Clinical and histopathological characterization of a large animal (ovine) model of collagen-induced arthritis.

Collagen induced arthritis (CIA) is the most studied and used rheumatoid arthritis (RA) model in animals, as it shares many pathological and immunological features of the human disease. The aim of this study was to characterize clinical and immunological aspects of the ovine CIA model, and develop lameness and histopathological scoring systems, in order to validate this model for use in therapeutic trials. Sheep were sensitized to bovine type II collagen (BCII), arthritis was induced by injection of bovine collagen type II into the hock joint and the response was followed for two weeks. Clinical signs of lameness and swelling were evident in all sheep and gross thickening of the synovium surrounding the tibiotarsal joint and erosion on the cartilage surface in the arthritic joints. Leucocyte cell counts were increased in synovial fluid and there was synovial hyperplasia, thickening of the intimal layer, inflammation and marked angiogenesis in the synovial tissue. There was a large influx of monocytes and lymphocytes into the synovial tissue, and increased expression of TNF-α and IL-1ß in arthritic intima, angiogenesis and upregulation of VCAM-1. CIA in sheep appears to be an excellent large animal model of RA and has the potential for testing biological therapeutics for the treatment of rheumatoid arthritis.

Neonatal thymectomy identifies two major pools of sessile and recirculating peripheral T cells which appear to be under separate homeostatic control.

In this study the role of the thymus in the development of sessile T cell populations resident in spleen and lymph nodes (LN) was contrasted with the development of recirculating T cell populations trafficking between blood and lymph. Extensive analysis of the composition and the rate of growth of the secondary lymphoid tissues and recirculating lymphocyte pool coupled with neonatal thymectomy revealed that the sessile and recirculating T cell populations showed different degrees of thymic dependency and increased in size at different rates, suggesting these two populations might be under separate homeostatic control. Neonatal thymectomy also resulted in a much greater depletion of CD8+ and gammadelta TCR+ T cell subsets compared with CD4+ T cells in the sessile and recirculating T cell pools, and greatly reduced the number of T cells homing to peripheral lymph nodes compared with those homing to the gut.

Local immune responses to influenza antigen are synergistically enhanced by the adjuvant ISCOMATRIX.

The peripheral (draining) lymph node, as the primary site of immune induction, determines the course of systemic responses to an injected antigen. Lymphatic duct cannulation procedures in sheep were used to investigate local immunoreactivity to human influenza virus antigen (Flu ag) admixed with the adjuvant ISCOMATRIX (IMX). Compared to Flu ag or IMX alone, the co-administration of Flu ag and IMX (Flu ag+IMX) synergistically enhanced a number of immunological responses (lymphocyte and blast migration from the node, antigen-specific antibody levels and IL6 output in efferent lymph, and antigen-induced proliferation in cultured efferent lymph cells). Together, these results demonstrate that IMX is an immune modulator, and that lymphatic duct cannulation procedures may be used to evaluate antigen/adjuvant combinations for vaccine development.

Thymic export in aged sheep: a continuous role for the thymus throughout pre- and postnatal life.

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.

Biosynthesis of glycosaminoglycans and proteoglycans by the lymph node.

Previous studies of hyaluronan uptake and catabolism by lymph nodes indicated that the nodes might also add some HA of low molecular weight to the unabsorbed fraction that passes through from afferent to efferent lymph vessels. The ability of lymph nodes to synthesise HA and proteoglycans was therefore examined (i) by perfusion of [(3)H] acetate through an afferent lymph vessel in vivo, and recovery of labeled products from the efferent lymph vessel and from the node after perfusion; and (ii) by tissue culture of lymph nodes with [(3)H] acetate. Perfusion of lymph nodes with [(3)H] acetate in situ yielded: (a), in outflowing lymph, small amounts of chondroitin/dermatan sulfate within the first hour which continued to be produced for up to 24 h; heparin in the second hour and HA in the third. In the nodes removed 17 to 19 h later, equal amounts of hyaluronan and chondroitin/dermatan sulfate and heparan sulfate proteoglycans were detected. In the tissue culture of lymph nodes: (1) HA, heparin and proteoglycans of heparan sulfate and chondroitin/dermatan sulfate were released into the medium but in the cell extract only heparan sulfate proteoglycan was detected; and (ii) molecular weight of the released hyaluronan ranged widely but was mostly less than 4-5x10(5)D; heparan sulfate proteoglycan was 2.8x10(4) to 9.4x10(5)D; heparin 7.9x10(4)D and chondroitin sulfate 1.3x10(4)D, suggesting that the chondrotin sulfate were released from their proteoglycans core by enzymic degradation. It is concluded that lymph nodes can release HA, heparin, heparan sulfate and chondroitin/dermatan sulfate proteoglycans into efferent lymph but the amount of hyaluronan is likely to be small without immune or other stimulation and its molecular weight is lower than in other tissues.

