Summary of workshop findings for leukocyte antigens of sheep.
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Biomedical subjects
L Dudler
Publications and source records attributed to L Dudler.
Epitopes of the T19 lymphocyte surface antigen are extensively conserved in ruminants.
The reactivity of five monoclonal antibodies (mAbs SBU-T19, 197, IL-A29, CC-15 and CC-39) specific for the T19 molecule on sheep and cattle CD4-CD8- T cells was compared. MAbs SBU-T19 and 197 were shown to recognise separate epitopes on T19. All mAbs reacted with lymphocytes from several different ruminant species and the tissue distribution and frequency of positive cells was similar in each case. None of the mAbs reacted with horse, pig or camel lymphocytes. The extensive conservation of T19 epitopes in ruminants during the mammalian radiation could indicate an important role for this molecule in the ruminant immune system.
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Expression of the "T19" and "null cell" markers on gamma delta T cells of the sheep.
A 215 kDa molecule termed T19 marks CD4-CD8- T cells in sheep and cattle. In this report, we analysed the T19 or "null cell" panel of mAbs against gamma delta T cells of sheep, using a mAb specific for the gamma delta TCR. By two-colour immunofluorescence, all of the mAbs in the T19 panel reacted with gamma delta T cells or subsets thereof, although staining intensities and percentages of cells stained by the different mAbs indicated considerable heterogeneity for the T19 molecule. This probably results from differential expression of certain epitopes on T19. The reactivity of most of the mAbs for the 215 kDa T19 molecule was also confirmed by immunoprecipitation and SDS-PAGE.
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Naive and memory T cells show distinct pathways of lymphocyte recirculation.
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.
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Molecular cloning of sheep T cell receptor gamma and delta chain constant regions: unusual primary structure of gamma chain hinge segments.
The primary structure of sheep T cell receptor (TcR) gamma and delta chain constant (C) regions has been determined by cDNA cloning. A comparison of the nucleotide and deduced amino acid sequences of the sheep chains with known human and mouse sequences shows that the primary structure of the immunoglobulin, transmembrane and cytoplasmic C gamma domains and all of the C delta region has been substantially conserved. However, the hinge or connector region of sheep gamma chains differs significantly from all known TcR chains. Clones representing two different sheep C gamma genes were isolated and both contain additional sequence in this region, making them the longest TcR chains so far identified. The hinge region of both sheep C gamma sequences contains two additional cysteine residues and a motif of five amino acids (TTESP or TTEPP) which has been triplicated in one of the clones. Other repetitive segments of 13-17 amino acids could also be identified suggesting that, as in the human C gamma 2 gene, this region of the sheep genes could have arisen from an exon duplication or triplication event. Southern blot analysis of sheep DNA confirmed the presence of one C delta gene and at least two C gamma genes. A restriction fragment length polymorphism that is probably associated with allelic sequence variation in the sheep C delta gene was detected in DNA from different animals. Although the essential structure of the gamma/delta TcR appears well conserved through evolution, the marked heterogeneity evident in the hinge region of gamma chains both within and between species, and particularly the presence of additional cysteine residues in the sheep sequences, may be of structural and functional importance.
Amino Acid Sequence↗
Differential peripheral expansion and in vivo antigen reactivity of alpha/beta and gamma/delta T cells emigrating from the early fetal lamb thymus.
