monoclonal antibodies to the sheep analogues of human cd45 (leucocyte common antigen), mhc class i...

Veterinary Immunology and Immunopathology, 24 ( 1990 ) 331-346 331 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Monoclonal Antibodies to the Sheep Analogues of Human CD45 (Leucocyte Common Antigen), MHC Class I and CD5. Dif ferent ia l Express ion after Lymphocyte Act ivat ion In Vivo

JOHN HOPKINS and BERNADETTE M. -DUTIA

Department of Veterinary Pathology, University of Edinburgh, Summerhall, Edinburgh EH9 1QH (Great Britain)

(Accepted 12 October 1989)

ABSTRACT

Hopkins, J. and Dutia, B.M., 1990. Monoclonal antibodies to the sheep analogues of human CD45 (leucocyte common antigen ), MHC class I and CD5. Differential expression after lymphocyte activation in vivo. Vet. Immunol. Immunopathol., 24: 331-346.

This paper describes three anti-sheep monoclonal antibodies. The tissue distribution and apparent molecular weight of the antigens detected by these antibodies is consistent with them reacting with sheep leucocyte common antigen (CD45 (VPM18)), MHC class I (VPM19) and CD5 (VPM29). An ELISA method is described that permits the cross-reactivity of different antibodies to be assessed, this confirms the identity of the antigens detected by VPM18, VPM19 and VPM29. This method is also of value as either a positive or a negative screen in the construction of further monoclonals.

A study of the expression of these three antigens on efferent lymph small lymphocytes and antigen-activated lymphoblasts shows that the density of CD45 on lymphoblasts (activated either in vivo or in vitro ) is approximately half that of small lymphocytes whereas the density of MHC class I is the same in both populations. Furthermore, about 75% of small lymphocytes express CD5 but less than 10% of lymphoblasts are positive. Cell membrane CD5 expression is lost on lymphocyte activation. It does not seem to be linked to cell membranes via phosphatidylinositol and the loss is not due to the breaking of that link.

ABBREVIATIONS

FITC, fluorescein isothiocyanate; FSC, forward scatter; LCA, leucocyte common antigen; MCN, modal channel number; OVA, ovalbumin; PBL, peripheral blood leucocytes; PBM, peripheral blood mononuclear cells; PIPLC, phosphatidylinositol phospholipase C; PL-C, phospholipase C; PMSF, phenyl methyl sulphonyl fluoride; PPD, purified protein derivative of tuberculin; SDS- PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SSC, side scatter; TNT, tris- NaCI-Triton Xl00 lysis buffer.

0165-2427/90/$03.50 © 1990 Elsevier Science Publishers B.V.

332 J. H O P K I N S AND B.M. DUTIA

INTRODUCTION

Different functional cell populations can be defined by the different cell membrane glycoproteins they express. In both man and mouse these molecules have been biochemically defined using monoclonal antibodies and these reagents have provided much information on the relationship between various cell types and their maturation pathways. Increasingly they are becoming in- valuable tools for the study of modern veterinary immunology and pathology. In this paper we describe monoclonal antibodies to sheep analogues of CD45 (leucocyte common antigen), MHC class I and CD5. They are characterised by the distribution of their antigens in the sheep lymphoid system as well as the molecular weight of the molecules which they precipitate. We also describe a method for immunopurification of these antigens which has facilitated their characterisation and will be useful in the construction and screening of other monoclonals. These antibodies have been shown to define similar antigenic moieties to those described by the Melbourne group (Gogolin-Ewens et al., 1985; Mackay et al., 1985; Maddox et al., 1985b ), and are therefore important in confirming their specificity and extending the range of reagents available in the sheep.

This paper also illustrates our approach to the study of lymphoid cell biology in the sheep which integrates the use of characterised monoclonal antibodies with lymphatic cannulation. The main advantage of this system is that it al- lows access to in vivo fractionated cell populations from distinct lymphoid compartments as well as constant monitoring of the changes that occur in antigen-stimulated lymphoid tissue. Using this approach we show that cellular activation of efferent lymph cells draining in vivo challenged lymph nodes results in changes in cell phenotype. There are quantitative changes in the density of CD45 and MHC class I expression by blast cells. Cellular activation also results in an almost total loss of CD5 expression.

