Selection of monoclonal antibodies for the identification of lymphocyte surface antigens in the New World primate Saguinus oedipus oedipus (cotton top tamarin)

JOMWU. OF IMMUNOWGICAL METHODS

ELSEVIER

Journal of Immunological Methods 178 (199.5) 195-200

Selection of monoclonal antibodies for the identification of lymphocyte surface antigens in the New World primate Saguinus Oedipus Oedipus (cotton top tamarin) A.D. Wilson *, M. Shooshtari, S. Finerty, P. Watkins, A.J. Morgan Department of Pathology and Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1 TO, UK

Received 28 July 1994; revised 26 August 1994; accepted 16 September 1994

Abstract 32 monoclonal antibodies reactive with human CD antigens were tested against tamarin peripheral blood lymphocytes, ConA blasts and lymphoblastoid B cell lines derived from tamarin cells. Reagents that cross-react with MHC class I and II, B cells (CD20, -21 and -23), monocytes (CD14) and NK cells (CD16, -56) have been identified. In addition monoclonals that cross-react with T cells (CD2, CD3), the CD4/CD8 subsets of T cells and the IL-2 receptor (CD25) are reported. A monoclonal against the p chain of LFA-1 (CD181 cross-reacted strongly, but there was only a very poor cross-reaction with a monoclonal against the (Y chain of CDlla. Two monoclonals tested against ICAM-l(CD54) were negative. Keywords: Flow cytometry; Primate; CD antigen; Lymphocyte

1. Introduction The new world primate Saguinus Oedipus oedipus (cotton top tamarin) is susceptible to a number of pathological conditions of medical importance. As many as 60% of captive S. Oedipus develop spontaneous ulcerative colitis and 15% subsequently develop adenocarcinoma of the colon (Chalifoux and Bronson, 1981). S. Oedipus are also susceptible to a number of human virus

Abbreviations: EBV, Epstein-Barr virus: LCL, lymphoblastoid B cell line; NK, natural killer cell. * Corresponding author. Tel.: 01179-288604; Fax: 01179287896. 0022-1759/95/$09.50

pathogens including measles, retrovirus-induced sarcoma and Epstein-Barr virus (EBV) (Wolfe and Deinhardt, 1978). Following inoculation with EBV, S. Oedipus develop B cell lymphomas very similar to those occurring in immunosuppressed transplant recipients (Cleary et al., 1985). S. oedipus are resistant to tumour development following immunisation with the EBV envelope glycoprotein gp340 subunit vaccines or adenovirus expressing the gp340 gene (Morgan, 1992). We are currently analysing the mechanism of this immune mediated protection as part of a program of EBV vaccine development. In order to determine the phenotype of the cells involved in this immune response we have screened a panel of monoclonal antibodies reactive with human CD

0 1995 Elsevier Science B.V. All rights reserved

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antigens to determine which cross-react with lymphocytes from S. Oedipus and can be used in studies of immune function in this species. These monoclonal antibodies will also be useful in determining the immunopathology associated with colitis and adenocarcinoma in this species.

2. Materials

and methods

L. Young (University of Birmingham). Anti-CR2 (CD21) and Leu-llb were purchased from Becton Dickinson. Anti-CD4 was purchased from Alpha Laboratories, Boston, MA. MT910, UCHTl, MT310, DK25, TUlK4, DJ130c, HD37, Blyl, lF8, Act-l, T199, CR3/43,MMH23. MHM24,6.5B5. were from Dako, High Wycombe, Bucks. H50, EBV/CD4, LAl, TUl and B230, were a gift from D. Crawford (London School of Hygiene and Tropical Medicine).

2.1. Lymphocyte preparation

2.3. Flow cytometry Animals were sedated with 5 mg/kg ketamine hydrochloride (Park Davies, 201 Tabor Road, Morris Plains, NJ) and bled from the medial femoral vein. Peripheral blood lymphocytes were isolated from heparinised blood by discontinuous density gradient centrifugation over Ficoll/diatrizoate 1.0117 g/l. (lymphocyte separation medium ICN, Flow ICN Biomedicals, Thame, Oxfordshire, UK). 2 ml of whole blood were diluted with an equal volume of calcium magnesium-free Hanks’ balanced salt solution (HBSS, ICN Flow) and underlayed with 4 ml lymphocyte separation medium. The tubes were spun at 500 x g for 30 min and the cells at the interface collected and washed three times with HBSS. For culture cells were suspended at a final concentration of 106/ml in RPM1 supplemented with 10% foetal bovine serum, L-glutamine, penicillin and streptomycin (ICN Flow), or in PBS 0.1% azide for flow cytometry. 2.2. Antibodies Monoclonal anti-MHC class I BB7.7, OKT3, OKT4 and OKT8 were acquired from the American Type Culture Collection (ATCC Rockville Maryland), and cross-reaction of BB7.7 in S. Oedipus has already been described (Watkins et al., 19881. DA6.231 anti-MHC class II came from European Type Culture Collection (Porton Down, Wlits., UK). Purified SP34 was a gift from Cox Terhorst of the Dana Faber Cancer Institute, Boston. Purified MT1014 and MT122 were provided by P. Reiber (Institut fur Immunologie, Universitat Miinchen). BU35 came from The Binding Site, Birmingham, UK. BU36 was from

