Lisbeth Dudler

TCR gamma delta+ cells are prominent in normal bovine skin and express a diverse repertoire of antigen receptors.

More 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 γδ 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 γδ TCR. A highly diverse repertoire of γδ TCR was expressed in skin due mainly to the usage of multiple Vδ segments and to extensive sequence variation at the junctions of both TCRγ and TCRδ chains. However, a single receptor isotype was used. Transcripts encoding several new components of the bovine γδ TCR were identified, including three new Vγ segments, the Cγ5 region and 13 new functional Vδ segments. Taken together with earlier findings, these results emphasize that ruminant γδ T cells express exceptionally diverse antigen receptors and suggest they may have a more elaborate recognitive capacity than do their counterparts in other species.

Engraftment of human cord blood-derived stem cells in preimmune ovine fetuses after ultrasound-guided in utero transplantation.

OBJECTIVE The fetal sheep in utero transplantation model has developed into an important tool to study the efficacy of human in utero stem cell transplantation and gene therapy because of similarities in both the scale and development of immunocompetence relative to gestational age. The aim of this study was to determine whether human stem cells can be successfully transplanted to the first-trimester ovine fetus by use of a newly developed minimally invasive technique. STUDY DESIGN Human cord blood-derived, CD34(+)-enriched stem cells were injected into the peritoneal cavity of 45- to 60-day-old ovine fetuses by using ultrasound-guided transabdominal percutaneous needle puncture. Engraftment was determined 1 to 3 months after birth by flow cytometry with use of human-specific anti-CD45 antibodies. RESULTS In contrast to previous studies that used surgical techniques, we observed a fetal loss rate of 24%, significantly below previous values and only marginally higher than natural loss. Successful human cell engraftment was achieved in 18% of lambs available for analysis. Engraftment levels of human cells in bone marrow of the recipient were up to 0.8% of total nucleated cells. CONCLUSION Ultrasound-guided percutaneous transplantation of stem cells to fetal sheep in the first trimester is feasible. Although we were unable to observe a significant improvement in the level of engraftment of human cells in sheep, the decreased fetal loss rate associated with this technique allows greater use for further studies that use this model of in utero transplantation.

Subset-Specific Regulation of the Lymphatic Exit of Recirculating Lymphocytes In Vivo

The blood-to-lymph recirculation of lymphocytes is required for the maintenance of immune surveillance and the dissemination of memory. Although the ability of lymph-borne cells to recirculate has been well documented, relatively less is known about the migration capacity of PBLs. We have found a clear preference for PBLs to recirculate through s.c. rather than intestinal lymph nodes. This preference could be directly attributed to the migratory characteristics of γδ-T cells. γδ-T cells were found to express significantly higher levels of L-selectin than other subsets, suggesting that at least some of this preferential migration could be attributed to their interaction with ligands on vascular endothelium. More detailed experiments showed that γδ-T cells migrated through lymph nodes with greater efficiency than αβ T cells or B cells, which clearly indicated an enhanced ability of γδ-T cells to exit lymph nodes in the efferent lymph independent of entry from the blood. This hypothesis was supported by histological examination, where γδ-T cells were found almost exclusively in the interfollicular traffic areas within lymph nodes. These data indicate that γδ-T cells are the most active recirculating lymphocyte subset in ruminants and suggest new mechanisms to regulate the traffic of lymphocyte subsets through normal lymph nodes.

A role for lymphatic endothelium in the sequestration of recirculating γ δ T cells in TNF‐α‐stimulated lymph nodes

