Individual antigens of goats

Veterinary Immunology and Immunopathology, 27 ( 1991 ) 121-131

121

Elsevier Science Publisllers B.V., Amsterdam

6.1 Individual antigens of goats W.C. D a v i s a a n d J.A. Ellis b

aDepartment of Veterinary Microbiology~Pathology, 14"ashingtonState University. Pullman. WA 99164-7040, USA bDepartment of Veterinary Sciences, College of Agriculture, Universityof 14~voming,Jackson, WY82070, USA

INTRODUCTION

The studies of monoclonal antibodies (mAbs) reactive with goat leukocytes were less extensive than those of cattle and sheep. Insufficient data on immunefluorescent staining of cell lines and tissues were available to enable statistical clustering of mAbs to be performed. Consequently, a different approach was used to obtain useful information on specificity. MAbs that had been clustered or identified on bovine and ovine cells were assessed for reactivity on caprine peripheral blood leukocytes and, in some cases, preparations of thymocytes. They were also examined for staining of frozen sections of tissue. The percentage of stained cells as assessed by flow cytometry (FC), the staining protiie produced, and the distribution of stained cells in sections of caprine lymphoid tissues were examined and compared with the results obtained in cattle and sheep. Those mAbs that gave results in goats that were consistent with those obtained with bovine and ovine tissues were assigned to the appropriate cluster or workshop designations. Further dual fluorescent (FC) studies were used to confirm that the differentiation molecules defined exhibited comparable patterns of expression on individual cell types. The remainder of workshop mAbs that reacted with goat leukocytes but did not form clearly defined clusters in cattle or sheep were evaluated for specificity by single and dual FC. MATERIALS AND METHODS

Monoclonal antibodies The mAbs listed in Table 1, Section 1.3 of this report were tested on caprine cells. 0165-2427/91/$03.50

© 1991 n Elsevier Science Publishers B.V.

122

W.C. DAVIS AND J.A. ELLIS

Cell types Peripheral blood leukocytes were collected from Saanen goats 2 years of age. Thymocytes and tissues for immunohistoiogy were collected from a lyear-old Saanen goat. Flow cytometry A Becton Dickinson FACS Analyzer upgraded with a FACSLite laser was used to assess immunofluorescent staining (Becton Dickinson Immunocytometry System, Mountain View, CA, USA). Preparations of 5 × 105 cells were reacted with 50 gl of each mAb ( 15/zg/ml) and 100/zl of a l/200 dilution of FITC conjugated goat anti-mouse Ig (heavy and light chain specific, Caltag, South San Francisco, CA, USA). LYSYS software was used for data analysis (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA). Detailed methods are given in the Appendix (Davis et al., 1987; Davis et al., 1990). Immunohistochemistry Immunohistochemistry was performed as previously described (Davis et al., 1988; Ellis et al., 1988; MacHugh et al., 1988 ). RESULTS

Caprine CD1 The six mAbs that formed the BoCD l cluster and mAb 20-27 all reacted with caprine thymocytes as assessed by FC and immunocytochemistry (Table l ). Comparative analysis showed the FC profiles obtained with each mAb on thymocytes were similar (Fig. i ). Examination of tissue sections demonstrated that the mAbs react with thymocytes primarily localised in the thymic cortex. Only a few positive cells were found in the medulla. As noted in cattle mAb 20-27 stained 13-29% of PBM; hence this mAb may recognise a different form of the CD l molecule in goats. The low percentage of cells stained with VPM5 made it difficult to distinguish clearly between nonspecific and specific staining. Further studies are needed to determine if this mAb detects a different molecule. The firm data indicate that the six mAbs and 20-27 recognise epitopes conserved on the bovine, ovine and caprine orthologues of CD l (Goodman et al., 1987; Fitch, 1970). Caprine CD2 Eleven of the 14 mAbs in the BoCD2 cluster reacted with caprine lymphocytes (Table 1 ). As in cattle, the FC profiles obtained will BoCD2 mAbs could be distinguished from those obtained with mAbs reactive with other T-ceU molecules (compare profiles in Fig. l ). Comparative dual fluorescence studies demonstrated that CoCD4 + and CpCD8 + cells coexpress CpCD2, CoCD5