MATERIALS AND METHODS

Animals and surgery 1-2-year-old Finnish-Landrace sheep were obtained from the Moredun Re-

search Institute. Sheep were antigenically primed by intradermal (i.d.) injection of 5 human doses of BCG (Glaxo, Greenford, Middx., U.K. ) and by subcutaneous injection of 1 mg (ovalbumin (OVA) in complete Freunds adjuvant. Cannulation of efferent lymphatics draining sheep prefemoral lymph nodes was as described by Hall (1967). Lymph was collected quantitatively into sterile, plastic bottles containing 100 iu heparin. Sheep were allowed at least 7 days post-operative recovery prior to the start of the experiment. Antigenic stimu- lation of the cannulated prefemoral was by i.d. injection of 50 ]tg of either PPD (Batch 297; Central Veterinary Laboratories, New Haw, Weybridge, Surrey)

MONOCLONAL ANTIBODIES TO SHEEP ANALOGUES OF HUMAN CD45, MHC CLASS I AND CD5 333

or OVA in sterile PBS. Female BALB/c mice were obtained from the Dept. of Veterinary Pathology breeding colony.

Cell and tissues Sheep thymus, spleen, lymph nodes and ileal Peyer's patches from 10-month-

old lambs were taken post-mortem. Thymocytes were prepared by teasing thymus tissue into a single-cell suspension and purified using Lymphoprep (Nygaard, Oslo). Peripheral blood leucocytes were isolated by ammonium chloride lysis of heparinised venous blood (Mishell and Shiigi, 1980). In vivo activation was carried out by culturing Lymphoprep purified peripheral blood mononuclear cells (at 1 X 106/ml) for 24 and 48 h with the mitogen concana- valin A (Sigma, Poole, Dorset) at 5 pg/ml.

Production o[ monoclonal antibodies 8-week-old BALB/c mice were immunised by subcutaneous injection of

2 × 106 sheep thymocytes emulsified in complete Freunds adjuvant and boosted with 1 × 106 cells intraperitoneally 3 times at 2-week intervals. Four days be- fore the fusion they were injected with 1 × 10 ~ cells intravenously. Fusion of spleen cells with the myeloma NSO was as described by Galfre et al. (1977). After HAT selection supernatants were screened for antibody by indirect im- munofluorescence. Selected lines were cloned, first by soft agar and then by limiting dilution methods.

Immunoglobulin isotype analysis Isotype analysis of the twice cloned monoclonal antibody supernatants was

by double immunodiffusion precipitation using class- and subclass-specific anti- mouse Ig antisera (Serotec, Bicester, U.K. ). All three monoclonal antibodies were shown to be IgG1 isotype.

Immunohistology 8 pm cryostat sections were cut from fresh tissues and snap-frozen in liquid

nitrogen. Immunohistology of efferent lymph cells was done using cytospin smears. Air-dried sections or smears were fixed in dry acetone and stained with the antibodies using the standard indirect immunoperoxidase method. SBU- T4 and SBU-T8 {Maddox et al., 1985a) were used to detect CD4 and CD8 positive cells, VPM8 (a mouse monoclonal to sheep light chains) was used to detect B cells.

Flow cytometry 2 × 106 washed efferent lymphocytes or peripheral blood leucocytes were in-

cubated for 60 min at 20 ° C with 50 pl of 1/1000 dilution of monoclonal ascitic fluid (in 0.1% BSA, 0.01 M sodium azide in PBS). Cells were washed and incubated for a further 60 min in the appropriate dilution of FITC-conjugated


sheep anti-mouse Ig (in PBS/BSA/azide) . Double staining experiments were performed by using biotinylated SBU-T4 and SBU-T8 (IgG2~ isotype) with phycoerythrin-conjugated streptavidin (Serotek) and VPM29 (IgG1 isotype) with FITC-conjugated anti-mouse IgG, The experiments to examine the quantitative expression used directly FITC-conjugated IgG of each monoclonal antibody at 10 #g/ml. Quantitation of antigen expression was as described by Hopkins et al. (1989) using a FACScan cell analyser (Becton-Dick- enson). Small lymphocytes and lymphoblasts were analysed separately by 'live gating', being distinguished by their FSC/SSC profiles (Fig. 1 ). The gain set- ting for FL1 was adjusted when analysing the two different cell populations so that the auto fluorescence peaks obtained with the 1/500 normal mouse serum controls were exactly coincident.

