut, although only memory T cells migrated through the skin. However, when T cells from the gut were labeled with fluorescein isothiocyanate and assessed for their migration back to the gut, it was the memory population which showed a tropism for the gut. Gut-tropic memory T cells migrated poorly through the skin, indicating that these cells were distinct from skin-tropic memory T cells. This was confirmed by phenotypic analysis. Gut memory T cells expressed very low levels of the alpha 6 and beta 1 integrins, in contrast to skin memory T cells which expressed high levels. There was no evidence for heterogeneity within the naive T cell population, which migrated preferentially to lymph nodes. This migration pattern could be explained in part by the high expression of the L-selectin (lymph node homing receptor, LAM-1) on naive T cells, in contrast to memory T cells from gut or skin which were mostly L-selectin negative. These results in sheep indicate that subsets of alpha/beta memory T cells show tissue-selective migration patterns, which probably develop in a particular environment following encounter with antigen.

Antigen challenge has profound effects on a regional lymph node (LN); it leads to an increase in blood flow to the node, and a marked increase in lymphocyte output through the efferent lymphatics. We used the isolated LN model developed in the sheep to see if antigen challenge in a LN resembled inflammation in peripheral tissues. Following stimulation with an antigen (purified protein derivative of tuberculin), lymphocyte output from the LN showed the typical periods of "lymphocyte shutdown" and "recruitment". The shutdown phase, when cell numbers in efferent lymph dropped by approximately 80%, affected almost exclusively the naive-type (adhesionlo, L-selectin+) T cell population. The large increase in T cell traffic through the node during the recruitment phase was mostly due to CD4+ memory-type T cells and, moreover, the majority of these T cells were L-selectin-, indicating that these cells were crossing from the blood by a molecular mechanism other than L-selectin interaction with its ligand, the "lymph node vascular addressin" (MECA-79). Examination of LN high endothelial venules revealed the presence of vascular cell adhesion molecule-1 (VCAM-1), an endothelial adhesion molecule which has been reported to bind preferentially memory-type T cells in inflammatory lesions. Within the skin, antigen challenge also induced the rapid expression of VCAM-1 on vascular endothelium. It was purely memory-type T cells (beta 1+, L-selectin+/-) that collected in lymph draining from this tissue. However within chronically inflamed skin, the MECA-79 determinant appeared on vascular endothelium, and a small proportion of T cells draining from chronically inflamed skin were of naive-type. The present results illustrate that there are similarities in the cellular and molecular events that characterize antigen stimulation of a LN and inflammation in a peripheral tissue.

SUMMARYMore than 80% of T cells in bovine skin localized in the superficial 0·5 mm of the dermis. Only 3% occurred within the epidermis or made contact with the stratum basale while the remainder occupied deeper dermal sites. The cd T-cell receptor ( TCR) was expressed by 44% of T cells in skin and 39% and 35% expressed, respectively, the CD4 and CD8 markers. Some cells co-expressed CD8 and the cd TCR. A highly diverse repertoire of cd TCR was expressed in skin due mainly to the usage of multiple Vd segments and to extensive sequence variation at the junctions of both TCRc and TCRd chains. However, a single receptor isotype was used. Transcripts encoding several new components of the bovine cd TCR were identified, including three new Vc segments, the Cc5 region and 13 new functional Vd segments. Taken together with earlier findings, these results emphasize that ruminant cd T cells express exceptionally diverse antigen receptors and suggest they may have a more elaborate recognitive capacity than do their counterparts in other species. INTRODUCTIONreflect an ordered programme of gene rearrangement in the thymus, although this possibility has not been examined Several properties of ruminant cd T cells distinguish them directly. A further question that then arises is whether or not from their counterparts in other species. In calves and lambs, specific subsets of ruminant cd T cells with a restricted TCR cd T cells may comprise up to 30-50% of circulating T cells in repertoire either localize or recirculate preferentially to particuthe first few weeks after birth.1,2 Circulating sheep cd T cells lar types of body surfaces. As a first approach to clarifying are derived from a thymus-dependent pathway of diÂeren-this question, we have assessed the frequency and TCR tiation and are almost totally and permanently depleted by diversity of cd T cells in bovine skin, a tissue where these cells removing the thymus about half-way through fetal gestation.3,4 localize prominently.1,11 Our results show that in normal A majority of ruminant cd T cells specifically express a unique bovine skin, cd T cells account for at least 44% of resident accessory molecule, a member of the scavenger receptor family T cells. The cd T cells express an extremely diverse TCR termed WC1, that is encoded by a large and complex family variable-region repertoire but have a restricted usage of one of genes.1,5,6 Finally, ruminant cd T cells express a diverse receptor isotype. repertoire of T-cell receptors ( TCR) encoded by at least five diÂerent Cc segments, around 15-20 diÂerent Vc genes and perhaps as many as 40-50 diÂerent Vd gene segments.7,8 MATERIALS AND METHODS In mice, cd T cells localizing in diÂerent types of mucosal Collection of bovine skin surfaces express a distinctive and usually restricted repertoire Pieces of skin approximately 15×10 cm were collected from of antigen receptors. The populations of cd T cells that colonize the hides of yearling Brown Swiss cattle after slaughter at an diÂerent epithelia arise at distinct stages of fet...

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