123

INDIVIDUAL ANTIGENS OF GOATS

and CpCD6. B and W C 1 + lymphocytes, monocytes/macrophages, and granulocytes did not express CpCD2. Immunohistochemical studies with representative mAbs demonstrated that the cells which express CpCD2 are distributed in thymus, spleen and lymph nodes as noted in other species. The OvCD2 mAbs did not react with caprine cells. Caprine CD4

Five of the 12 mAb~ that reacted with the bovine and/or ovine CD4 antigen reacted with caprine lymphocytes. Each of the five mAbs yielded a pattern of labelling indistinguishable from that observed when the mAbs were used to label bovine and ovine cells (Fig. 1 ). Dual fluorescence studies with the broadly cross-reactive mAb, GC50A1, demonstrated that the mAbs reactive with species restricted and conserved determinants reacted with the same population of cells in each species (Section 3.5, Table 1 ). Immunohistochemical studies verified that the cells expressing CpCD4 have the same distribution as noted in other species (Fig. 2 ). Caprine CD5

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Fig. 1. Representative flow cytometric profiles of caprine thymocytes and peripheral blood mononuclear cells labelled with different monoclonal antibodies. Frame A, second step reagent alone: frame B, thymocytes labelled with VPM5 ( ! 1%); frame C, thymocytes labelled with 2027, CpCDI (15%): frame D, PBM labelled with BAQ95A, CpCD2 (45%): frame E, PBM labelled with GC50AI, CpCD4 (23%); frame F, PBM labelled with CC! 7, CpCD5 (73%): frame G, PBM labelled with BAQ91A, CpCD6 (41%): frame H, PBM labelled with CACT80C, CpCD8 (21%).

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TABLE 1 Reactivity of workshop monoclonal antibodies with caprine cells MAb

Ig isotype

Sample

Code

CD i

VPM 5 20-27 CC14 TH97A CC20 CC 13 IL-A26 CH 132A CACT31A ST- 11 CC42 8-1 E3 CH 128A BAQ95A BAT 18A BAT42A

CD2

135-A

CD4

CD5

CD6

IL-A42 IL-A43 BAT76A 16-1El0 IL-A45 GC50AI CACT83B CACT87A CC30 CACT 138A SBU-T4 (44-97) ST-4a IL-A 11 IL-A 12 CC8 GC 1A 1 SBU-T4 (44-38 ) CC 17 79-5 25-91 CC29 BLT- 1 8CII IL-A67 ST- I a IL-A28 IL-A57 CC38 NAM3 CACT 141A BAQ82A IL-A27 BAQ91A

% of PBM stained G25

IgM IgG 1 lgGl IgG2a IgG2a lgG3 IgM IgM IgM IgM IgG 1 lgG 1 IgG 1 IgG 1 IgG 1 IgG 1 IgG 1 IgG2a IgG2a IgG2a lgG2a IgG2b lgM lgM lgM !gG ! IgG 1 IgG 1 IgG 1 IgG2a lgG2a IgG2a IgG2a IgG2a IgG 1 IgG 1 lgG 1 lgG 1 IgG2a lgG2a lgG 1 IgG2a lgM lgG2a lgG2b IgG2a lgG2b IgM lgG 1 IgGl

G36

G44

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37 38 40 44 35 41

44 48 49 46 36 48

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29 25 70 36

28 25 66 32

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INDIVIDUAL ANTIGENS OF GOATS

125

TABLE 1 (Continued) MAb

Ig isotype

Sample

Code

CD8

IL-A51 CC58 CC63 38-65 CACT80C CACT88C BAT82A ST-8 BAQI I iA CACT 130A IL-AI 5 ( C D I Ib) C 5 B 6 ( C D I lc) NAM 4 ( C D I lc) F150(LFA-I )CDI l? SBU-25-32 SBU-LCA 151 VPM 18 LCA SBU-20-96 IIITS(LFA-3) IL-A29 CC 15 CC39 19(19-19) BAQ4A BAQ 128A CACTB31A NAM 2 197 BAQ90A B7AI BAQ89A BAQ 159A CACTB32A CACTB6A CACTB81A CC21 CC37 CC51 IL-A65 CC57 CC55 IL-A54 IL-A55 72-875 IL-A87 s 72-376

