Veterinary Immunology and Immunopathology, 39 (1993) 49-59 49 0165-2427/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All fights reserved

Section 5

Summary of workshop findings for sheep

5.1 Summary of workshop findings of leukocyte antigens in sheep

John Hopkins*, Alan Ross, Bernadette M. Dutia Department of Veterinary Pathology, University of Edinburgh, Edinburgh, UK

Introduction

The complete range of monoclonal antibodies (mAbs) submitted to the Second Workshop on Differentiation Antigens on Bovine and Ovine Leuko- cytes was screened for reactivity to sheep leukocytes. The mAbs were tested by flow cytometry, using sheep peripheral blood lymphocytes and granulo- cytes, alveolar macrophages, afferent lymph lymphocytes and dendritic cells and efferent lymph small lymphocytes, and by immunohistology using thy- mus, lymph nodes, spleen and Peyer's patch. Of the 189 mAbs submitted to the workshop 89 reacted with sheep leukocytes. In addition, selected mAbs submitted to the First Workshop on Leukocyte Differentiation Antigens in Cattle, Sheep and Goats ( 1989 ) were included as comparisons.

Conventional cluster analysis was not possible on sheep tissues alone as only two laboratories (INRA and UEDIN ) presented data on sheep and only one (UEDIN) submitted full flow cytometry data. The cluster analysis in sheep therefore utilized the cattle cluster analysis, using those mAbs which cross-reacted between the two species. In addition, specific well-characterized antisheep mAbs were used which had been included, and clustered, in the First Workshop.

Fourteen well-defined clusters of differentiation (OvCD) were identified as were six workshop clusters (OvWC). In addition, there were more than 40 mAbs which could not be clustered.

*Corresponding author.

50 Z Hopkins et aL / Vet. ImmunoL ImmunopathoL 39 (1993) 49-59

Materials and methods

Monoclonal antibodies (mAbs)

The mouse mAbs were submitted to the workshop in a number of forms: crude ascitic fluid, purified antibody or tissue culture supernatant. The dilu- tions used for each antibody were as directed by the submitting laboratory.

Cells and tissues

Peripheral blood was obtained by percutaneous jugular venipuncture using heparin sulphate at 5 iu ml- 1 as anticoagulant. Leukocytes were isolated by ammonium chloride lysis (Mishel and Shiigi, 1980). Efferent and afferent lymph was collected by chronic cannulation of the prefemoral efferent (Hall, 1967) and pseudoafferent lymphatics respectively (Hopkins et al., 1985). Alveolar macrophages and lymphoid tissues were obtained from sheep postmortem.

Flow cytometry

Washed cells ( 10 6) were incubated for 60 min at 4°C with the appropriate dilution of mAbs (in 0.1% bovine serum albumin, 0.01 M sodium azide in phosphate-buffered saline). Ceils were washed and incubated for a further 60 min in the appropriate dilution of fluorescein isothiocyanate-conjugated sheep antimouse Ig (Scottish Antibody Production Unit, Carluke, Lanark, Scot- land). Then 10 4 cells were analysed using a FACScan cell analyser (Becton Dickenson, Mountain View, CA, USA). Different cell populations were ana- lysed separately by 'live gating', being distinguished by their scatter (forward scatter/side scatter) profiles (Hopkins and Dutia, 1990).

Immunohistology

Cryostat sections (8/tm) were cut from fresh tissue and snap frozen in liquid nitrogen. Immunohistology of cell suspensions was done using cytos- pin smears. Smears or sections were air-dried for 30 min, fixed in dry acetone at -20°C for 30 min and stained using standard alkaline phosphatase-con- jugated antimouse Ig antiglobulin.

Results

Table 1 summarizes the reactivities of the Second Workshop mAbs with sheep leukocytes.

J. Hopkins et al. / Vet. lmmunoL lmmunopathol. 39 (1993) 49-59 51

Table 1 Summary of reactivities of Second Workshop monoclonal antibodies with sheep leukocytes

Cluster Monoelonal antibody

Workshop Clone number number

Specificity (similarity to First Workshop mAbs)

OvCD 1

OvCD2

OvCD4

OvCD5

OvCD6

OvCD8

OvCD 11 a

OvCD1 lb

OvCD1 lc

OvCD 18

OvCD25

OvCD44

OvCD45

7 CC90 12 CC43 13 CC20 39 CC40 37 CC132 40 CC118 43 CC122 38 20-27

92 306F

133 GC50A1

25 CC17 97 JPID4

132 BAQ91A

8 CC63 134 BATg2A

44 72.87 85 IL-A99 94 FI0-150 99 MD1H11

100 MD2B7 178 BAQ 30A

11 CC126 23 CC125 62 IL-A 15

105 OM1 180 BAQ 153A

102 MF 13F5 103 MFI4B4

54 IL-AI 11 171 CACT 109A 172 CACTI 16A

61 IL-A118 93 25-32

CD 1 homologous to human CD 1 b (similar to CC 13, CC 14, VPM5 and TH97A)

