Workshop Cluster Research Articles

Porcine ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1/CD203a): Cloning, transcription, expression, mapping, and identification of an NPP1/CD203a epitope for swine workshop cluster 9 (SWC9) monoclonal antibodies

Swine workshop cluster 9 (SWC9) antibody identifying a porcine epitope on macrophages and thymocytes was used to precipitate and characterize the molecule from biotinylated macrophages and to obtain peptide sequence by mass spectrometry. The protein was identified as ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1/CD203a). The porcine NPP1/CD203a encoding gene was mapped to chromosome 1 using a radiation hybrid panel, and transcription was investigated by RT-PCR analysis of several tissues. The cDNA was cloned and introduced into COS7 cells resulting in expression of functionally active enzyme and verification of the specificity of an SWC9 reacting monoclonal antibody. The antibody was used for immunohistochemical examination of various porcine tissues. Most prominent expression of NPP1/CD203a was found in lung macrophages and liver sinusoids.

Comparative gene expression by WC1+ γδ and CD4+ αβ T lymphocytes, which respond to Anaplasma marginale, demonstrates higher expression of chemokines and other myeloid cell-associated genes by WC1+ γδ T cells

The functions of gammadelta T cells are enigmatic, and these cells are often considered as evolutionary remnants of well-characterized alphabeta T cells. However, their conservation throughout evolution suggests that gammadelta T cells are biologically unique. In ruminants, gammadelta T cells expressing the workshop cluster 1 (WC1) scavenger receptor comprise a large proportion of circulating lymphocytes, suggesting these cells are biologically relevant and functionally different from alphabeta T cells. In fact, bovine WC1(+) gammadelta T cells can act as APC for alphabeta T cells, indicating they may express genes encoding proteins associated with innate immunity. The present study was designed to compare immune function gene expression profiles of clonal populations of WC1(+) gammadelta and CD4(+) alphabeta T cells derived from the same animal, which respond to major surface protein 2 (MSP2) of the intraerythrocytic rickettsial pathogen of cattle, Anaplasma marginale. Gene expression profiles of activated T cell clones were compared using a microarray format, and differential gene expression was confirmed by real-time RT-PCR and protein analyses. We demonstrate that although MSP2-specific alphabeta and gammadelta T cell clones express many of the same genes, gammadelta T cell clones express high levels of genes associated with myeloid cells, including chemokines CCL2, CXCL1, CXCL2, CXCL6, and surface receptors CD68, CD11b, macrophage scavenger receptor 1, macrophage mannose receptor, and galectin-3. It is important that many of these genes were also expressed at higher levels in polyclonal WC1(+) gammadelta T cells when compared with CD4(+) alphabeta T cells selected from peripheral blood.

Workshop cluster 1, a γδ T cell specific receptor is phosphorylated and down regulated by activation induced Src family kinase activity

Workshop cluster 1 + gamma delta (WC1 +γδ) T cells have been shown to play important roles in the immune response to infections. WC1 is a transmembrane glycoprotein, uniquely expressed on the surface of γδ T cells of ruminants and pigs. A role for WC1 in inducing a reversible growth arrest of γδ T cells has been previously demonstrated. WC1-induced growth inhibition has been shown to be overcome following γδ T cell activation with Concanavalin A (Con A). However, molecular mechanism(s) by which WC1 signalling might be modulated following activation have not been elucidated. In this paper we show that Con A activation of bovine lymphocytes induces the tyrosine phosphorylation of WC1 in a Src-family kinase-dependent manner. Src family kinases also phosphorylated WC1 in a COS-7 co-transfection system. Furthermore, a glutathione- S-transferase (GST)-WC1 cytoplasmic domain fusion protein was directly phosphorylated by recombinant Lck (rLck) in vitro. The Y 1303 of WC1 was identified by mutational analysis as the only one of the five WC1 tyrosine residues to be critical for Src family phosphorylation. The importance of activation-induced Src family activity for WC1 function was investigated with the Src-family specific inhibitor PP2. These studies show that the surface levels of WC1 are down regulated in a Src-family-dependent manner following activation of bovine lymphocytes. Down regulation of surface WC1 was accompanied by a Src-family-dependent accumulation of intracellular WC1. These data show that WC1 is modulated by activation-induced tyrosine phosphorylation thus providing a new insight into the signalling mechanisms by which WC1 and γδ T cell activation are regulated in this important and unique cell population.