Regulation of T cell homeostasis during fetal and early postnatal life.

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.

L-selectin expression on thymic emigrants defines two distinct tissue-migration pathways.

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.

The ontogeny of T cell recirculation during foetal life.

Studies on the ontogeny of the immune system in the sheep foetus, which remains immunologically naive until after birth, indicate that a large scale recirculation of T cells is just as much a feature of the foetal immune system as it is in animals after birth. An extensive recirculation of T cells and dendritic cells through peripheral tissues-including the gastrointestinal tract and skin-develops early in foetal life, although a population of gut-homing memory T cells does not develop until postnatal life. Current evidence suggests that two populations of thymic emigrants with distinct tissue-homing specificities to spleen and lymph node contribute to the development of the foetal peripheral T cell pool. CD8(+) thymic emigrants mostly target spleen while CD4(+) thymic emigrants home predominantly to lymph nodes. The lifespan of thymic emigrants is uncertain, although cells entering lymph are long-lived and form the basis of a diverse pre-immune repertoire of recirculating T cells. The life history and growth rates of non-recirculating T cells in spleen and lymph nodes during foetal life are at present unknown.

Cellular requirements for the activation and proliferation of ruminant gammadelta T cells.

Requirements for the activation and proliferation of gammadelta T cells were investigated. Maximum numbers of gammadelta T cells expressed the IL-2R alpha-chain after 6-h Con A stimulation in peripheral blood, efferent lymph, and afferent lymph. In comparison, IL-2R alpha-chain expression on CD4 T cells only reached maximum levels in response to Con A stimulation in peripheral blood and afferent lymph populations. Analysis of enriched gammadelta T cells demonstrated that Con A-induced expression of the IL-2R alpha-chain was independent of APC. Together, these data suggest that the requirements for gammadelta T cell activation are less stringent than those for alphabeta T cell activation. Unfractionated peripheral blood, efferent lymph, and afferent lymph cell populations proliferated in response to Con A alone. In contrast, enriched gammadelta T cells (CD4/CD8 depleted) from efferent lymph did not proliferate in response to Con A alone, but required the addition of IL-2. This requirement for exogenous IL-2 could be overcome by the addition of dendritic cells purified from afferent lymph. These results suggested that gammadelta T cells required costimulatory signals provided by APC to ensure the production of sufficient IL-2 to drive proliferation. CD28 and CTLA-4 mRNA were detected in efferent lymph and afferent lymph populations containing CD4 and CD8 T cells stimulated with Con A and IL-2 or with Con A alone, respectively. In contrast, negligible levels of these mRNA species were detected in efferent and afferent lymph populations devoid of CD4 and CD8 T cells. These results suggest that ovine gammadelta T cells may use alternative costimulatory pathways.

An immune system switch in T cell lifespan at birth results in extensive loss of naive fetal T cells during the first week of postnatal life.

Lymphocyte recirculation is critical to maximize the efficiency of immunological surveillance and is an absolute requirement for the development of systemic memory. The consensus view of the lifespan of peripheral T cells holds that naive T cells are long-lived cells and most memory T cells are short-lived cells, although the question of the lifespan of peripheral T cells is not yet fully resolved. We have studied the lifespan of T cells circulating in efferent lymph draining lymph nodes (LN) in the immunologically naive sheep fetus and in postnatal lambs immediately following birth by examining the in vivo incorporation of [3H]thymidine by newly formed T cells during continuous administration of [3H]thymidine. We report that authentically naive fetal T cells are long-lived cells which continue to recirculate between blood and lymph during fetal life. At birth, however, a process is triggered whereby fetal T cells circulating through LN are rapidly lost from the peripheral T cell pool and are replaced by freshly arriving T cells which have been formed since birth. Our results indicate that by the end of the first week of postnatal life, around three-quarters of the T cells circulating through peripheral LN have been formed since birth.

An immune system switch at birth triggers a change in the lifespan of peripheral T cells.