CDI 1

CD44 CD45

CD45R CD58 WC 1

WC2 WC3

WC4 WC5

Other mAbs

IgG 1 IgGl IgG2a IgG2a IgG l IgG3 IgG l IgM IgM IgG3 IgGl lgGl IgGl IgGl IgGl IgG2a IgG2a IgG 1 lgG 1 IgG2b lgG 1 IgG2a lgG 1 IgGl IgG 1 IgG 1 IgG2b IgG2a igG2b IgG3 IgM IgG 1 IgG 1 IgG 1 IgM lgG 1 IgG 1 IgG 1 IgG2b IgG2b lgG 1 IgG 1 IgG 1 IgG2a IgG2a IgG2a lgG 1

% of PBM stained G25

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23 22 11 19 22 19 2l 21 P -

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84

614

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10 864 30 25 29 28 24 22 25 27 22 24 26 27 21 23 8 11 15 12 30 !3 20 90 -

~ND = Not done. MAbs received after the primary study was performed on the animals indicated. 2? = Unreliable results. Further studies needed to verify reactivity of mAbs indicated. 3p = Polymorphic determinant. 4Gated to include granulocytes and monocytes. SThought to recognize CD 11 a or CD 18. 6May recognise CD45R in other species.

W.C. DAVIS AND J.A. ELLIS

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Fig. 2. Representative patterns of staining of different tissues with workshop mAbs reactive with bovine, ovine and caprine leukocyte differentiation molecules. (A) follicle/paracortex of a caprine prescapular lymph node stained with mAb GC50AI (CpCD4); (B) follicle/paracorrex ofa caprine prescapular lymph node stained with mAb CACT88C (CpCD8); (C) follicle/ paracortex of a caprine prescapular lymph node stained with mAb BAQ 128A (WC l ).

INDIVIDUAL ANTIGENS OF GOATS

127

a pattern of labelling in FC clearly identical to that obtained with bovine calls (Fig. l ). The mAb reacted with 63% to 75% of PBM (Table 1 ). As in cattle (Howard et al., 1989; Howard and Leibold, this report), two populations of cells expressing high and low levels of the antigen could be distinguished; CD2 + lymphocytes expressed the CD5 antigen at a higher intensity than WCI + T cells or B cells. The OvCD5 mAb (ST-1A) was not tested with the animals used in this study; however, previous studies with other animals had demonstrated that the mAb is not reactive with goat cells (Table 1 ).

Caprine CD6 Two of the seven BoCD6 reacted with caprine lymphocytes (Table 1, Fig. l ). However, only one of these, BAQ91A, yielded consistent labelling. This mAb reacted with a determinant that is apparently conserved on the BoCD6 and CpCD6 molecules. The determinant is polymorphic in sheep (see Appendix; Davis, unpublished observations). The ovine cells used in the comparative analysis (Section 3.5, Table l ) were negative for the determinant. As noted above, comparison of FC profiles demonstrated that the patterns of labelling obtained with bovine and eaprine cells were similar and clearly distinct from those obtained with CD2 mAbs and other mAbs that recognise caprine T-cell subpopulations. Dual fluorescence studies showed that CpCD6 + cells coexpress CpCD2 and CpCD5 and either CpCD4 or CpCD8. The present studies did not provide any evidence that the caprine orthologue is expressed on a subpopulation of B-cells as noted in humans (Knapp et al., 1989 ). Caprine CD8 ~c~tt~tcu .1,-,o5 , ~ f o r ' ~ -ou,~.~o P'° .... " - J with caprine iymphocytes. Nine ,,,~-,h.. IO !'" ^~ . .~. .v. ~ ,:r:... Of these, eight reacted with one or more determinants conserved on bovine, ovine and caprine orthologues of CD8 (Section 3.5, Table 1 ). One of the mAbs (BAQI 11A) reacted with all bovine PBM samples tested but recognised a polymorphie determinant on the CpCD8 molecule. The FC profiles obtained with each of the mAbs were similar in each species and distinct from those obtained with other workshop mAbs (Fig. 1 ). Dual fluorescence studies demonstrated that the BoCD4 and BoCD8 mAbs react with mutually exclusive populations of CpCD2 +, CpCD5 +, CpCD6 ÷ T-cells. ~,.$1 I b l l ~