The concentrations of different lymphocyte subsets in the blood of lambs which had been thymectomized (Tx) in utero between days 67-75 of fetal gestation were measured at birth and at various intervals during the first year of life. Compared to thymus-intact (Ti) controls, Tx lambs were severely depleted of both alpha/beta and gamma/delta T cells at birth (less than 10% of control levels). The majority of the residual alpha/beta T cells present in Tx lambs at birth were CD4+CD8-. As the Tx lambs aged, the concentration of alpha/beta T cells in blood increased steadily to reach levels around 50% of control values. In contrast, the circulating gamma/delta T cells did not expand in Tx animals and remained barely detectable throughout the observation period, although these cells accounted for 30%-60% of the T cells in the blood of Ti lambs. The expansion of alpha/beta but not gamma/delta T cells was also reflected in changes in the cellular composition of solid lymphoid organs in Tx lambs. B cell numbers were similar in both groups at birth but Tx lambs were persistently B lymphopenic from 3 weeks of age onwards. The alpha/beta T cells that had expanded in Tx lambs responded to stimulation with bacterial antigens in a way that was qualitatively similar to the response in Ti lambs. By contrast, the few gamma/delta T cells in Tx lambs responded abnormally. Our results show that although sheep alpha/beta and gamma/delta T cells are equally thymus dependent during ontogeny, the early fetal thymic emigrants which establish the two T cell lineages in the periphery have strikingly different antigen reactivities and capacities for self-renewal and expansion.
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Characterization of lymphatic and venous emigrants from the thymus.
Lymphocytes leaving the thymus via two different pathways were examined as to their morphology and phenotype. Cells leaving the thymus via lymphatics were obtained by a direct cannulation of thymic lymphatics and those leaving via the thymic vein were labelled within the thymus using an extracorporeal perfusion system and identified subsequently as fluorescent cells in the draining vein. In both cases the cervical thymus in lambs was used, since it is located in the neck region and ensures easy access to both blood and lymphatic vessels of the thymus without subjecting the animal to a major trauma or stress. Cells obtained from the thymic lymphatics or lymphatic emigrants were found to have distinct features different from peripheral T cells in terms of their surface morphology and expression of the MHC antigens. Venous emigrants were also slightly different from peripheral T cells in MHC expression. Estimation on the rate of thymocyte emigration into the periphery suggested that neither venous nor lymphatic emigrants represent a major fraction of de novo synthesized cells in the thymus of this animal species, as has been suggested in the mouse.
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T cell receptor gene expression in sheep: differential usage of TcR1 in the periphery and thymus.
We have isolated cDNA clones for each chain of the sheep T cell receptor (TcR) and used these to analyze TcR gene expression in thymocyte and peripheral T cell subsets. Outer cortical thymocytes expressed a low level of mature message for all TcR chains suggesting that intrathymic precursors for the alpha/beta and gamma/delta lineages occur in this population. Inner cortical and medullary thymocytes expressed high levels of mature alpha/beta transcripts, low levels of mature delta transcripts but no detectable gamma message. Mature alpha/beta transcripts were detected in peripheral CD4+ lymphocytes and these as well as CD8+ cells expressed a surface heterodimer of 85 kDa which resolved into 40- and 50-kDa subunits after reduction. Peripheral CD4-CD8-lymphocytes, which in sheep are marked by the T19 antigen and may account for up to 60% of T cells in blood, expressed a surface heterodimer of 75 kDa. The T19+ cells had high levels of mature delta and truncated beta transcripts in their cytoplasm but did not express the C gamma gene detected in DN thymocytes, although they seem to share V gamma and/or J gamma elements. Both forms of the sheep TcR are associated with CD3 molecules on the cell surface. These results show that (a) in contrast to the situation in rodents and humans, a large proportion of peripheral sheep lymphocytes use TcR gamma/delta; (b) the proportion of T cells in the periphery which use TcR gamma/delta is greater than in thymus; and (c) CD4-CD8- cells in thymus and periphery (T19+) use the same C delta gene but appear to use different C gamma genes, suggesting that in sheep there may be more than one lineage of lymphocytes expressing TcR gamma/delta.
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Ontogeny, morphology and tissue distribution of a unique subset of CD4-CD8- sheep T lymphocytes.