The CD4+ T cell immunodominant Anaplasma marginale major surface protein 2 stimulates γδ T cell clones that express unique T cell receptors

Major surface protein 2 (MSP2) of the bovine rickettsial pathogen Anaplasma marginale is an abundant, serologically immunodominant outer membrane protein. Immunodominance partially results from numerous CD4+ T cell epitopes in highly conserved amino and carboxy regions and the central hypervariable region of MSP2. However, in long-term cultures of lymphocytes stimulated with A. marginale, workshop cluster 1 (WC1)+ gammadelta T cells and CD4+ alphabeta T cells proliferated, leading to a predominance of gammadelta T cells. As gammadelta T cells proliferate in A. marginale-stimulated lymphocyte cultures, this study hypothesized that gammadelta T cells respond to the abundant, immunodominant MSP2. To test this hypothesis, gammadelta T cell clones were isolated from MSP2 vaccinates and assessed for antigen-specific proliferation and interferon-gamma secretion. Seven WC1+ gammadelta T cell clones responded to A. marginale and MSP2, and three of these proliferated to overlapping peptides from the conserved carboxy region. The gammadelta T cell response was not major histocompatibility complex-restricted, although it required antigen-presenting cells and was blocked by addition of antibody specific for the T cell receptor (TCR). Sequence analysis of TCR-gamma and -delta chains of peripheral blood lymphocytes identified two novel TCR-gamma chain constant (Cgamma) regions. It is important that all seven MSP2-specific gammadelta T cell clones used the same one of these novel Cgamma regions. The TCR complementarity-determining region 3 was less conserved than those of MSP2-specific CD4+ alphabeta T cell clones. Together, these data indicate that WC1+ gammadelta T cells recognize A. marginale MSP2 through the TCR and contribute to the immunodominant response to this protein.

Genetic Variation in Bovine Mononuclear Leukocyte Responses to Dexamethasone

The objective of this study was to determine whether bovine mononuclear leukocytes exhibit genetic variability prior to and after a glucocorticoid hormone challenge in vivo. Test animals included 60 pedigreed Holstein bulls treated on 3 consecutive days with dexamethasone and 5 untreated control bulls. Eight indicator traits of leukocyte responsiveness to dexamethasone included the percentages of circulating B cells, T cells (CD4, CD8, and workshop cluster 1 molecule expressed by bovine γδ T cell), major histocompatibility complex (MHC) I and II expressing cells, and mean expressions of surface MHC I and MHC II on circulating cells. Blood for this work was collected from each test bull 10 times before, during, and after dexamethasone administration, with corresponding samples taken for control bulls. Random regression models with treatment-specific serial correlation were applied to the leukocyte data sets to estimate genetic and nongenetic sources of variation in baseline and recovery aspects of the traits. All traits responded predictably to glucocorticoid challenge. Genetic variation was observed in baseline measurements of all traits, with heritability estimates ranging from 0.21±0.03 to 0.60±0.06. Genetic variation in linear recovery from nadir values following dexamethasone administration was significant only for percentage CD4, percentage CD8, and for surface expression of MHC II. The genetic covariance between basal and linear recovery was positive and significant for percentage CD4, percentage CD8, and MHC II expression. The bovine lymphocyte antigen DRB3.2 locus accounted for significant proportions of total variation in percentage MHC II cells and MHC I expression. These results suggest that genetic variability exists for important basal and glucocorticoid-modified phenotypes of bovine mononuclear leukocytes, implying that immunocompetence traits impacted by this stress hormone may be enhanced by genetic selection.