Caprine CD1 l b The mAb that was considered to react with the BoCD 11 b antigen, IL-A 15, reacted with cells from sheep and goats (Section 3.5, Table 1 ). In goats, ILA I 5 reacted predominantly with granulocytes and monoeytes in a pattern similar to that obtained with bovine cells. Insufficient data were obtained to assess whether the pattern of labelling, as assessed by FC, was distinct from other mAbs that labelled caprine granuloeytes and monocytes.

! 28

W.C. DAVIS AND J.A. ELLIS

Caprine CD 1lc One of the three mAbs that formed the CD 1 l c cluster, NAM-4, reacted with caprine ceils. However, the pattern of labelling was not as distinct as that obtained on bovine cells. Thus it was not possible to assess whether the mAb reacted with the caprine orthologue of CD 11c.

CD44 MAb 25-32, reactive with ovine CD44, reacted with cells from goats and exhibited a pattern of labelling that was distinct from that obtained with other mAbs that react with all leukocytes. The pattern of labelling was similar on bovine, ovine and caprine cells.

Leukocyte common antigen (CD45) None of the anti-CD45 mAbs reacted with caprine cells (Section 3.5, Table 1 ). A mAb believed to be specific for a restricted form of CD45 in sheep (2096) reacted with one of the goats tested. Further studies are needed to establish whether this indicates the determinant is polymorphic in goats.

CD58 The anti-CD58 mAb (T 11TS) submitted to the workshop only reacted with ovine cells (Section 3.5, Table 1 ).

Workshop cluster I (WC1) The 14 mAbs that formed BoWCI reacted with caprine cells (Table 1 ). Dual FC demonstrated the cells that express WC 1 molecules express CpCD5 but not CpCD2, CpCD4, CpCD6 or CpCD8. The analysis also demonstrated that the cells do not express any of the monocyte/granulocyte, B, or MHC class II molecules detected with the other workshop mAbs. As noted with bovine cells, there was considerable variation in the percent of cells labelled with each mAb suggesting differences in affinity or the possibility that more than one molecule is detected by this set of mAbs (Fig. 3 ).

Workshop cluster 2 (WC2) One of the two mAbs in the bovine workshop cluster 2 reacted with caprine cells. Dual FC analysis revealed CACTB6A reacts with a subpopulation of WCI ÷ cells in goats. In addition, the analysis revealed a subpopulation of WC2 ÷ cells express the CpCD2 (see Sopp et al., pp. 163-168 for review of similar findings in cattle).

Workshop cluster 3 (WC3) Two of the four mAbs, CC21 and CC37, that comprise BoWC3 reacted with caprine lymphocytes (Section 3.5, Table 1 ). Comparative dual FC demonstrated the mAbs only react with B lymphocytes.

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Fig. 3. Representative flow cytometric profiles of caprine PBM labelled with mAbs that recognise WCI and WC2 molecules. Frame A, PBM laoelled with B7A1, WCI (35%);frame B, PBM labelled with CACTB32A, WCI (8%); frame C, PBM labelled with BAQ89A, WCI (25%); frame D, PBM labelled with CACTB6A, WC2 ( 13% ). The variations in the concentration of labelled cells were consistent for each mAb. MAbs CACTB32A and BAQ89A always labelled fewer cells than mAbs B7AI and other WCI mAbs such as IL-A29 and 19-19. WC2 labelled fewer cells than B7AI. Analysis also showed WC2 labelled some but not all CACTB32A and BAQ89A positive cells.