CD1 homologous to human CDlc

CD2 (similar to 135/A)

CD4 (same as ST-4a, 44-38 and 44-97)

CD5 (similar to 25-91, ST-la, VPM29 and 79-5)

CD6 (similar to CC38)

CD8 (similar to CC58, 38.65, ST8 IL-A51 and BAT82A)

CD 11 a (LFA- 1 ) al. heavy chain associated with//2- integrin (CD 18 ) receptor for ICAM 1 and ICAM2

CDI lb (Mac-1 or CR3) a m heavy chain associated with ]/2-integrin (CD 18 ) receptor for iC3b

CD 1 lc (P 150,95 ) ax heavy chain associated with fl2- integrin (CD18) (similar to IL-A46)

CD18 (fl2-integrin) (light chain ofCD1 la, 1 lb and 1 lc)

CD25 (a chain of the IL-2 receptor)

CD44 (pgp I/Hermes)

113 TD 14 CD45/LCA all isoforms (similar to VPM 18, 151, 1.28 ) 114 TD15

52 J. Hopkins et al. / Vet. lrnmunoL Immunopathol. 39 (1993) 49-59

Table 1 (Continued)

Cluster Monoclonal antibody Specificity (similarity to First Workshop mAbs)

Workshop Clone number number

OvCD45R 3 CC76 28 CC77

109 Bo42 154 GS5A 155 GX18A 156 BAG36A 158 GC6A

CD45R restricted to the higher Mr isoforms of CD45 (similar to 73-37, 73B and 20-96)

OvWC 1 9 CC101 30 CC117 31 CC115 45 CC15

146 CACTB 1A 147 CACTB 15B 149 BAQ89A 150 CACTB7A 151 BAG25A

T 19 molecules present on the CD4- CD8- , TCR 1 ÷ T ceils (similar to 19-19 and ST 197 )

OvWC2 (?/~ TCR1 ) 136 CACTB6A 141 CACT71A 145 CACTB44A

TCR1 (7 /~TCR) (similar to 86D)

OvWC3 34 CC70 152 BAQ 15A

Peripheral B cells

OvWC4 17 CC57 ?CD 19 peripheral B-cells

OvWC6 42 CC98 57 IL-A114 66 IL-A53

OvWC9 82 IL-A96 ?CD9

Unclustered 86 J3 95 17A3

174 CACTB52A

Non-lineage

135 TH 14B MHC Class II control 165 CACT101A

10 CC85 59 IL-A56

182 DH16A

Non-lineage

126 VPM30 153 GC65A 175 CACT65A

Peripheral B cells, cortical thymocytes and activated CD4 + T cells

J. Hopkins et al. / Vet. Immunol. Immunopathol. 39 (1993.) 49-59 53

Cluster Monoclonal antibody

Workshop Clone number number

Specificity (similarity to First Workshop mAbs)

Sheep-specific

64 IL-A24 181 DH59B

101 MD7C7 183 BAG 18A

188 CAPP2A

90 P13

83 IL-A97 84 IL-A98

106 OM2 116 TD19

2 CC69 29 CC28 68 IL-A56

52 IL-AI09 56 IL-A113 89 PI0

157 BAS21A

57 IL-A114 66 IL-A53 96 26A9

148 BAQ72A 166 CACT26A 167 CACT77A

69 IL-A58 14 CC70

119 TD22 124 TD9

107 OM3 120 TD25 121 TD26 123 TD28 127 VPM32 128 VPM33

Non-lineage

Macrophages, monocytes and lymphocytes

Platelets

T cells, negative B cells

Macrophages and monocytes

Macrophages and monocytes

T cell subset

Non-lineage

Peripheral B cells and follicular DC

Markers on TCR1 + 7/8 T cells

Anti-Ig ?CD21

Macrophages

Macrophages and monocytes Macrophages, monocytes and lymphocytes

54

OvCD1

~L Hopkins et al. / Vet. ImmunoL lmmunopathoL 39 (1993) 49-59

Eight mAbs clustered together as CD 1 (T6), but these can be split into two subclusters. The C D l b subcluster consists of seven mAbs including one (CC20) which formed part of the ovine CD l b in the First Workshop and is equivalent to CD 1 b in man. All these mAbs had a similar cellular distribution to the other First Workshop mAbs, CC 13, CC 14, VPM5 and TH97A. At pres- ent no discrimination can be made between the reactivities of these mAbs in sheep. The second subcluster consists of only one mAb, 20-27 (SBU-T6). Although this mAb cross-reacts with ovine CD 1 b (Dutia and Hopkins, 1991 ), its cellular distribution is similar to human CD 1 c. Alternatively it reacts with a framework determinant of sheep CD1 and binds to all the sheep CD1 isoforms.