TNF‐α is one of the most potent immunoregulatory molecules in vivo. In addition to important regulatory effects, it is also a potent inducer of extravascular lymphocyte infiltration. To examine the dynamic changes that are induced in local lymphocyte migration through regional lymph nodes following TNF‐α injection, we used a protocol of direct lymphatic cannulation to quantitatively and qualitatively examine the traffic of lymphocytes through regional lymph nodes. We observed that local TNF‐α injection reduced the output of lymphocytes from lymph nodes up to 90 % within 6 – 10 h following stimulation. TNF‐α also altered the specificity of migration of lymphocyte traffic through subcutaneous lymph nodes. In addition to the decreased output, phenotypic analysis demonstrated decreases in the concentration of γ δ T cells by up to 30 % following TNF‐α injection. Histological examination showed that the γ δ T cells were found in close association with VCAM‐1‐expressing cells in TNF‐stimulated lymph nodes, at least some of which appeared to be lymphatic endothelium. These data indicate that TNF‐α is capable of altering the number and specificity of lymphocytes recirculating through stimulated lymph nodes by selectively altering the entry of lymphocytes into the efferent lymphatics of inflamed lymph nodes in vivo.

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 Peyers 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.

Structure and expression of ovine complement receptor type 2

The structure of sheep complement receptor type 2 (CR2) was characterised by cDNA cloning, protein sequencing and immunoprecipitation. The primary structure of sheep CR2 is similar to known mammalian homologues but the higher-order structure is unusual. Two distinct CR2 isoforms occur, one of which is ubiquitinated in the cytoplasmic domain, and the two molecular forms are expressed at the cell surface as non-covalently associated dimers. The percentage of sheep B-cells that express CR2 changes during development and varies between different body compartments. CR2+ and CR2 B-cell subsets also differ in the expression of other surface markers and in functional properties. Differential expression of CR2 may, therefore, delineate B-cells that arose by alternative developmental pathways, or it could be a marker for B-cells at different phases of antigen exposure.

Sequence analysis of sheep T-cell receptor β chains

Virtually all of our present understanding of repertoire development in c~/3 T-cells, particularly at the molecular level, has come from studies on humans and mice but there has been very little work done on other species (reviewed by Davis and Bjorkman 1988; Strominger 1989). In some instances, the use of alternative species, especially larger animals, would afford significant experimental advantages such as providing a longer time span over which to study fetal T-cell ontogeny, and also by allowing better access to lymphocytes recirculating through specific regions of the body, both in adult animals and in fetuses. However, the lack of sequence information for T-cell receptor (Tcr) segments and the unavailability of homologous probes has prevented a molecular analysis of the T-cell repertoire in these animals. We have begun to alleviate this in the case of sheep, an animal which offers both of the experimental advantages mentioned above, and have recently reported the basic molecular characterization of the sheep Tcr,, Tcr~ and Tcr~ chains (Hein et al. 1990, 1991). We have now analyzed a group of Tcrt~ chain cDNA clones derived from a postnatal thymus and from recirculating CD4 + T cells and in this communication report the sequences of a number of C, V, and J region segments. Two cDNA libraries were screened during the present work. The methods used during library preparation and screening have been described in detail previously (Hein et al. 1989; Grossberger et al. 1990). The first library was prepared in the Xgtl 1 vector from RNA isolated from the thymus of a single eight-week-old lamb. A second library was constructed in the XZAP vector from RNA prepared from a pool of CD4 ÷ T cells that had been purified by panning from the efferent lymph of the prescapular nodes

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.

Expression of the “T19” and “null cell” markers on γδT cells of the sheep

Abstract 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 γδT cells of sheep, using a mAb specific for the γδTCR. By two-colour immunoflourescence, all of the mAbs in the T19 panel reacted with γδT 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 ffor the 215 kDa T19 molecule was also confirmed by immunoprecipitation and SDS-PAGE.

Diversification of sheep immunoglobulins.

The sheep antibody repertoire was assessed by sequence analysis of Vlambda Vkappa and V(H) cDNA clones. In fetuses, all immunoglobulin chains were essentially in germline configuration, although limited diversity occurred at the junctional regions of Vkappa chains. Fetal B cells expressed 12 distinct Vlambda and six Vkappa gene segments. V(H) gene segments were also in germline configuration in fetuses but became extensively mutated with advancing post natal age. Two distinct types of mutations, point mutations and block mutations, were detected in V(H) transcripts and these localised differently along the gene. This raises a question as to whether a novel mutational mechanism may be involved in V(H) diversification in sheep.