Type-A CpG oligonucleotides activate exclusively porcine natural interferon-producing cells to secrete interferon-alpha, tumour necrosis factor-alpha and interleukin-12.

Natural interferon-producing cells (NIPC), also referred to as immature plasmacytoid dendritic cells (PDC), constitute a small population of leucocytes secreting high levels of type I interferons in response to certain danger signals. Amongst these signals are those from DNA containing unmethylated CpG motifs. The present work demonstrated that the CpG oligonucleotides (CpG-ODN) 2216, D32 and D19 induce high amounts of interferon-alpha (IFN-alpha), tumour-necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12 in porcine peripheral blood mononuclear cells (PBMCs). Swine workshop cluster 3 (SWC3)1ow CD4high cells, with high IL-3-binding activity, representing NIPC, were the exclusive cytokine-producing cells responding to the CpG-ODN. These cells did not express CD6, CD8 or CD45RA. Importantly, monocyte-derived DC did not respond to CpG-ODN by secretion of IFN-alpha or TNF-alpha or by the up-regulation of costimulatory molecule expression. CpG-ODN up-regulated MHC class II and CD80\86 expression on the NIPC, but were unable to promote NIPC survival. Interestingly, certain CpG-ODN, incapable of inducing NIPC to secrete IFN-alpha or up-regulate MHC class II and CD80\86, did promote NIPC viability. Taken together, the influence of CpG-ODN on porcine NIPC, monocytes and myeloid DCs relates to that observed with their human equivalents. These results represent an important basis for the application of CpG-ODN as adjuvants for the formulation of novel vaccines and demonstrate the importance of the pig as an alternative animal model for this approach.

DEC-205 expression on migrating dendritic cells in afferent lymph.

Previous studies have identified a 210 000-molecular weight molecule expressed at a high level on the surface of dendritic cells (DCs) in afferent lymph of cattle and evident on cells with the morphology of DCs in lymphoid tissues. Expression is either absent from other immune cells or is present at a lower level. The molecular weight and cellular distribution suggested that the molecule, called bovine WC6 antigen (workshop cluster), might be an orthologue of human DEC-205 (CD205). To establish whether this was the case, the open reading frame of bovine DEC-205 was amplified, by polymerase chain reaction, from thymic cDNA (accession no. AY264845). The cDNA sequence of bovine DEC-205 had 86% and 78% nucleic acid identity with human and mouse molecules, respectively. COS-7 cells transfected with a plasmid containing the cattle DEC-205 coding region expressed a molecule that stained with WC6-specific monoclonal antibody, showing that ruminant WC6 is an orthologue of DEC-205. Two-colour flow cytometry of mononuclear cells from afferent lymph draining cattle skin, and from blood, confirmed the high level of expression on large cells in lymph that were uniformly DC-LAMP positive and major histocompatibility complex class II positive. Within this DEC-205+ DC-LAMP+ population were subpopulations of cells that expressed the mannose receptor or SIRPalpha. The observations imply that DCs in afferent lymph are all DEC-205high, but not a uniform population of homogeneous mature DCs.

Porcine peripheral blood dendritic cells and natural interferon-producing cells.

Peripheral blood contains two major particular infrequent dendritic cells (DC) subsets linking the innate and specific immune system, the myeloid DC and plasmacytoid DC equivalent to the natural interferon-producing cells (NIPC). The functional characterization of these cells demands large volumes of blood, making a large animal model more appropriate and beneficial for certain studies. Here, two subsets of porcine blood mononuclear cells expressing swine workshop cluster 3 (SWC3, a SIRP family member), are described and compared to monocytes. The blood DC specialized in T-cell stimulation were major histocompatibility complex (MHC) class II+, CD80/86+, CD1+/-, CD4-, and in contrast to monocytes CD14-. A CD16- and a CD16+ subset could be discriminated. Granulocyte-macrophage colony-stimulating factor and interleukin-3 were survival factors for this DC subset, and culture induced an up-regulation of MHC class II and CD80/86. The second subset described, are porcine NIPC, typically CD4++, MHC class IIlow, CD80/86low, CD1-, CD8-/low, CD16-/low and CD45RA-/low. Porcine NIPC had high interleukin-3 binding capacity, and survived in response to this cytokine. Their unique function was strong interferon type I secretion after virus stimulation. Both subsets were endocytically active when freshly isolated, and down-regulated this activity after in vitro maturation. Taken together, the present report has delineated porcine blood DC and NIPC, permitting a more detailed understanding of innate immune defences, particularly in response to infections.