OvCD2

Only one mAb is specific for ovine CD2 (T11 or LFA-2 ), 36F, which is the workshop control (Mackay et al., 1988a; Hein et al., 1991 ). This antibody has an identical cellular distribution to a First Workshop mAb, 135A (Hein et al., 1991 ). These mAbs react with a 50 000 MW glycoprotein which acts as the cellular receptor for CD58 (LFA-3) and is involved in T cell adhesion to accessory cells and in T cell activation.

OvCD4

Only one Second Workshop mAb, GC50A, reacted with ovine CD4 (L3T4 or T4). This had an identical distribution to three fully characterized antish- eep CD4 mAbs, ST4A, 44-38 and 44-97 (Hopkins, 1991 ). Sheep CD4 is a 56 000 MW glycoprotein which acts as a cellular receptor for MHC Class II and also as a coreceptor for TCR2 (ot/fl T cell receptor) in T cell activation.

OvCD5

Two mAbs clustered as ovine CD5, one of which (CC 17 ) is the workshop control. These mAbs are identical in their reactivity to 25-9 l, ST 1 a and 79-5 (Keech and Brandon, 1991 a). Unlike cattle there is no evidence for allelic variations in ovine CD5. Sheep CD5 (Lyl or T1 ) is approximately 67 000 MW in size and is expressed at a high level on all T cells and at a lower level by many B cells, expression is lost on cellular activation (Hopkins and Dutia, 1991).

J. Hopkins et al. / Vet. lmmunol, lmmunopathol. 39 (1993) 49-59 55

OvCD6

Only one mAb (BAQ91A) clustered as CD6 and reacted with sheep. The other antibovine CD6 mAbs did not cross-react with sheep. The reactivity of BAQ91A is comparable with the First Workshop anti-CD6 mAb, CC38. CD6 (T 12) is a single chain glycoprotein of about 100 000 MW present on thy- mocytes and the majority of T cells.

OvCD8

Two mAbs reacted with ovine CD8 (Ly2 or T8 ), one being mAbs the work- shop control, CC63. These reacted in the same way as the First Workshop mAbs CC58, 38-65, ST8, I1-A51 and BAT82A (Keech and Brandon, 199 lb) . CD8 is a transmembrane dimeric glycoprotein consisting of two polypeptide chains of 35 000 and 33 000 MW (or and fl chains, respectively). It exists as a combination of an ot/fl heterodimer or an a/ t~ homodimer. It is present on 20-25% of T cells and functions both as the cellular receptor for MHC Class I and a coreceptor molecule in T cell activation. In both cattle and sheep (Maddox et al., 1985; MacHugh and Sopp, 1991 ) there is evidence for the existence of the two CD8 isoforms although there is no evidence for the dif- ferential reactivity of the CD8 cluster mAbs in sheep.

OvCD l l a

Six mAbs clustered as antiovine CD 11 a (leukocyte functional antigen-1 ) including the workshop control 72-87 and F 10-150 which had previously been described as CD1 la specific (Mackay, 1990). These mAbs reacted with a 180 000 MW c~ chain associated with the CD 18fl chain (fl2-integrin).

OvCD l l b

Three mAbs clustered as recognizing ovine CD 11 b (Mac 1 or CR3 ) includ- ing the workshop control IL-A 15. These antibodies recognize the 170 000 MW

chain associated with the CD 18fl chain (fl2-integdn) which acts as the cel- lular receptor for the complement component iC3b and is intimately in- volved in leukocyte-endothelial interaction.

OvCD l l c

CD1 lc is represented by two mAbs, BAQ153A and OM1. The C D l l c workshop control, NAM4, does not cross-react with sheep. OMI (Pepin et al., 1992) did not cluster with CD 11 c as it does not react with bovine antigen,

56 z Hopkins et aL / Vet. ImmunoL lrnmunopathoL 39 (1993) 49-59

but its reactivity is consonant with it being CD1 lc specific (Gupta et al., 1993 ). These mAbs recognize the 150 000 MW ot chain associated with the CD 18fl chain (fl2-integrin). CD 11 c (also known as p 150,95 ) acts, like CD 11 b, as a cellular receptor for the complement component iC3b and is involved in the margination of granulocytes.