Lymphocyte recirculation is an essential element in the integration of immune responses and is an absolute requirement for the development of systemic memory in postnatal animals. During foetal life a large pool of recirculating T cells develops and migration pathways of naive T cells to skin and peripheral tissues as well as LN are established. At birth a process is triggered whereby naive fetal T cells are rapidly lost from the circulating pool and are replaced by newly arriving T cells which have been formed since birth. At present our data suggest that the thymic export in the fetus creates a pool of long-lived naive T cells of wide diversity. The situation in neonatal lambs is more complex since the thymus is exporting large numbers of short-lived thymic emigrants which enter a peripheral T-cell population where many T cells are dividing.

Origin and development of the gamma delta T-cell system in sheep: a critical role for the thymus in the generation of TcR diversity and tissue tropism.

Ruminant gamma delta T cells are concentrated at epithelial surfaces and share many features in common with species such as mice and humans which contain relatively fewer gamma delta T cells than ruminants. To date no gamma delta T cells with invariant TcR have been found in sheep and the generation of gamma delta TcR diversity which is thymic dependent follows a developmentally regulated sequence. Analysis of thymic export of gamma delta T cells shows that emigration of gamma delta T-cell subsets increases markedly during fetal life and after birth suggesting intrathymic processes leading to mature gamma delta T cells may change during development. Skin homing gamma delta T cells acquire their tissue tropism inside the thymus and pathways of recirculation of gamma delta T cells to skin are laid down during fetal development independent of antigen and remain stable through into adult life.

ITA, a vertebrate homologue of IAP that is expressed in T lymphocytes.

Apoptosis plays a crucial role in both the development and the control of the immune system. During T lymphocyte development, thymocytes undergo apoptosis as part of the process of elimination of self-reactive clones. Mature T cells also undergo apoptosis following antigen-stimulated proliferation as part of a mechanism that controls the immune response. Apoptosis also provides a defense mechanism against viruses whereby the rapid death of virus-infected cells reduces virus spread. Viruses, on the other hand, often express proteins that inhibit apoptosis of their host cells, thereby enhancing their infectivity. We have isolated a novel gene, ita (inhibitor of T cell apoptosis), which is a vertebrate homologue of the viral apoptosis inhibitor IAP. Expression of ita appears to be restricted to cells of the T lymphocyte lineage, and high levels of ita mRNA are induced within 4-8 hr of T cell activation. Immunohistologic studies show that medullary and cortical thymocytes express detectable levels of ITA. ITA is a 69 kDa protein that contains a C-terminal ring-finger motif that is found in several oncogenic proteins and N-terminal repeat elements that have only been reported in other apoptosis inhibitors. These findings suggest that ITA may play a role in controlling apoptosis in T cells.

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.

Virgin alpha beta and gamma delta T cells recirculate extensively through peripheral tissues and skin during normal development of the fetal immune system.

Current models of T cell migration place severe restrictions on the recirculation of virgin T cells, condemning them to migrate exclusively via high endothelial venules in lymph nodes until they either die or acquire the capacity to migrate to skin and peripheral tissues as memory cells following stimulation with antigen. We have demonstrated in the sheep fetus (which is immunologically virgin until after birth) that virgin T cells and dendritic cells circulate through skin and peripheral tissues during fetal life in the same non-random manner as adult T cells but in much larger numbers than they do in adult animals. Our data also showed that T cells do not discriminate between peripheral tissues and skin or lymph nodes on the basis of virgin or memory CD45R phenotype, or CD2, CD58 or CD44 phenotype, and with the possible exception of CD11a/CD18, that it is not mandatory for lymphocytes to be activated to adhesion moleculehi status in order to home to fetal skin. Our results indicate that unique tissue-homing specificities for extra-lymphoid tissues can be imprinted on virgin T cells independent of foreign antigen. Virgin T cells have previously been thought to be denied access to peripheral tissues; however, the large-scale traffic of virgin T cells through extra-lymphoid tissues in the fetus reported here provides a mechanism whereby direct virgin T cell interactions with self-antigens expressed only on tissues outside the thymus can occur repeatedly during development of the fetal immune system.

Role of the thymus in the generation of skin-homing alpha beta and gamma delta virgin T cells.