100 ~1 of monoclonal antibody at optimal dilution were added to cells at 1-2 x 106/ml in PBS 0.1% azide 10% normal goat serum and incubated at 4°C for one hour. The cells were washed twice with PBS 0.1% azide. 100 ~1 of a l/250 dilution of goat anti-mouse FITC labelled, affinity purified, F(ab’), fragments adsorbed against human serum proteins (Sigma) were then added. In addition the conjugate was prepared in PBS azide with 10% normal tamarin plasma and incubated at 4°C for 1 h prior to the addition of cells to remove any anti-tamarin cross-reacting antibody. After a second incubation of 1 h the cells were washed in PBS azide and kept at 4°C until analysed using a Becton Dickinson FACScan and Lysis II software. Control samples were either incubated with conjugate alone or with an isotype-matched non-reactive monoclonal antibody. Two colour samples were processed and stained in a similar manner, except that two primary monoclonals of different isotype were added simultaneously. Isotype-specific FITC and phycoerythrin conjugates were used (Southern Biotechnology, Birmingham, AL) for the second step, which were also absorbed by preincubation for 1 h with an equal volume of tamarin serum immediately before use. Appropriate dual negative, phycoerythrin only, and FITC only controls were used. 2.4. Cell culture Lymphocytes were cultured in RPM1 10% FCS supplemented with glutamine penicillin and streptomycin. Cultures containing lo6 cells/ml

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were stimulated with ConA at 10 pg/ml for 72 h at 37°C in 5% CO*. After culture they were harvested and dead cells removed by centrifugation through a solution of 18% metrizamide (Sigma) prepared in PBS, 4% foetal calf serum as described by Taylor et al. (1987) the cells were then stained as before. In some cases the expression of CD markers on EBV-transformed lymphoblastoid cell lines (EBV-LCL) was assayed, these cell lines were prepared as previously described (Finerty et al., 1988).

3. Results Table 1 shows a list of the monoclonal antibodies that have been tested and indicates those which reacted with tamarin cells. Evidence for the specificity of the staining comes from the number, intensity and distribution of staining of lymphocyte subsets in tamarins versus human cells. All tamarin lymphocytes were strongly MHC I positive (Fig. la). By comparison there was a range of intensity of MHC II staining from negative to strongly positive with no distinct cut-off point (Fig. lb). Many other antibodies such as CD2 and CD3 (T cells) CD20 and CD21 (B cells) and CD14 (monocytes) stained discrete subpopulations of cells, e.g., CD14 in Fig. lc and CD2 in Fig. Id. The average percentage of peripheral blood lymphocytes from six tamarins that were positive for T cell, B cell and monocyte markers are shown in Table 2. Cells expressing CD16 and CD56 were not frequently present in normal peripheral blood lymphocytes isolated using lymphocyte separation media with a density of 1.077 g/ml. Positive staining for CD16 was present if cells were isolated on Ficoll with a density of 1.088 g/ml., polymorphonuclear neutrophils (PMN) were detected by light microscopy in this denser fraction indicating that CD16 is present on tamarin PMN. CD56 and CD16 were also shown to react with a cell line derived from Tamarin lymphocytes stimulated in vitro with a tamarin EBV-LCL, and these CD56 positive cells had NK activity as measured by killing of K562 targets (Shooshtari et al., manuscript in preparation.). Increased

Table 1 A list of the monoclonal antibodies tested by flow cytometry on tamarin peripheral blood lymphocytes, LCL. ConA blasts or T cell lines, indicating those which were found to be reactive and where possible a source or supplier CD number