Cross-blocking studies These experiments were done to establish if VPM18, VPM19 and VM29

reacted with the same epitopes as the existing anti-sheep monoclonals. 1 × 106 sheep efferent lymphocytes were incubated at room temperature with 25 ,ul of a titration (diluted in HBSS, 1% BSA, 0.01% sodium azide) of competing monoclonal antibody ascitic fluids. After 30 min, 25 ~l of FITC-conjugated test monoclonal IgG at 10 #g/ml was added. The samples were incubated for a further 30 min, washed three times and then fixed with 0.25% paraformalde- hyde. Cells were analysed by flow cytometry as described above. The competing monoclonal antibodies tested were: SBU-LCA (anti-CD45), SBU-I (anti- MHC Class I) and SBU-T1 and ST la (anti-CD5) {Beya et al., 1986).

Phospholipase treatment of efferent lymphocytes This was done in order to assess if VPM29 antigen was linked to the cell

membrane via a phosphatidyl inositol linkage. Efferent lymphocytes were washed twice in RPMI1640/ l% BSA/0.01% sodium azide and incubated for 60 min at 37 °C with either phosphatidyl inositol phospholipase C (Sapporo Breweries Ltd, Shizuoka, Japan) or phospholipase C (Type XIV Sigma). Phenylmethylsulpholyl fluoride (PMSF) was present at 0.2 mM during the digestions to help prevent non-specific serine-protease activity. The cells were at 2 × 107/ml and were incubated with 5 un i t s /ml of enzyme. After incubation the cells were washed, stained with the antibodies as described above and then analysed by flow cytometry. The control antibodies used were VPM19 and VPM56. VPM56 is an IgG1 monoclonal antibody specific for resting sheep T cells that is linked via phosphatidyl inositol.

Immunoprecipitation of VPM18 and VPM19 antigens Thymocytes and splenocytes were prepared from fresh sheep thymus and

spleen by centrifugation over Lymphoprep. A mixture of 2 × 107 thymocytes and 2 × 107 splenocytes were resuspended in 0.1 ml 0.1 M borate buffer, pH 9.0, 0.1 M NaC1 and iodinated by the iodogen method using 500 ttCi 125I. After


completion of the reaction, the cells were lysed on ice in 0.5 ml 20 mM Tris- HC1, pH 8.0, 150 m M NaC1, 0.5% Triton X100 (TNT) incorporating PMSF at 0.2 mM, the nuclei were pelleted and the lysate was fractionated on Sepha- dex G25 to remove free iodine. The lysate was precleared by incubation overnight with Sepharose 4B beads and immunoprecipitations containing 2 X 10 ~ cpm lysate and 5 ~1 VPM18, 19 or normal mouse ascitic fluid were incubated overnight at 4 o C. Immune complexes were precipitated with sheep anti-mouse Ig conjugated to Affigell0 (Biorad). The precipitates were washed ×5 with TNT, and eluted with SDS gel sample buffer, (125 m M Tris-HC1, pH 6.8, 20% glycerol, 2% SDS, 5% 2fl-mercaptoethanol, 0.04% bromophenol blue) and fractionated on 5-15% or 5-20% polyacrylamide gels (Laemmli, 1970). Gels were stained using Coomassie blue, dried and visualised by autoradiography at - 70 ° C. Electroblotting of SDS-PAGE separated cell proteins was done as described by Hopkins et al., 1986.

Affinity purification and ELISA of CD5, LCA and class I Purification of the antigens recognised by VPM18, 19 and 29 was carried

out as described by Turkewitz et al. (1983) and Hopkins et al. (1986). Eluted antigen was mixed with an equal volume of SDS elution buffer, boiled for 5 min then fractionated on SDS-polyacrylamide gel electrophoresis as above. Gels were visualised by silver staining (Morrisey, 1981 ). For the ELISA antigen was coated onto ELISA plates (Dynatech) by 18 h incubation at 4°C of the eluates diluted at least 1/10 in 0.1 M N a H C Q pH 9.5. After washing in PBS/0.1% Tween 20 the plates were blocked for 30 min in 1% BSA in P B S / Tween. Monoclonal antibody (dilutions of tissue culture supernatant) was added and incubated for 60 min at 20 ° C. After washing the plates were incubated in the appropriate dilution of peroxidase coupled sheep anti-mouse Ig for 60 min. The reaction was finally visualized using orthophenylenediamine/ H 2 0 2 .