Infection and transformation of dendritic cells from bovine afferent lymph by Theileria annulata.

Following incubation with sporozoites of the protozoan parasite Theileria annulata, dendritic cells (DC), extracted from bovine afferent lymph, became infected and transformed into large, rounded, continuously proliferating cell lines. Phenotypic analysis of the transformed cells by immunostaining and flow cytometry revealed that they expressed MHC class I and II antigens, the myeloid marker MyD (SIRP alpha) and the bovine WC6 (workshop cluster 6) molecule. Transformed DC cell lines differed from those produced from infection of macrophages and B cells in that some lines expressed CD21 and a proportion of cells continued to express the antigen stained by the mAb CC81, a marker which defines a subpopulation of DC in afferent lymph. Both of the main populations of DC that have been identified in bovine afferent lymph appeared to be equally permissive for infection and transformation with T. annulata. These findings raise the possibility that the transformed proliferating cells characteristic of in vivo infections could be derived from DC as well as macrophages. This could have consequences for understanding the pathogenesis of the disease and for developing methods to manipulate immune responses to eliminate the parasite.

Associations between immune system, growth and carcass variables in cattle

A range of haematological and immunological variables was measured in calves to assess relationships between these factors and effects of maternal and pre-weaning supplementation on meat quality. The ability of immunological and haematological measurements to predict both weight loss during transport and meat quality was also examined. Calves from supplemented cows had higher counts of lymphocytes bearing the γδ T cell receptor [workshop cluster (WC)1+ lymphocytes]. When cultured in vitro, lymphocytes from calves supplemented directly produced higher levels of the cytokine interferon (IFN)γ. These changes may reflect a greater resilience to the effects of stress associated with routine handling for weighing and blood sampling, or earlier maturation of the immune system in supplemented calves. Two calves from unsupplemented treatments died of calf diphtheria before weaning. During feedlot finishing, the prevalence of disease was low and did not differ between groups. The prevalence of cluster of differentiation (CD)4+ lymphocytes in blood was correlated with growth rates from weaning to slaughter and during feedlot finishing. Lymphocyte counts and the ratio of neutrophils to lymphocytes in blood collected either before or after transport was correlated with weight change during transport. These variables were not correlated with meat quality, however the numbers of lymphocytes after transport in the subset bearing the CD8 marker was correlated with the meat quality traits, tenderness, juiciness, flavour, overall likability, meat quality (MQ) 4 score and Meat Standards Australia (MSA) grade ratings. The results demonstrate that lymphocyte subpopulations are associated with growth rate, weight loss during transport, and meat quality. Their utility in predicting performance, stress tolerance, and meat quality therefore, warrants further investigation.

Intermediate stages in monocyte-macrophage differentiation modulate phenotype and susceptibility to virus infection.

The kinetics of monocyte-macrophage differentiation was analysed using two Swine Workshop Cluster (SWC) CD molecules: SWC1 and SWC9. Myeloid cells were selected by labelling for the common myeloid antigen, SWC3. Confirmation of macrophage identification used acid phosphatase and phagocytosis activities. During differentiation, SWC1 was gradually lost. SWC9 was absent on monocytes but up-regulated early. Consequently, monocytes were SWC1+ SWC9- and macrophages were SWC1- SWC9+. An additional, intermediate, cell population was identified as SWC1+ SWC9+. Size and granularity characteristics mirrored the monocyte, macrophage and intermediate-cell phenotypes. Overall, SWC9 up-regulation was central in macrophage differentiation and dependent on plasma factors. The concomitant loss of SWC1 was independent of these factors, but always associated with mature macrophages. Upon up-regulation of SWC9, the SWC1+ SWC9+ intermediate monocytic cells became susceptible to African swine fever virus infection. These results demonstrate the heterogeneity of monocytic cell differentiation and the importance of these characteristics for interaction with monocytotropic viruses.

