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Background and Aim: The development and use of experimental models using lymphatic cannulation techniques have been hampered by the lack of high-quality colour imaging of lymphatic vessels in situ. Most descriptions of lymphatic anatomy in sheep have historically depended on schematic diagrams due to limitations in the ability to publish colour images of the lymphatic vessels with decent resolution. The aim of this work was to encourage more widespread use of the ovine cannulation model by providing clear photographic images identifying the location and anatomical layout of some major lymphatic ducts and their in situ relationship to surrounding tissues., Methods: The cadavers of the sheep were collected after they had been euthanized at the end of animal trials not associated with this study. The lymphatics were dissected and exposed to show their appearance in the surrounding tissues and their relationship to other organs. Patent Blue was used to locate lymphatic vessels in exploratory preparations. However, in order to present the natural appearance of the vessels, we used minimal dissection and dye was not used for the photographed examples. Instead, we have indicated the course of the vessels with lines where their position is less clear., Results and Conclusion: In this paper, we have used sheep specimens as examples to show characteristic images of lymphatic vessels. The images of in situ lymphatics and lymph nodes combined with schematic summaries provide a concise illustration of the lymphatic drainage scheme in sheep., Competing Interests: The authors have declared that no competing interests exist.

Lymphatic cannulation models are useful tools for studying the immunobiology of the lymphatic system and the immunopathology of specific tissues in diseases. Sheep cannulations have been used extensively, as models for human physiology, fetal and neonatal development, human diseases, and for studies of ruminant pathobiology. The development of new and improved cannulation techniques in recent years has meant that difficult to access sites, such as mucosal associated tissues, are now more readily available to researchers. This review highlights the new approaches to cannulation and how these, in combination with advanced omics technologies, will direct future research using the sheep model., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A diverse repertoire among peripheral T cells is established in early life by thymic export when the naive T cell pool is first formed. In contrast, during adult life the thymus has been thought to play only a minor role in T cell homeostasis. As individuals age there is an increasing proportion of peripheral T cells bearing a memory phenotype, as well as a corresponding decrease in the number of naive T cells. The change in the composition of the peripheral T cell pool with age is thought to occur as a result of reduced or completely curtailed thymic export following thymic involution at puberty together with the antigen-driven expansion of naive T cells in the periphery. We examined thymic export throughout life in fetal, neonatal and aged sheep. We found that the thymus in adult animals showed an efficiency of production and export on a per gram basis equivalent to that observed for much younger animals, and continued to export substantial numbers of T cells long after puberty. The data demonstrate that naive T cells constantly enter the peripheral T cell pool at the same rate throughout fetal, neonatal and adult life, and that one in every 50 T cells in the peripheral lymphoid tissues of aged sheep had emigrated from the thymus in the previous 24 h. The data suggest that restoration by the thymus of a normal peripheral T cell repertoire in chronic T cell-depleting conditions should be possible in adult patients, provided the thymus is not damaged by disease or therapy.

Before parturition the fetal lamb develops a large pool of long-lived recirculating T cells which provides a large population of naive T cells with a diverse TcR repertoire. After birth and concomitant with exposure to environment antigens, fetal T cells are rapidly replaced by short-lived cells formed postnatally. The majority of thymic emigrants homing to spleen in postnatal lambs are short-lived, in contrast to emigrants targeting lymph nodes where a population appears to be long-lived. The lifespan of thymic emigrants in the fetus is unknown as in the relative importance of antigen-driven processes versus developmental programming in regulating T cell homeostasis in early postnatal life.

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.