R E S U L T S

Tissue distribution of VPM18, 19 and 29 antigens The tissue distribution of VPM18, 19 and 29 was investigated by flow cyto-

metry and immunohistology. VPM 18 and 19 always react with > 98% of lymphocytes isolated from either peripheral blood or efferent lymph while VPM29 stains 59% (49-66%) in PBM and 76% (68-89%) in efferent lymph. Double staining flow cytometry showed that both the SBU-T4 (anti-CD4) and SBU- T8 (anti-CD8) were coexpressed with VPM29 (data not shown). VPM18 and 19, in contrast to VPM29, also react with the high FSC/SSC granulocyte population in peripheral blood. RBCs are negative for all three antibodies.

Table 1 illustrates the distribution within lymphoid tissue of the antigens recognised by these monoclonals. VPM18 reacts with leucocytes in all tissues

336 J. HOPKINS AND B.M. DUTIA

TABLE1

Tissue distribution of antigens recognised by VPM18, 19 and 29, anti-sheep lymphocyte differentiation antigens

Tissue Percentage of cells labelled with:

VPM18 VPM19 VPM29

Thymus >98 (98-100) 15 (12-20) 85 (82-90) Spleen > 98 ( 98-100 ) > 98 (98-100 ) 47 (41-54) Lymph node > 98 (98-100) > 98 (98-100) 66 (61-70) Ileal Peyers patch > 98 (98-100) > 98 (98-100) < 5 (2-6) Efferent lymph > 98 (98-100) > 98 (98-100) 76 (68-89) PBL > 98 (98-100) > 98 (98-100) 39 (32-45) PBM > 98 (98-100) > 98 (98-100) 76 (68-89)

Single cell suspensions of thymus, spleen, prefemoral lymph node and ileal Peyer's patch were made by teasing cells into RPMI 1640 and purified using Lymphoprep. Peripheral blood leucocytes (PBL) were prepared by ammonium chloride lysis of heparinised blood and included high FSC/SSC scattering cells. Peripheral blood mononuclear cells (PBM) were prepared from defi- brinated blood purified using Lymphoprep and contained only low FSC/SSC scattering cells. The percentages of positive cells were assessed by flow cytometry, gating the 1/500 dilution of normal mouse serum negative control as less than 1% positive.

tested, non-leucocytes are negative. There is little anatomical localisation within the thymus, spleen and lymph nodes, although within ileal Peyers patches staining is limited to the follicles and in the lungs to the interstitial and alveolar macrophages. In contrast VPM19 is not leucocyte specific, reacting with all tissues indiscriminately with the exception of the thymic cortex and the brain. VPM29 reacts only with lymphocytes, recognising mainly cortical thymocytes, paracortical lymph node cells and the periarteolar lymphoid sheath of the spleen. VPM18, 19 and 29 show identical tissue distributions to SBU-LCA (anti-CD45), SBU-I (anti-MHC class I ) and SBU-T1 (anti-CD5), respectively. The reactivity of these antibodies was always compared with the panel of anti-sheep lymphoid monoclonals obtained from the University of Melbourne, Australia (Gogolin-Ewens et al., 1985; Mackay et al., 1985; Mad- dox et al., 1985b). We have no evidence for these monoclonals reacting with polymorphic determinants as they bind to cells obtained from all sheep so far tested.

Expression of VPM18, 19 and 29 antigens on antigen-activated cells The changes in lymph cell expression of the antigens recognised by VPM18,

19 and 29 were examined by enumerating the numbers of positive cells in efferent lymph following in vivo antigenic challenge. Different physical param- eters (FSC/SSC coordinates) were used to discriminate small lymphocytes and activated lymphoblast cells. Small lymphocytes are characterised by a low


FSC/SSC profile and activated blasts have a high FSC/SSC scatter profile (Fig. 1). Small lymphocytes have a diameter of approximately 6.5 #m (6-7 /~m) and blasts approximately 9/zm (6.5-11 #m). The surface area of the small lymphocytes and blasts is approximately 130/~m ~ and 260/~m ~.