Adhesion molecules and lymphocyte subsets in milk and blood of periparturient Holstein cows

Migration of leukocytes into the mammary gland is an essential element of resistance to infection which is likely influenced by expression of adhesion molecules. The contribution of subsets to mammary gland resistance remains unclear. Mononuclear cells from milk and blood of dairy cows were examined for variation in CD4 +, CD8 +, and WC1 + (Workshop Cluster 1; marker for γδ T cells) lymphocyte phenotypes and expression of LFA-1 and l-selectin at several time points during the periparturient period and at Week 16 of lactation. Proportions of CD4 + T cells were higher ( p ≤ 0.05) in blood than milk at all times between Week 0 and Week 16 relative to calving; the inverse was true of CD8 + cells. Expression of l-selectin was lower ( p ≤ 0.05) on CD4 + cells and higher on CD8 + cells from milk. The WC1 + subset was more frequent in blood than in milk except at calving when the opposite was true. After calving, proportions of l-selectin +WC1 + cells decreased steadily to Week 16. Expression of LFA-1 was examined on mononuclear cell populations and found to be lower on milk cells and did not vary over time. We conclude that proportions of T cells subsets differ significantly between blood and milk, particularly around calving. Corresponding variations in l-selectin expression may indicate a role for this molecule in regulating the movement of CD8 + and WC1 + T cells into the bovine mammary gland.

T-cell responses and the influence of dendritic cells in cattle

This chapter discusses the T-cell responses and the influence of dendritic cells in cattle. The major T-cell populations in cattle have been identified with monoclonal antibodies (mAbs) to differentiation antigens expressed by the cells produced and characterized in the laboratories of origin or within a series of international workshops that have been held. The nomenclature used follows the human canine distemper (CD) nomenclature where there is sufficient evidence to conclude that the homologous molecule is being identified in cattle and humans. If human CD homologs are not evident WC (workshop cluster) numbers were assigned to the mAb and molecules recognized. Thus cattle leukocytes are defined in terms of the CD or WC antigens expressed. A question, asked in relation to specific infections—that is central to vaccine design—is whether a particular T-cell population is primarily responsible for recovery from infection or immunity to reinfection. Differences among different infections would influence the appropriate strategy selected for immunization.

Porcine myelomonocytic markers: summary of the second international swine CD workshop

Forty five mAbs submitted to the Second International Swine CD workshop were analyzed by six different laboratories for their possible reactivity with porcine myelomonocytic cells using flow cytometry and immunohistochemistry. As a result of these analyses, a new swine workshop cluster, SWC9, composed of two mAbs that recognize an antigen selectively expressed on mature macrophages, was defined. In addition, several mAbs were identified, allowing the differentiation of granulocytes from monocytes/macrophages, or monocytes from macrophages. Further work is required to identify the antigen recognized by these mAbs. Nevertheless, they should already prove useful for the identification of different stages in the macrophage maturation/differentiation, and will certainly aid analyses on the complexity of the mononuclear phagocyte system in the pig. Finally, the cross-reactivity of three anti-human CD14 mAbs with porcine myelomonocytic cells was established in this workshop.