Following T cell activation with antigen or mitogens, there is an up-regulation of interleukin-2 receptor alpha (IL-2R alpha) chain expression. A high proportion of the IL-2R alpha chain is shed from the surface of the T cell in a soluble form following proteolytic cleavage, and thus determination of soluble IL-2R alpha (sIL-2R alpha) chain is an excellent measure of lymphocyte activation. A sandwich immunoassay for the detection of ovine sIL-2R alpha chain has been developed. Three monoclonal antibodies (mAbs) with specificity for the IL-2R alpha chain, demonstrated by immunoprecipitation of a 50 kDa protein from an ovine IL-2R alpha chain cDNA transfected Chinese hamster ovarian (CHO IL-2R) cell line, were analysed for additive and competitive binding to CHO IL-2R cells and Concanavalin A (Con A) activated ovine lymphocytes, respectively. Two non-competitive ovine IL-2R alpha chain specific mAbs were then used in a sandwich immunoassay to detect native sIL-2R alpha chain in the supernatant (SN) of Con A activated ovine lymphocytes and recombinant sIL-2R alpha chain in the SN of CHO IL-2R cells. Soluble IL-2R alpha chain could also be detected in complex biological fluid. In the efferent lymph of a cannulated ovine popliteal lymph node (LN), an increase in the level of sIL-2R alpha chain following local alloantigen LN activation was observed. This increase correlated with an increase in the output of activated T cell blasts.

We have examined the distribution of alpha beta and gamma delta T cells, together with B cells, in a range of lymph nodes and blood before and after birth. The proportions of blood-borne alpha beta and gamma delta T cells changed markedly during development with a wave of increasing numbers of blood-borne gamma delta T cells occurring in the fetus during the last month of gestation and early postnatal life. gamma delta T cells constituted 18% of T cells in the blood of fetal lambs one month before birth and 60% of T cells in the blood of lambs one month after birth. There were also changes in the numbers of alpha beta T cells circulating through lymph nodes after birth. The proportion of CD4+ and CD8+ cells in mesenteric nodes increased substantially after birth, whereas that of gamma delta T cells decreased. Although B cells were present in much larger numbers in ileal lymph nodes in both the fetus and lamb, there was a large increase in the concentration of B cells in all lymph nodes in lambs after birth. In addition to the differences in the distribution of lymphocyte subsets circulating in fetal and postnatal lambs, markedly different growth patterns were also observed between lymph nodes before and after birth.

Marked differences were found in the percentage and distribution of lymphocyte subsets (CD5, CD4, CD8, T19, CD45, and CD45R cells) in hemal nodes compared to those in mesenteric lymph nodes and blood. There were a greater number of T19 (gamma/delta)+ lymphocytes and fewer CD8+ lymphocytes in hemal nodes than in mesenteric nodes and a higher percentage of CD4+ in hemal and mesenteric lymph nodes than in blood. T19 (gamma/delta)+ lymphocytes were found throughout hemal nodes but were absent from the follicles. Of special interest was the finding that 20% of hemal node lymphocytes appeared not to express CD45 (leucocyte common antigen). According to these results, because of the unique distribution of lymphocyte subsets and the presence of germinal centers, hemal nodes may have a specialized role in the immune system.

The migratory properties of small, CD4+ and CD8+ T lymphocyte subsets have been examined in sheep under physiological conditions. Lymphocytes obtained free-floating in lymph were directly labeled with fluorochrome in vitro and returned to the blood of the same animal. Over the next 48 h the lymph collection bottles were replaced at various times. The cells in these collections, as well as an aliquot of the cells used for direct fluorescent labeling, were then phenotyped with monoclonal antibodies (mAbs) which define the mutually exclusive CD4+ and CD8+ T lymphocyte subsets in sheep. Binding of mAbs was detected by using secondary reagents labeled with a fluorochrome of a different colour. All cell samples were analyzed by flow cytometry and CD4/CD8 ratios were determined for the recirculated, fluorochrome-labeled population in each lymph collection. The mean CD4/CD8 ratio was then calculated and compared with the CD4/CD8 ratio of the intravenously infused starting population. In three experiments employing efferent prescapular lymph cells, three experiments employing efferent intestinal lymph cells and two experiments employing afferent intestinal lymph cells, the mean CD4/CD8 ratio of the recirculated, fluorochrome-labeled population was different from the CD4/CD8 ratio of the starting population, thereby indicating that non-random migration of these subsets had occurred. The finding that the CD4/CD8 ratio of the recirculated population was higher than the CD4/CD8 ratio of the transfused starting population in every case provides strong experimental support for the hypothesis that small, CD4+ T cells are extracted from the blood by specialized vascular endothelium with greater efficiency than small, CD8+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)