Fig. 2 compares the relative expression of the antigens recognised by VPM18, 19 and 29 on 'live gated' small lymphocytes and lymphoblasts present in efferent lymph 4 days after in vivo antigenic challenge. It is clear that the density of VPM18 antigen expressed on the blast cell population is considerably less than on small lymphocytes, the modal channel number (MCN) for the blasts being 54 (48-56 in four experiments) while the MCN for small lymphocytes is 132 (125-140). A difference in 76 channels represents a doubling in fluorescence intensity. The density of VPM 19 antigen expression on the two cell populations is approximately equal while the VPM 29 antigen is virtually absent from the blast cells. Less than 10% of blast cells express low levels of this molecule while greater than 70% of small lymphocytes are positive. Expression of CD4 and CD8 by the two populations remains relatively unaltered (data not shown). These experiments were also done using cells stimulated for 24 and 48 in vitro with Con A. In common with the in vivo activated cells, blasts produced in vitro lose cell surface CD5 although they retain CD4 and CD8 (data not shown). As with the tissue distribution these experiments were also done with SBU-LCA, SBU-I and SBU-T1 which gave almost identical results to VPM18, 19 and 29 respectively.

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L I N E A R F S C

Fig. 1. Flow cytometry scatter profile of sheep efferent lymph cells 4 days after in vivo antigen activation. Small lymphocytes are distinguished by their characteristic low forward scatter (FSC) and side scatter (SSC), lower box. Lymphoblasts are distinguished by their characteristic high FSC/SSC, upper box.

338 J . H O P K I N S A N D B . M . D U T I A

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Fig. 2. Flow cytometry histograms of efferent lymph cells taken 4 days after secondary in vivo ant igen challenge. Cells are stained with VPM18, VPM19 or VPM29. Solid line ( ) shows small lymphocytes, dot ted line ( - ' . ) shows lymphoblasts and dashed line ( --- ) is the 1/500 normal mouse serum control on blasts. The negative control peak for small lymphocytes is exactly coincident with the negat ive blast peak.

Cross-reactivity with other monoclonal antibodies Immunopurified antigen eluted at high pH in the presence of 0.5% sodium

deoxy-cholate (DOC) was used as antigen for a standard indirect ELISA as- say. Adherence of the antigen to the polystyrene ELISA plate was effective only after dilution of the eluate to at least 1/10 (i.e. DOC concentrations of


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Fig. 3. Antibody binding curves showing the binding of monoclonal antibodies to immunopurified VPM18 antigen immobilised onto ELISA plates. The binding of mouse monoclonal antibody was detected using peroxidase-conjugated anti-mouse Ig developed with OPD/H202.0D495 was read using Titertek Microelisa.

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Fig. 4. Autoradiograph of SDS-polyacrylamide gel electrophoresis gel showing immunoprecipitation from 125I-labelled spleen and thymus cells with the monoclonals VPM19 (gel A, 5-20% acrylamide gradient gel) and VPM18 (gel B, 5-15% acrylamide gradient gel). Molecular weight markers ( × 1000) are as indicated.

less than 0.05% ). After 18 h incubation of the antigen dilutions it was shown that homologous antibody reacted with the purified antigens, antibodies with obviously different specificities did not react. A comparison of VPM18, 19 and


29 with the Melbourne panel of antibodies showed clearly that SBU-LCA recognised the same antigen as that purified by VPM18, SBU-I was the same as VPM19 and SBU-T1 was equivalent to VPM29 (and STla) . The T cell specific monoclonals SBU-T4, T8 and T19 as well as the anti-MHC class II monoclonal antibodies SBU-II, SW73.2 (Hopkins et al., 1986) and VPM4, 16, 36, 37, 41 and 47 (Dutia et al., 1989) did not react to the VPM18, 19 and 29 antigens. Representative data showing the reactivity of VPM18 and SBU-LCA with purified VPM18 antigen is shown in Fig. 3.

Although VPM18, VPM19 and VPM29 react with the same molecules as SBU-LCA, SBU-I and SBU-T1 (and STla) respectively the cross-blocking experiments showed that they bind to totally different epitopes (data not shown).

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Fig. 5. Si lver s t a i n ed P A G E gel ( 5 - 2 0 % acry lamide g rad ien t ) of an t i gens i m m u n o p u r i f i e d f rom de te rgen t lysate of sheep sp lenocytes w i th t he monoc lona l an t ibodies , V P M 1 9 and V P M 2 9 . Mo- lecular weight m a r k e r s ( × 1000 ) are as indica ted .