The morphology, distribution and ontogeny of the 197+ T lymphocyte subpopulation is described. Cells were identified by immunofluorescent and immunoenzymic staining of cell suspensions and tissue sections. In the circulation, 197+ cells are small and regular, indistinguishable in size from peripheral CD4+ and CD8+ cells, with little cytoplasm and a large amount of condensed chromatin. In the post-natal animal they are unevenly distributed, being most common in the circulatory pathways, particularly in the blood. In the thymus they chiefly localize in the regions around Hassal's corpuscles and medullary blood vessels. They are totally absent from B cell areas in all tissues. They are the last of the T cells to appear in ontogeny, and are first detected in the thymus. Their frequency in blood is age-dependent with peak levels occurring perinatally. There is a post-natal decline in the frequency of 197+ cells in ileocaecal but not in prescapular lymph nodes. We conclude that these T cells differ from the more commonly described T cells, not only in their surface expression of the CD4, CD8 and T19 antigens, but also in the time of their first appearance, their age-related prevalence and their distribution between and within tissues.
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Surface expression of differentiation antigens on lymphocytes in the ileal and jejunal Peyer's patches of lambs.
The surface phenotype of lymphocytes in the ileal (IPP) and jejunal (JPP) Peyer's patches (PP) of lambs was compared using flow cytometry and immunohistology with a panel of monoclonal antibodies (mAb). The B-cell markers p220, BAS9A and surface Ig molecules were detected on 70-95% of cells from the IPP. T-cell markers were detected on less than 1% of IPP lymphocytes, confirming that the IPP in lambs contains virtually only B lymphocytes. The JPP contained a lower proportion of B cells and 16% T cells, nearly all of which expressed the CD4 molecule. Interestingly, the reactivity of a fourth B-cell markers, BAQ44a, differed from this pattern; only 12% of IPP lymphocytes were positive whereas 70% of JPP lymphocytes expressed this marker. A majority of both IPP and JPP lymphocytes (80-95%) expressed the cell adhesion molecules CD11a (LFA-1) and LFA-3. Other adhesion molecules, such as CD2 and CD44, were expressed by fewer cells from the IPP than from the JPP. MHC class I antigens were detected on more than 95% of lymphocytes from both the IPP and JPP. In the case of MHC class II antigens, more positive cells occurred in the IPP (greater than 95%) than in the JPP (80%). The in situ localization of cell-surface antigens was assessed by immunohistology. CD4+ T cells occurred in the interfollicular T-cell regions and in JPP follicles, whereas CD8+ T cells localized only in the interfollicular regions and were absent from follicles. The pattern of expression of B-cell markers, adhesion molecules and MHC antigens indicated that a gradient of increasing maturity of B cells existed within follicles from the base towards the dome region. The data presented here lend support to the notion that the IPP in lambs represents a novel B-cell lymphoid tissue with a function different from that of the conventional Peyer's patches found in the jejunum.
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A novel glycoprotein expressed on sheep T and B lymphocytes is involved in a T cell activation pathway.
We have produced a new mouse mAb that identifies a sheep T cell activation Ag. The mAb B5-5 is specific for low m.w. components on nearly all sheep thymocytes and peripheral T and B lymphocytes but does not label immature B cells in Peyer's patches or germinal centers. After cross-linking of target structures either directly by plastic-bound mAb or indirectly using anti-Ig reagents, peripheral T cells, but not thymocytes or peripheral B cells, were activated. IL-2 was secreted by T cells after cross-linking and activation was strongly augmented in the presence of PMA. The addition of soluble B5-5 mAb to mitogen-stimulated cultures of sheep lymphocytes resulted in a suppression of PHA responses and augmentation of PWM responses and had a variable effect on Con A responses but had no effect on LPS- or protein A-induced proliferation. When added to alloantigen-stimulated cultures, B5-5 augmented the proliferative response. The B5-5 membrane component consists of 14- to 19-kDa glycoproteins but the banding patterns obtained during SDS-PAGE analysis of 125I-labeled Ag differed between thymocytes, peripheral T cells, and peripheral B cells. On the basis of its range of expression on lymphoid cells and known biochemical and functional properties, we conclude that the B5-5 component on sheep lymphocytes is different from T cell activation Ag in other species.