Workshop cluster 4 (WC4) None of the mAbs that comprise BoWC4 reacted with caprine lymphocytes.

Workshop cluster 5 (WC5) Both of the mAbs that comprise BoWC5, II-A55 and I1-A55, reacted with caprine lymphocytes (Section 3.5, Table 1 ). Dual FC demonstrated that the mAbs only react with B lymphocytes in PBM.

MHC class H molecules One anti-MHC class II mAb (S-MHC-II) was submitted to the workshop. It was not included in the general study. However, it was used in conjunction with one of our anti-MHC mAbs (THI 4B) (Davis et al., 1987) as a standard to determine if any of the other submitted mAbs reacted with MHC class II molecules. Comparison of FC profiles revealed that BLMo-4 most likely reacts with a MHC class II molecule in goats (see Appendix).

Undefined workshop mAbs Additional mAbs submitted to the workshop reacted with caprine leukocytes (see summary in the Appendix). MAb 72-87, possibly directed against CD 11 a reacted with cells from goats but proof of homology with the human antigen remains to be established. MAb IL-A87, possibly to CD 1 la or CD 18, did not react with goat cells. A number of other mAbs yielded distinct patterns of labelling but did not resemble those obtained with the mAbs listed above. Dual FC demonstrated that some of the mAbs detect lineage-specific molecules. For example, BAS9A, BAQ44A, BAQI55A and VPM30 reacted with molecules present on B lymphocytes (Table 1 ). Other mAbs reacted with

! 30

W.C. DAVIS AND J.A. ELLIS

molecules expressed on one or more lineages of cells. Further studies are needed to characterise these molecules. DISCUSSION AND SUMMARY

Many of the leukocyte differentiation molecules first defined in rodents and humans are highly conserved with respect to their antigenic composition and expression on leukocytes. This has permitted the use of FC to compare the patterns of expression of mAb-defined molecules on leukocytes across species and to ascertain their apparent specificity prior ~o biochemical and functional ~nalysis. It has also allowed mAbs to conserved determinants to be used to establish that the same molecules have indeed been identified in different species. MAbs to MHC class I and class II molecules and surface immunoglobulin were established in previous studies by these methods (Davis et al., 1987). From the FC and immunohistological data obtained in the workshop, it can be concluded that the mAbs that form clusters with bovine and/or ovine differentiation molecules that cross-react with caprine leukocytes define the caprine orthologues of CDI, CD2, CD4, CDS, CD6, CD8, CD44 and possibly CD45R. In addition, the techniques have been useful in identifying new differentiation molecules, some of which have as yet no identified CD equivalents; as, for example, the mAb-defined WC l molecule(s). The continued use of the comparative approach to the study of leukocyte differentiation molecules should greatly facilitate definition of the immune system in ruminants and the constellation of molecules that define individual populations of ceus. Another finding is that the lymphoid system of goats, as in the other two species examined, differs in composition from that noted in rodents and humans, a finding of major importance to the understanding of immune regulation in ruminants. Studies presented here and elsewhere have shown the caprine lymphoid system is composed of three antigenically distinct populations of cells: T-cells defined by the expression of orthologues of CD2, CD4, CDS, CD6 and CD8, B-cells defined by the expression of sIgM and a set of Bcell specific molecules described in the present workshop, and a third population of cells initially described as a subpopulation of T-cells in sheep and cattle lacking T-cell and B-cell markers (Mackay et al., 1988; Davis et al., 1987; Morrison et al., 1988). Comparative studies have shown that the latter population is defined by a set of mAbs that recognise high molecular weight molecules expressed on all or subpopulations of cells that form cluster 1 in the workshop. No information is available on the relation of the molecules recognised by WC1 mAbs to known CD molecules. Subpopulations of these WCI + cells express the 37/47 kDa antigen detected by the WC2 mAbs. The molecule identified by the WC2 mAbs appears to be similar to that recog-