Clone number

Source

Activity

CD2 CD3 CD3 CD3 CD4 CD4 CD8 CD8 CD8 CD8 CD1 la CD14 CD16 CD16 CD18 CD19 CD20 CD21 CD21 CD21 CD23 CD23 CD23 CD23 CD23 CD25 CD54 CD56 MHC I MHC II MHC I1

MT910 0KT3 UCHTI SP34 Anti-CD4 MT301 OKT8 DK25 MT1014 MT122 MHM24 TUK4 DJ130c Leu llb MHM23 HD37 B-Lyl 1FS BU35 BU36 HSO LA1 TUI B230 EBV/CD4 ACT- 1 65.B5 T199 BB7.7 CR3/43 DA6.231

Dako ATCC Dako Dr. C. Terhorst Alpha Laboratories Dako ATCC Dako Dr. P. Rieber Dr. P. Rieber Dako Dako Dako Becton-Dickinson Dako Dako Dako Dako Binding site Dr. L. Young Dr. D. Crawford Dr. D. Crawford Dr. D. Crawford Dr. D. Crawford Dr. D. Crawford Dako Dako Dako ATCC Dako EACC

+

-

+ + +

+ + f /- (blasts) + + + + + + -

+ +

+ + +-

numbers of CD56 positive cells were also seen in peripheral blood from tamarins challenged with EBV in vivo; these results are consistant with CD16 present on PMN and CD56/CD16 present on NK cells as is the case in humans. Dual staining was also helpful in verifying the specificity of the monoclonal antibody staining. For example, simultaneous staining with MT301 IgGl anti-CD4 and MT1014-IgM anti-CD8 demonstrated that these antibodies stained mutually exclusive populations of lymphocytes in a ratio of roughly 2 CD4/1 CD8 with only a few double positive cells detected (Fig. 2a). Similarly, dual staining with anti-B cell antibodies Blyl

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lb1

[aI %

of Immunological Methods I78 (I 995) 195-200

7

Con

Con

4

a] Unstained

VI

[Cl

“i

b] CD4 vs. CD8

cells

Con

%

Con

CD2

L L CD 14

1

1

1

1

1

1

1

1

4 1

1

Fig. 1. Histograms of tamarin peripheral blood lymphocytes stained with a selection of monoclonal antibodies chosen to illustrate different patterns of reactivity. The solid area represents unstained control cells and the open area stained cells. a: MHC class I; b: MHC class II; c: CD14: d: CD2.

(anti-CD20 IgGl) and BU-35 (anti-CD21 IgG2b) showed that as expected both of these markers were present on the same subset of lymphocytes (Fig. 2b). Many CD markers are not present on resting peripheral blood lymphocytes or are only expressed at low levels or on a small number of cells. Fig. 3 shows the detection of CD antigens on activated ConA blasts. Cells recovered after 3 days in ConA stimulated culture were 90% CD3 positive (Fig. 3a) and showed an increase in the proportion of cells expressing CD25 (IL-2 receptor) at high intensity (Fig. 3b). Activated lympho-

c] CD20 vs. CD21

Fig. 2. Dual staining of peripheral blood lymphocytes. a: two colour dot plot of CD4 phycoerythrin ( y axis) versus CD8 FITC (X axis), showing the two discrete subsets with only a few dual positives. b: two colour dot plot of CD20 phycoerythrin and CD21 FITC demonstrating that the majority of CD20 B cells are also CD21 positive. CD21 is not present on CD20 negative cells.

cytes are also expected to express higher levels of intercellular adhesion molecules. We therefore tested monoclonal antibodies to CDlla and CD18 the (Y and p chain of LFAl. Two monoclonals against CD54 (ICAMl) the complementary ligand of LFAl were also tested. Monoclonal antiCD18 gave a strong positive signal on 99% of cells. CDlla was consistently negative on resting PBL and positive on activated blasts. However, the intensity of staining was poor by comparison with human cells and we concluded that this

Table 2 The average percentage of normal tamarin peripheral blood lymphocytes found to react with monoclonal antibodies representing the main cell types, including T cells (CD2 and CD3), the helper (CD4) and suppresser/cytotoxic subsets, B cells (CD201 and monocytes (CD14)

Mean Range

to CD antigens (CD81 T cell

CD2

CD3

CD4

CD8

CD14

CD20

MHC I

MHC II

56% 47-65

56% 46-65

38.5% 25-46

24% 17.5-35

20% 11-24

17% 13-24

99% 96-99.9

64% 48-74

All cells were MHC class I positive and many expressed MHC class II. Each value is the mean of six individuals and minimum percentage positive cells are shown in row three.