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Analysis of cell division among subpopulations of lymphoid cells in sheep. I. Thymocytes.
The number, distribution and surface phenotype of dividing cells in the thymus, and differences between the cell cycle status of thymocyte subpopulations, were studied in fetal and post-natal lambs using double-labelling techniques. Dividing cells were labelled in vivo for various periods with 5-bromo-2-deoxyuridine (BrdU). The proportions of constituent thymocyte subpopulations that had synthesized DNA during the labelling period were measured by flow cytometry or immunohistochemistry using a panel of monoclonal antibodies (mAbs) specific for sheep lymphocyte differentiation antigens and MHC class I and class II antigens in conjunction with an anti-BrdU mAb. The proportion of thymocytes that incorporated BrdU during a 1-hr labelling period varied with age, and levels of 30%, 13% and 9% were measured, respectively, in 40- and 125-day-old fetuses and 8-week-old lambs. Eight percent of the thymocytes in lambs were synthesizing DNA, with 4% entering the G2 phase per hour, and a substantial number of thymocytes (21%) had a G2 + M phase DNA content. A small subset of thymocytes (1-3%) recognized by mAb E-79 localized to the subcapsular region of the cortex and displayed the highest level of BrdU incorporation. Cortical-type thymocytes (CD1+) comprised 50-70% of thymocytes; however, few of these incorporated BrdU and the proportion in the G2 + M phase of the cell cycle was higher than for other thymocyte subpopulations. The 197+CD4-CD8- T cells also showed no evidence of in vivo division.
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Analysis of cell division among subpopulations of lymphoid cells in sheep. II. Peripheral lymphocytes.
The number, distribution and surface phenotype of dividing cells in lymph nodes and blood and differences between the cell-cycle status of lymphocyte subpopulations were studied in lambs using double-labelling techniques. Dividing cells were labelled in vivo for various time periods with 5-bromo-2-deoxyuridine (BrdU). After removal of lymphoid tissues, the proportions of constituent lymphocyte subpopulations which had synthesized DNA during the labelling period were measured by flow cytometry or immunohistochemistry using a panel of monoclonal antibodies (mAbs) specific for sheep lymphocyte differentiation antigens and MHC class I and class II antigens in conjunction with an anti-BrdU mAb. There was a higher overall level of cell division in the ileocaecal lymph node than in either the prescapular or parathymic lymph nodes. In all three lymph nodes, the majority of lymphocytes which incorporated BrdU occurred in B-cell follicles or germinal centers. CD4+ and CD8+ T cells had a higher level of cell division (LI 5-14%) than those recognized by mAb 197 (CD4- CD8- subset) (LI less than or equal to 3%).
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A murine anti-sheep T8 monoclonal antibody, ST-8, that defines the cytotoxic T lymphocyte population.
A mouse monoclonal antibody (mAb), ST-8, reactive with a subpopulation of sheep T lymphocytes was investigated by tissue distribution analysis and functional studies of the antigen-bearing (ST-8+) cells. Two other mouse mAb, ST-1 and T-80 that recognize all sheep T cells and a T-cell subset, respectively, were also examined for comparison. The ST-8+ cells represented 61-69% of thymocytes and about 10-30% of peripheral T lymphocytes. Histologically, ST-8+ cells were found mainly in the thymic cortex, T-dependent areas of the peripheral lymphoid tissues and the splenic red pulp and marginal zone, but not in B-dependent areas such as germinal centers of lymph follicles of the Peyer's patches. ST-8 mAb plus complement treatment completely abolished both the induction phase and the effector phase of alloreactive cytotoxic T lymphocytes but did not affect proliferative responses to T-dependent mitogens and allogeneic antigens. ST-8 mAb also blocked the cytotoxic T lymphocyte function of lysing specific targets in the absence of complement. ST-8 mAb immunoprecipitated an antigen from the surface of sheep T lymphocytes under reducing conditions with an apparent molecular weight of 33-35 kilodaltons. Therefore the antigen recognized by the ST-8 mAb showed striking similarities to human T8/Leu-2a and mouse Lyt-2 antigens not only in the tissue distribution and function of antigen-bearing cells but also the molecular weight. We conclude that the ST-8 antigen is the ovine homologue of the human T8 and mouse Lyt-2 antigens.