INDIVIDUAL ANTIGENS OF GOATS

131

nised by n o n - w o r k s h o p mAbs 86D a n d 96C ( M a c k a y et al., 1988, 1989; M a c k a y and Hein, 1989) which have been shown to be specific for the ),/~ T C R in sheep and cattle. ACKNOWLEDGEMENTS These studies were s u p p o r t e d in part by grants from the U S D A (86-CRSR2-2913, 89-37265-4537), N I H N C I ( R O 1 CA50141 ), a n d gifts from V M R D Inc. ( P u l l m a n , W A ) , Becton Dickinson I m m u n o c y t o m e t r y Systems ( M o u n tain View, C A ) , N o r d e n L a b o r a t o r i e s Inc. (Lincoln, N B ) , Ribi I m m u n o chem. Research Inc. ( H a m i l t o n , M T ) a n d C o s t a r Corp. (Cambridge, M A ) . REFERENCES Davis, W.C., Marusic, S., Lewin, H.A., Splitter, G.A., Perryman, L.E., McGuire, T.C. and Gorham, J.R., 1987. The developmenl and analysis of species-specific and cross-reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species. Vet. Immunol. Immunopathol., 15: 337-376. Davis, W.C., Ellis, J.A., MacHugh, N.D. and Baldwin, C.L., 1988. Bovine pan T-cell monoclonal antibodies reactive with a molecule similar to CD2. Immunology, 63: 165-167. Davis, W.C., Larsen, R.A. and Monaghan, M.L., 1990. Genetic markers identified by immunogenetic methods. International Symposium and Educational Workshop on Fish-marking Techniques. Am. Fish. Soc. Symp., 7:521-540. Ellis, J.A., Davis, W.C., MacHugh, N.D., Emery, D.L., Kaushal, A. and Morrison, W.I., 1988. Differentiation antigens on bovine mononuclear phagocytes identified by monoclonal antibodies. Vet. Immunol. Immunopathol., 19: 325-340. Fitch, W.M., 1970. Distinguishing homologous from analogous proteins. Syst. Zool., 19: 99113. Goodman, M., Miyaraoto, M.M. and Czelusniak, J., 1987. Pattern and process in vertebrate phylogeny by coevolution of molecules and morphologies. In: C. Patterson (Editor), Molecules and Morphology in Evolution: Conflict or Compromise? Cambridge University Press, Cambridge, UK, pp. 142-176. Howard, C.J., Parsons, K.R., Jones, B.V., Sopp, P. and Pocock, D.H., 1989. Two monoclonal antibodies (CCI 7, CC29) recognising an antigen (Bo5) on bovine T lymphocytes, analogous to human CD5. Vet. Immunol. Immunopathol., 19:127-139. Knapp, W., Rieber, P., Dorken, B., Schmidt, R.E., Stein, H. and Born, A.E.G. Kr. v.d., 1989. Towards a better definition of human leucocyte surface molecules. Immunol. Today, 10: 253-258. MacHug~2, N.D., Bensaid, A., Davis, W.C., Howard, C.J., Parsons, K.R., Jones, B. and Kaushal, A., 1988. Characterisation of a bovine thymic differentiation antigen analogous to CDI in human. Scand. J. Immunol., 27: 541-547. Mackay, C.R., Hein, W.R., Brown, M.H. and Matzinger, P., 1988. Unusual expression of CD2 in sheep: implications for T cell interactions. Eur. J. Immunol., 18:1681-1688. Mackay, C.R., Beya, M-F. and Matzinger, P., 1989. ~,/t~T cells express a unique surface molecule appearing late during thymic development. Eur. J. Immunol., 19:1477-1483. Mackay, C.J. and Hein, W.R., 1989. A large proportion of bovine T cells express the 7t~T cell receptor and show a distinct tissue distribution. International Immunol., 1: 540. Morrison, W.I., MacHugh, N.D., Bensaid, A., Goddeeris, B.M., Teale, A.J. and McKeever, D.J., 1988. A monoclonal antibody which reacts specifically with a population of bovine lymphocytes lacking B cell and T cell markers. Adv. Exp. Med. Biol., 237:591-596.