Current models of lymphocyte traffic suggest that homing specificities of T cells to tissues such as skin are generated outside the thymus as a result of activation of naive T cells by antigen in lymph nodes. Virgin T cells are thought to home to high endothelial venules in lymph nodes, but are thought to be unable to home to extra-lymphoid tissues such as skin. We used the technique of in situ labeling of the thymus with fluorescein isothiocyanate to examine the homing specificities of authentically naive T cells in vivo, immediately after their export from the thymus. We report that homing specificities for skin as well as lymph node are imprinted on T cells inside the thymus, independent of antigen. We also show that both alpha beta and gamma delta emigrant T cells exhibit homing patterns to skin and lymph nodes which are identical to those of mature T cells. Our findings demonstrate a key role for the thymus in the induction of skin-homing specificities on T cells indicating that skin-homing specificities of T cells are not generated solely outside the thymus as a result of the activation of virgin T cells by antigen. The migration of thymic emigrants to extra-lymphoid tissues within a few hours of leaving the thymus may have implications for mechanisms of peripheral self-tolerance. This pathway provides an opportunity for direct virgin T cell interactions with self components only expressed in the periphery at a time when emigrants may be more susceptible to tolerance induction than mature circulating T cells.

Antigen-independent maturation of CD2, CD11a/CD18, CD44, and CD58 expression on thymic emigrants in fetal and postnatal sheep.

We have compared the expression of CD2, CD11a/CD18, CD44, and CD58 and alpha beta and gamma delta T cells emigrating from the fetal and postnatal thymus. We report that both gamma delta and the CD4+CD8- and CD4-CD8+ subsets of alpha beta T cells express mature levels of the adhesion molecules CD11a/CD18, CD44, and CD58 upon emigration from the thymus. Whereas CD44 is up-regulated on gamma delta + thymocytes prior to export, down-regulation of both CD11a/CD18 and CD58 occurs prior to emigration from the thymus, suggesting that down-regulation of these molecules may be a final maturational step taken by developing gamma delta T cells before their export from the thymus. In contrast, there is continued up-regulation of CD2 on gamma delta and alpha beta T cells upon emigration from the thymus and as they move into the mature peripheral T-cell pool. There was also a marked reduction in the number of CD2+ gamma delta T cells exported during fetal development that was associated with a marked reduction in the percentage of CD2+ gamma delta thymocytes exported. The postthymic maturation of CD2 and the other changes in adhesion-molecule expression appear to be independent of extrinsic antigen, as the same changes were observed in the antigen-free environment of the fetus as in the postnatal lamb, which has been exposed to extrinsic antigen. It thus appears that these changes in adhesion-molecule expression are as a result of the normal maturation pathway from a developing thymocyte to a mature peripheral T cell.

Differences in the tissue-specific homing of alpha beta and gamma delta T cells to gut and peripheral lymph nodes.

Tissue-selective streaming of T cells is considered to be a critical element in the integration of normal immune responses in intact animals. The results presented in this paper show that while there were major subsets of gut-homing T cells present in intestinal lymph, there were considerable differences in the tissue tropism of T cell populations circulating in lymph draining gut and peripheral lymph nodes. Thus, while CD4+ cells returned preferentially to their tissue of origin, gamma delta T cells showed a strong migratory preference for peripheral lymph nodes regardless of their tissue of origin. In contrast, although a population of gut-homing CD8+ cells was present in ileal lymph, CD8+ T cells from peripheral lymph nodes homed equally well to gut and lymph nodes. There were also considerable differences in the expression of L-selectin on T cells circulating in the two compartments. L-selectin was down-regulated on alpha beta T cells present in ileal lymph but not on gamma delta T cells which expressed the highest levels of L-selectin of all T cell subsets. It is suggested that gut-homing alpha beta T cells which have down-regulated L-selectin are formed in the gut-associated lymphoid tissues in response to gut antigens while the migratory properties of gamma delta T cells are ontogenetically determined, independent of antigen.

Lymph node homing cells biologically enriched for gamma delta T cells express multiple genes from the T19 repertoire.

Sheep gamma delta T cells have been shown serologically to express T19, a membrane protein of 180-200 kDa which is a member of the scavenger receptor superfamily. Previous work from this laboratory resulted in the detection of a multigene family of T19-like genes in the sheep genome. In this study nucleotide sequences from several T19 genes were determined and are reported along with the corresponding segments of a number of expressed mRNA molecules. A segment of a single sheep T19-like gene was sequenced and these data, along with the corresponding sequences from cloned T19-like cDNA molecules from sheep and cow, were used to design an oligonucleotide primer system suitable for amplification of corresponding segments of many T19 genes and their cDNAs. Between 30 and 40% of cloned T19 genes were amenable to amplification using the selected primers, and sequence analysis of cloned PCR products confirmed that different T19 genes encode unique amino acid sequences. The expression of multiple T19 genes was established using cDNA molecules obtained from a single sample of sheep lymphocyte mRNA. The possible role of the T19 family of genes is discussed.