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Numbers and phenotype of lymphocytes emigrating from sheep bone marrow after in situ labelling with fluorescein isothiocyanate.
In normal young lambs the bone marrow was selectively labelled with fluorescein isothiocyanate by a temporary perfusion of one hind-leg. One day later, the incidence of bone marrow emigrants in different lymph nodes, spleen, Peyer's patches, thymus, non-perfused bone marrow and blood was determined. The emigrants were also phenotyped by the use of monoclonal antibodies and classified into monocytes or lymphocyte subsets. Large numbers of lymphocytes left the bone marrow of the perfused leg during 1 day. Considerable numbers of cells migrated to other bone marrow compartments. Varying numbers of mononuclear emigrants were found in peripheral lymphoid organs, with labelling indices ranging from 1.06% in the blood to 0.004% in the thymus. In the spleen, comparable numbers of B- and T-lymphocyte emigrants from the bone marrow were found, whereas in the blood, lymph nodes and jejunal Peyer's patches many more emigrants were T lymphocytes than B lymphocytes. In the prescapular lymph nodes, for instance, 90.4% of emigrants were T cells but only 9.6% were B cells. Based on the large numbers of lymphocytes emigrating from the bone marrow, their phenotypes and their entry into other bone marrow compartments, it it can be concluded that the bone marrow of young lambs is an integral part of the migratory route of lymphocytes.
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Studies on the differentiation of T lymphocytes in sheep. II. Two monoclonal antibodies that recognize all ovine T lymphocytes.
Two mouse monoclonal cytotoxic antibodies (ST-1a and ST-1b) recognize an antigen present on the large majority of thymocytes and all T cells in the periphery, but not B cells or other haemopoietic cells in sheep. Examination of frozen sections of various fetal tissues revealed that the cells expressing this antigen first appeared in the thymus, and these cells markedly increase in numbers in the peripheral lymphoid tissues after mid-gestation. Large accumulations of positive cells were located in the paracortex of lymph nodes, the periarteriolar lymphoid sheath of the spleen, and interfollicular areas of jejunal Peyer's patches, all of which are known to be T-dependent areas. Treatment of lymphocytes with ST-1a and complement resulted in the abrogation of T-proliferative responses, but the response to a B-cell mitogen, lipopolysaccharide, was not reduced. Neither ST-1a nor ST-1b cross-reacted to lymphocytes obtained from other species of animals (man, monkey, mouse, rat, guinea-pig, chicken, frog, pig, horse, goat and cattle). Based on these findings, it was concluded that the expression of the antigen recognized by ST-1a and ST-1b is restricted to the T-cell lineage of sheep, and that all ovine T cells express this antigen. Furthermore, ST-1a and ST-1b were determined to recognize the same antigen by reciprocal blocking experiments.
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Differentiation of B lymphocytes in sheep. II. Surface phenotype of B cells leaving the 'bursa-equivalent' lymphoid tissue of sheep, ileal Peyer's patches.
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Distribution of membrane-associated actin in sheep lymphocytes. Functional implications.
Using the DNAase I inhibition assay, we titrated two forms of actin, monomeric (G) and total (G + F) actin of various populations of lymphocytes in adult and fetal sheep. Lymphocytes leaving a lymph node always showed higher actin activities than lymphocytes resident in the lymph node at the time of examination. T cells had significantly higher actin activities than B cells. A subpopulation of T cells which is present in the blood but not in the lymph showed much lower activities than total T cell populations in the blood or efferent lymph. Thymocytes showed the lowest actin activities among the lymphoid cell populations examined. A proposal is made that DNAase I inhibitions per cell can be considered as an intrinsic parameter of lymphocyte recirculating abilities.
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