M O N O C L O N A L A N T I B O D I E S T O S H E E P A N A L O G U E S O F H U M A N CD45, M H C C L A S S I A N D CD5 341

Immunochemical characterisation of antigens The antigen immunoprecipitated from 125I-labelled mixed spleen and thy-

mus cell lysate with VPM18 had at least four distinct bands with apparent molecular weights between 180 000 and 220 000 (Fig. 4B). VPM18 did not recognise antigen immobilised on nitrocellulose after electroblotting from SDS- PAGE, although polyclonal antiserum from mice immunised with immunopurified VPM18 antigen blotted to bands of the same molecular weight as those seen by immunoprecipitation (data not shown). In contrast the molecules immunoprecipitated by VPM19 had apparent molecular weights of 44 000 and 12 000 (Fig. 4A). By electroblotting VPM19 was found to be specific for the 44 000 molecular weight MHC class I heavy chain.

Consistent immunoprecipitation results using 125I-labelled cells were not obtained with VPM29. The immunochemical characterisation of VPM29 antigen was investigated by using silver stained gels of immunopurified antigen. Fig. 5 shows that the VPM29 antigen has an apparent molecular weight of 67 000, using the same methods VPM19 antigen has the same molecular weights as

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Fig. 6. Flow cytometry profiles of sheep efferent lymph lymphocytes stained with the monoclonal antibodies VPM29 and VPM56. The anti-class I antibody VPM19 is used as the control. Cells had been incubated either PIPLC or PL-C prior to reaction with antibody.


that seen using radiolabelled lysate (Fig. 4). VPM 29 does not recognise blotted antigen.

Phospholipase treatment of lymphocytes Many of the cell surface glycoproteins that cease to be expressed on cellular

activation (e.g. Thy-1, TAP and LFA-3) are linked to the cell membrane via phosphatidyl inositol and are lost because of the action of the specific phospholipase, phosphatidyl inositol phospholipase C (PIPLC). Indeed the action of this enzyme and the loss of the molecules can be the initial trigger for activation (Low and Kincade, 1985). The loss of sheep CD5 by activated T cells is similar to that of Thy-1 and TAP and specific enzyme digestions were done to investigate if CD5 is linked via phospatidyl inositol. Cells were incubated with PIPLC, phospholipase C (PL-C) or in the absence of enzyme and then examined for the expression of CD5. Fig. 6 shows that neither enzyme had any significant effects on CD5 expression. In contrast the VPM56 antigen was lost by the action PIPLC but not PL-C showing that the PIPLC had active enzy- matic activity. CD5 does not seem to be linked to cell membranes via phosphatidyl inositol.

DISCUSSION

This paper describes three mouse monoclonal antibodies that define distinct cell surface molecules of sheep lymphoid cells. The tissue distribution of VPM18, VPM19 and VPM29, together with the biochemical characteristics of the antigens with which they react, suggests that they are specific for the sheep analogues of human CD45 (leucocyte common antigen ), MHC class I and CD5 (T1) respectively.

The antigen recognised by VPM18 is present on all cells of the lymphocyte, granulocyte, dendritic cell and macrophage lineages but is absent on non-mar- row derived cells. The VPM18 epitope seems to be common to all the CD45 molecules as at least four bands are immunoprecipitated by this antibody, which have apparent molecular weight of between 180 000 and 220 000. VPM18 does not discriminate between the higher and lower molecular weight members of the family. This is very similar to the 'conventional' anti-CD45 described in other species (Fabre and Williams, 1977; Scheid and Triglia, 1979; Dalchau et al., 1980; Maddox et al., 1985b).

The evidence that the antigen recognised by VPM19 is MHC class I is three- fold: firstly this monoclonal reacts with most cells in the body with the exception of the thymic cortex and the brain; secondly it immunoprecipitates molecules of apparent molecular weight 44 000 and 12 000 which are equivalent in size to the heavy chain of class I and to fl2-microglobulin respectively, and lastly immunopurified VPM19 antigen is recognised specifically by the other anti- sheep MHC class I monoclonal, SBU-I (Gogolin-Ewens et al., 1985). VPM19


seems to react with a monomorphic determinant of class I as it has reacted with the cells of every sheep so far tested.