Monoclonal antibodies defining differentiation antigens of swine lymphoid and myeloid cells

Monoclonal antibodies recognising swine leucocyte antigens were identified and the corresponding antigens were characterised by determining their tissue distribution and molecular weight as well as immunohistochemical staining. On the basis of these data, we suggest that two antibodies are specific for a molecules within a procine orthologue of one of the human CD11 CD18 complexes. We suggest that two others recognise swine wCD21, that one may recognise swine wCD6 and that another recognises an antigen designated by the International Swine CD Workshop cluster SWC9. Other antibodies were obtained which are specific for swine macrophages or B cells, but CD or cluster assignments for these antigens have not been made. Detailed analysis of the antibodies is presented. It is proposed that these antibodies will be useful in studies of the pig immune system and of virus infection.

Identification of two distinct populations of dendritic cells in afferent lymph that vary in their ability to stimulate T cells.

Immunofluorescent staining and flow cytometric analysis of dendritic cells from cattle afferent lymph has established that within the afferent lymph veiled cells (ALVC) there are two phenotypically distinct, major populations. One is CD11a+, CD5+, CD21- and expresses the bovine WC10 (workshop cluster 10) molecule and the Ag recognized by mAb CC81 but is not recognized by mAbs CC149 and IL-A24. The second ALVC subpopulation is CD11a-, CD5-, CD21+/-, workshop cluster 10- and is not recognized by mAb CC81 but is recognized by mAb CC149. Thus, the two populations, which can be identified by staining for CD11a, are defined by the differential expression of a number of Ag. The ALVC populations had differing capacities to stimulate T cells. CD11a- ALVC were more effective at stimulating proliferative responses in allogeneic CD4+ T cells and CD8+ T cells. This was not related to binding of CTLA4Ig or CD40L fusion proteins, implying similar levels of expression of their ligands, CD80 and CD86 or CD40. Both subsets were able to present OVA to resting memory CD4+ T cells, indicating that both were able to take up and process soluble native protein. In contrast, the CD11a- ALVC were more effective in presenting respiratory syncytial virus Ag to resting CD4+ T cells. Considering the central role of dendritic cells in the initiation of immune responses in naive animals, the two cell types may have different roles in the induction of primary responses induced following infection or immunization.

Comparison of E-selectin-binding glycoprotein ligands on human lymphocytes, neutrophils, and bovine gamma delta T cells.

We compared E-selectin-binding cell surface ligands on bovine gamma delta T cells and human leukocytes using an E-selectin/Ig chimera. The chimera worked well in flow cytometric studies and showed that bovine gamma delta T cells were the only lymphocyte population in newborn animals that bound E-selectin chimera. Furthermore, the chimera blocked gamma delta T cell rolling on E-selectin. Chimera reacted with four potential glycoprotein ligands of 180, 200, 250, and 300 kDa in Western blot analysis of gamma delta T cell detergent lysates, and it specifically precipitated at least two of these E-selectin ligands (200 and 250 kDa) from lysates of cell surface biotinylated gamma delta T cells. Preclearing bovine gamma delta lysates of GD3.5 Ag and workshop cluster 1 did not abrogate E-selectin ligand precipitation, suggesting that these surface markers do not represent E-selectin ligands. Human neutrophils possessed three E-selectin-binding ligands of approximately 80 to 90, 130, and 230 kDa, while human lymphocytes variably possessed three ligands of 120, approximately 220 to 240, and 260 kDa. Cross-precipitation experiments confirmed the results of others that neutrophil L-selectin serves as the 80 to 90-kDa E-selectin ligand. The human lymphocyte approximately 220 to 240-kDa and 260-kDa ligands may be analogous to the bovine gamma delta T cell molecules, whereas the 120-kDa is unique to human cells. The identities of the human and bovine lymphocyte E-selectin ligands are unknown. Finally, E-selectin ligand-1 apparently may have a minimal role, if any, in lymphocyte/E-selectin interactions, since a polyclonal anti-E-selectin ligand-1 serum stained a minimal number of cutaneous lymphocyte-associated Ag-positive human lymphocytes and bovine gamma delta T cells.

Porcine bone marrow myeloid cells: phenotype and adhesion molecule expression.