OvCD18

Two mAbs react with sheep CD18 (Gupta et al., 1993). CD18 is the fl2- integrin molecule which acts as the fl chain for the CD 11 ol chains. This mol- ecule has a MW of approximately 95 000 and coprecipitated with the three different-sized CD 11 molecules.

OvCD25

Three mAbs cluster to form the ovine CD25 cluster. CD25 is the ot chain of the cellular receptor for the T cell cytokine interleukin 2 (IL-2). This mol- ecule (of 55 000 MW) is expressed after T cell activation and associates with the constitutively expressed 75 000 MW fl chain to form a high affinity recep- tor for IL-2.

OvCD44

Two mAbs define the ovine CD44 cluster including one, 25-32, with a pub- lished specificity for ovine CD44 (Mackay, 1988b). This molecule is a highly glycosylated cell membrane polypeptide which in man is present in two dis- tinct isoforms (of90 000 and 130 000 MW). There is no evidence for these different isoforms in the sheep. CD44, also known as pgpl and Hermes, is expressed by a wide variety of cell types and is involved in cell adhesion and T cell activation.

OvCD4 5

Two mAbs cluster to form the sheep CD45 cluster, TD 14 and TD 15. These two mAbs recognize all the different isoforms of CD45 (leukocyte common antigen) with MWs ranging from 180 000 to 220 000. These mAbs have iden- tical reactivities to the First Workshop antibodies 151 and 1-28, and with VPM18 which has a published specificity for CD45 (Hopkins, and Dutia, 1990).

OvCD45R

There are seven mAbs which form the CD45R cluster (Dutia et al., 1993a). These seven can be subdivided into four subgroups based on their cellular

J. Hopkins et aL / Vet. Immunol. Immunopathol. 39 (1993) 49-59 57

distributions: subgroup 1 containing antibodies CC76 and CC77; subgroup 2 containing Bo42; subgroup 3 with BAG36A; subgroup 4 with GS5A, GX18A and GC6A. In addition there are two First Workshop mAbs, 73B and 20-96, which form a fifth subgroup (Hein and Mackay, 1991 ).

OvWC1

Nine mAbs are in the sheep WC 1 cluster. In the sheep there is evidence for only two distinct isoforms (equivalent to N3 and N4 subgroups) whereas in cattle there seem to be four subgroups, mAb CC 15 recognizes all WC 1 (T 19 ) molecules in the sheep (Davis et al., 1990). This cluster also contains the antisheep WC1 mAbs 19-19 and ST197 submitted to the First Workshop (Mackay et al., 1991 ).

OvWC2 (?/O TCR1)

Six mAbs cluster as the sheep WC2. This cluster defines the 7/J T cell re- ceptor (TCR1) originally characterized by the 86D (Mackay et al., 1989). mAbs CACT26A and CACT77A are not specific for the TCR but react with an activation marker on TCR 1 + ceils.

OvWC3

Two mAbs in the Second Workshop reacted with sheep WC3. These mAbs also reacted with bovine WC3 which contains six mAbs, including three from the First Workshop., This molecule is of 145 000 MW and reacts with periph- eral B-cells and follicular dendritic cells. The cellular distribution and molec- ular size are consistent with WC3 being the ruminant homologue of CD21 (CR2).

OvWC4

Only one mAb forms the sheep WC4 cluster. This mAb reacts with a 90 000 MW polypeptide present on the majority of mature, peripheral B cells as well as follicular dendritic cells. It is possibly homologous to CD 19 (Mukwedeya et al., 1993 ).

OvWC6

This cluster is defined by three mAbs originally thought to react with the B220 molecule. Although precipitating polypeptides of apparent MW 220 000 and 180 000-190 000 it does not cluster with CD45R and is antigenically unrelated to CD45 (Dutia et al., 1993b).

58 ,L Hopkins et aL / Vet. lmmunol, lrnmunopathoL 39 (1993) 49-59

OvWC9

This cluster contains just one mAb, IL-A96, which possibly reacts with the sheep homologue of CD9.

Other mAbs

There remain other mAbs which could not be assigned into either well- defined CD clusters or into WC clusters. These are also listed in Table 1. In addition, there are six mAbs which remain unclustered mainly because they show no cross-reactivity to cattle antigens.

References

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Dutia, B.M., Ross, A.J. and Hopkins, J., 1993b. Analysis of the monoclonal antibodies com- prising WC6. Vet. Immunol. Immunopathol., 39:193-199.

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