and the maximum

A. D. Wilson et al. /Journal

of Immunological Methods I78 (I 995) 195-200

Fig. 3. Histograms of CD antigens on ConA blasts. a: the blasts are 80% CD3 positive showing an increase over resting PBL. 6: the majority of blasts express CD25 (IL-2 receptor) , this antigen is only expressed on a few (l-2%1 of resting cells. c: blasts all express CD18 (LFAlpJ: the anti-CDlla (LFAla) monoclonal gives a weak reaction with blasts, but not with resting cells suggesting that it is upregulated on these cells.d: at present no monoclonal anti-CD54 (ICAMI) which is the ligand of LFAl in cell/cell interactions has reacted positively with tamarin cells.

antibody was at best only weakly cross-reactive (Fig. 3~). Neither CD54 antibody tested stained any of the tamarin cells tested (Fig. 3d).

4. Discussion Several features

of the immune system of S. Watkins et al. (1988) demonstrated an unusually restricted number of MHC class I poymorphisms in this species, in conjunction with relatively normal polymorphic MHC class II products. Letvin et al. (1983) also demonstrated that monoclonal antibodies directed at human cell surface markers cross-react with primate lymphocytes. The frequency of cross reaction was inversely proportionate to the phylogenetic distance between species, thus cross-reactions between tamarinspecific and human-specific monoclonals were less

Oedipus have already been described,

199

frequent than those between antibodies used to compare humans and chimpanzees or humans and macaques. Monoclonal antibodies recognising CD2 (Tll) and CD4 (T4a) but not CD8 epitopes of S. Oedipus were described. The data presented here extend the range of reagents to cover the most common lymphocyte subset markers. Indeed, wherever sufficient numbers of reagents have been tested we have always found a match (e.g., CD23 one out of five). There is a remarkable similarity between human and tamarin lymphocytes in the number and distribution of lymphocyte subsets in blood. In particular the CD4:CD8 ratio of 2:l is the same as that reported for humans, but differs from than that of 0.5:1 reported for cynomologous monkey Macaca fascicularis (Bleavins et al., 1993). The cotton top tamarin has been proposed as a model for human disease with respect to both colitis and EBV (Chalifoux and Bronson, 1981; Morgan, 1992). Given that the immune system has a key role in the pathogenesis of these diseases it is essential that methods for the study of tamarin immunology keep pace with advances in the field. The extended range of monoclonals detecting tamarin CD markers will permit detailed analysis of the immune responses associated with EBV and ulcerative colitis in this species. References Bleavins, M.R., Brott, D.A., Alvey, J.D. and De la Inglesia, F.A. (1993) Flow cytometric characterisation of lymphocyte subpopulations in the cynomolgus monkey (Macaca fascicularis) Vet. Immunol. Immunopathol. 37. l-13. Cleary, M.L., Epstein, M.A., Finerty, S.. Dorfman, R.F., Bornkamm, G., Kirkwood, J.K., Morgan, A. and Sklar, J. (1985) Individual tumours of multifocal EB virus induced malignant lymphoma in tamarins arise from different B cell clones. Science 288, 722-724. Chalifoux, L.V. and Bronson, R.R. (1981) Colonic adenocarcinoma associated with chronic colitis in cotton top marmosets. Gastroenterology 80, 942-946. Finerty, S., Scullion, F.T. and Morgan, A.J. (1988) Demonstration in vitro of cell mediated immunity to Epstein-Barr virus in cotton-top tamarins. Clin. Exp. Immunol. 73, 181185. Letvin, N.L., King, N.W., Reinherz, E.L.. Hunt. R.D., Lane, H. and Schlossman, S.F. (1983) T lymphocyte surface antigens in primates. Eur. J. Immunol. 13. 345-347.

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Morgan, A.J. (1992) The development of Epstein-Barr virus vaccines. Vaccine 10, 564-571. Taylor, P.M., et al. (1987) In: C.G.B. Klaus (Ed.), Lymphocytes a Practical Approach. IRL Press, Oxford. Watkins, D.I., Hodi, S. and Letvin, N.L. (1988) A primate with limited major histocompatibility class I polymorphism. Proc. Natl. Acad. Sci. USA 8.5, 7714-7718.

Wolfe, L.G. and Deinhardt, F. (1978) In: E.I. Goldsmith and F.J. Moor-Jankowsskir (Eds.), Primates in Medicine: Marmosets in Experimental Medicine. Karger, New York, pp. 96-118.