The purpose of this study was the discrimination of porcine bone marrow hematopoietic cell (BMHC) subpopulations and cell maturation stages by using cluster of differentiation (CD) and Swine Workshop Cluster (SWC) molecules, combined with cell size, granularity, and the capacity to generate an oxidative burst. SWC3 was the earliest myeloid marker identified in the differentiation pathways. Through SWC3/SWC8 double labeling, three populations committed to the myeloid lineage were discriminated: early myeloid progenitors, which were SWC3lowSWC8-; cells committed to the granulocytic lineage, which were SWC3+SWC8+; monocytic cells, which were SWC3+(-)SWC8-. The SWC3lowSWC8- myeloid progenitors expressed high levels of CD44 and CD49d, but no CD14. Among the SWC3+SWC8+ granulocytic cells, the most immature were CD14-CD11a/18-CD44low, an intermediate stage was CD14+CD11a/18lowCD44-, and the most mature were CD14+CD11a/18high-CD44high. Only the latter could generate a rapid oxidative burst. Moreover, during granulocytic maturation, a constant decrease in CD49d/29 expression was observed. In contrast, the SWC3+SWC8- monocytic cells were mainly CD14+ and expressed high levels of the adhesion molecules. These results define porcine hematopoietic differentiation and maturation stages. Similarities in human and porcine adhesion molecule expression have been identified, suggesting common functionalities in the regulation of hematopoiesis and the potential of the pig as a model for hematopoietic studies.

5531943 Method of making a carbon/metal composite

Further studies were carried out on the monoclonal antibodies (mAbs) from the First International Swine CD Workshop which react with γδ Null T-lymphocytes, defined by the binding of mAb w020141 (MAC320) as Swine Workshop Cluster number SWC6. Studies were also carried out on several other mAbs from the same workshop which identify other CD antigens, but whose binding is not restricted exclusively to γδ Null T-lymphocytes. The first group consists of 11 mAbs (w021, w022, w059–w065, w105 and w117) and the second group of 18 mAbs (w008, w026, w056, w067–w071, w080, w091–w094, w110, w111, w118, w119 and w121). All mAbs were characterised by binding to peripheral blood lymphocytes (PBL) from normal, sham-thymectomized (STx) and thymectomized (Tx) pigs of the Babraham SLAbb line and by their overlap, using two-colour immunofluorescence with biotinylated mAb MAC320 (w020141, which identifies all γδ Null cell T-lymphocytes and the Null cell subpopulation identified by MAC319 (w021). The Null cell-specific mAbs were also used in inhibition studies of MAC319 and MAC320 binding and by staining PBL with pairs of mAbs together with either MAC319 or MAC320. Based on these data we suggest a putative relationship of the Null cell subsets defined by these mAbs with each other.

5597074 Packing tray of semiconductor devices and a method for manufacturing the same: Ko Seung S Seoul, Korea

Further studies were carried out on the monoclonal antibodies (mAbs) from the First International Swine CD Workshop which react with γδ Null T-lymphocytes, defined by the binding of mAb w020141 (MAC320) as Swine Workshop Cluster number SWC6. Studies were also carried out on several other mAbs from the same workshop which identify other CD antigens, but whose binding is not restricted exclusively to γδ Null T-lymphocytes. The first group consists of 11 mAbs (w021, w022, w059–w065, w105 and w117) and the second group of 18 mAbs (w008, w026, w056, w067–w071, w080, w091–w094, w110, w111, w118, w119 and w121). All mAbs were characterised by binding to peripheral blood lymphocytes (PBL) from normal, sham-thymectomized (STx) and thymectomized (Tx) pigs of the Babraham SLAbb line and by their overlap, using two-colour immunofluorescence with biotinylated mAb MAC320 (w020141, which identifies all γδ Null cell T-lymphocytes and the Null cell subpopulation identified by MAC319 (w021). The Null cell-specific mAbs were also used in inhibition studies of MAC319 and MAC320 binding and by staining PBL with pairs of mAbs together with either MAC319 or MAC320. Based on these data we suggest a putative relationship of the Null cell subsets defined by these mAbs with each other.