By the same criteria VPM29 reacts with sheep CD5. It is specific for sheep thymocytes and the majority of peripheral lymphocytes and its antigen has an apparent molecular weight of 67 000. Although immunoprecipitation of 12~I- labelled antigen proved unreliable with VPM29 its antigen was successfully purified by affinity chromatography and examined by standard protein staining of PAGE gels. This technique is very valuable as it yields purified antigen. Immunopurified antigen from VPM18, 19 and 29 affinity columns was used to assess the relative identify of these monoclonals with the Melbourne panel of antibodies. By this method it was found that the following pairs of antibodies recognised identical antigens; VPM18 and SBU-LCA, VPM19 and SBU-I, VPM29 and SBU-T1.

The possession of immunopurified antigen also facilitates the production of additional monoclonal antibodies. This can be by either negative or positive selection. ELISA based on abundant antigens (e.g. CD45, MHC class I and CD5 ) can be used in secondary screening assays of fusions in order to eliminate monoclonal antibodies with these specificities, concentrating time and effort on those monoclonals with different and unknown specificities. Alternatively, monoclonal antibodies are required that have certain subspecifities. For ex- ample, immunopurified CD45 (using a 'conventional' anti-CD45 antibody like VPM18) can be used as immunising and primary screening antigen for the production of antibodies to both the higher (CD45R) and lower (UCHL1 equivalent) molecular weight molecules that define particular functional lymphocyte subsets (Morimoto et al., 1985; Mossman et al., 1986; Dalchau et al., 1987; Smith et al., 1986). In addition antibodies with definite characteristics (e.g. for use in immunoblots or reaction with paraffin-embedded tissue) can be selected for particular diagnostic purposes.

We have also demonstrated by flow cytometry that there is differential expression of these antigens on resting and activated cells. It seems that there is no de novo expression of CD45 after activation and as a consequence of activation (and hence increase in cell size) the density of cell surface CD45 expression is reduced. This is reflected in the MCN of the CD45 positive small lymphocytes cells being approximately 70 channels greater than that of the activated blasts (double the fluorescence intensity). The small lymphocytes and blasts have mean diameters of about 6.5/lm and 9/~m respectively. The surface area of the blasts is therefore about twice that of small lymphocytes. In direct contrast is the expression of MHC class I, where the density of expression is approximately the same for the two cell populations. The significance of this is that there has been a two-fold increase in class I expression on cellular activation. This is very similar to that found for MHC class II (Hopkins et al., 1989). The situation with CD5 is very different. Activated peripheral T cells lose CD5 expression, so that there are large proportions of CD4 + C D 5 - and


CD8 + CD5 - blast cells in efferent lymph after in vivo activation of the lymph node. Furthermore, there is also loss of the CD5 antigen after in vitro stimu- lation of PBM with Con A. These data show that the CD5 molecule in sheep is lost on cellular activation. It may explain why the total numbers of CD4 +, CD8+ and T19+ (Mackay et al., 1985) in the peripheral blood of Border disease infected sheep (Burrells et al., 1989) or in lesions produced by the parasites Haemonchus (Gorrell et al., 1988a) and Trichostrongylus (Gorrell et al., 1988b) far exceed the numbers of CD5 + cells whereas in resting lymphoid populations CD4÷, CD8÷ and T19÷ cells all express CD5 (Mackay et al., 1988). This loss of CD5 expression by mature activated T cells contrasts with the expression of CD5 by proliferating immature, cortical thymocytes. CD5 on thymocytes has apparent molecular weight of 62 000 as well as the more usual 67 000 (Mackay et al., 1985) which suggests that CD5 exists in two forms. Although the loss of CD5 expression after activation is similar to TAP (Reiser et al., 1986), Thy-1 (Low and Kinkade, 1985) and LFA-3 (Dustin et al., 1987) it differs by not being linked to cell membrane via phosphatidylinositol.

These three new monoclonal antibodies to sheep lymphocyte differentiation antigens confirm the data obtained with existing reagents. In this study they have been used to study the physiological changes which occur in normal tissue, similar studies are now being done to analyse immune responses in disease states.

ACKNOWLEDGEMENTS

Our thanks to Mr. Alan Ross, Mr. Brian Kelly and Mrs. Esme Mills for excellent technical assistance. We are grateful to Prof. Ian McConnell for discussion on the data. This work was funded by the Agricultural and Food Re- search Council (Linked Research Group 31) and The Wellcome Trust.

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monoclonal antibodies to the sheep analogues of human cd45 (leucocyte common antigen), mhc class i...

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