Journal of immunological Methods. 153 0992) 85-91
85
~) 1992 Elsevier SciencePublishers B.V. All rights reserved 0022-1759/92/$05.00
J1M06390
A monoclonal antibody recognising an epitope associated • 41,~ ~ ~. i°cccp~ors with pig m,er.,,,.k...-,, M i c h a e l Bailey, Karin Stevens, Paul W. Bland and C h r i s t o p h e r R. Stokes Department of Veterinary Medicine. Unirersity of Bris.~ol,Langford Hovw. Lang[ord. Bristol. UK
(Received I May 1991,revised received 13 March 1992.accepted 31 March 1992
A monoclonal antibody is described which recognises an epitope associated with a receptor for interleukin-2 (IL-2) on pig lymphocytes. The monoclonal antibody inhibits high affinity binding of radiolabelled recombinant human IL-2 (rhlL-2) by pig lymphoblasts and also non-competitively inhibits both pig-TCGF and rhlL-2 maintained proliferation. By flow cytometry the antigen is apparently not present on freshly isolated blood lymphoeytes but is detectable on small cells between 6 and 12 h after activation and on large cells by 24-h. These findings are comparable with those obtained using monoclonal antibodies recognising the 55 kDa a chain of the human and mouse IL-2 receptor (p55, TAC) expressed on activated cells in vivo and in vitro. However, the molecular weight of the porcine antigen is between 65 and 70 kDa. Key words: lnterleukin-2receptor; Monoclonalantibody;(Pig)
Introduction
In both mouse and man binding studies have demonstrated three classes of cell surface IL-2 receptor with dissociation constants of approximately 30 nM, 1 nM and 20 pM (Robb et al., 1981, 1984; Dukovich et al., 1987; Sharon et al., 1987). Recent studies in both species suggest that the high affinity receptor represents a complex of, at least, the two lower affinity receptors (Robb
Correspondence to: M. Bailey, Department of Veterinary Medicine, Langford House, Langford, Bristol BSI8 7DU, UK. Abbreriations: rhlL-2, recombinant human interleukin-2; PBL, peripheral blood lymphocytes;aMM, a-methyl mannoside; TCGF, T cell growth factor, ConA supernatant; FCS, foetal calf serum; FALS, forward angle light scatter; GFL, green fluorescence.
et al., 1987; Tsudo et al., 1987) now termed the a (p55, CD25, TAC) and /3 (p70, p75) chains. Signal transduction and internalisation is apparently dependent upon the medium affinity /3 chain (Kd = 1 nM), which may be expressed by a number of resting cell types (Bich-Thuy et al., 1987; Siegal et al., 1987; Tanaka et al., 1988; Sharon et al., 1988), and a recently identified y chain (Sugamura et al., 1990). Several other possible components of an 1L-2 receptor complex have been described (Saragovi et al., 1990; Sharon et al., 1990). The a chain is expressed following activation and co-operates in the formation of high affinity receptors on activated cells. Monoclonal antibodies have been characterised which recognise, and interfere with activation through, mouse and primate receptors (Robb et ai., 198Z: Osawa et al., 1984; Takeshita et al., 1989) and have been used in vivo for suppression of immune
responses following transplantation (Hahn et al., 1990). Pig iymphoblasts are responsive to both native and recombinant human IL-2 (Eqglish et al., 1985; Fong and Doyle, 1986; Stott et al., 1986). It appears, therefore, that pig iymphoblasts can express a receptor capable of IL-2-binding and signal transduction. However, covalent cross-linking using IZSl-labelled recombinant human IL-2 (rhlL-2) has identified only a single 70 kDa surface molecule (Takamatsu et al., 1989). Despite the use of pigs in transplantation studies (Ricordi et al., 1990), no monocionals specific for the components of the pig IL-2 receptor have been described although one report found no cross-reactivity between pig and human IL-2 receptors (Aasted et ai., 1988). This report describes a monoclonal antibody which recognises a 65-70 kDa molecule expressed on activated, but not resting pig iymphocytes, and which inhibits the functional binding of IL-2.
Materials
and methods
Lymphocytes PBL were prepared by centrifugation (700 × g, 40 min)of peripheral blood diluted 1/2 in Hanks' balanced salt solution (HBSS, Flow Laboratories, Rickmansworth) on a cushion of Ficoll-Hypaque s.g. 1.077 (Ficoll-Paque, Pharmaeia, Milton Keynes). Lymphocytes were recovered from the interface, washed three times in HBSS and resuspended in RPMI 1640 medium (Dutch modification, Flow Laboratories) supplemented with 10% foetal calf serum (FCS, Gibco, Uxbridge), 10 -5 M 2-mercaptocthanol, 200 IU penicillin and 100 #g streptomycin/ml (complete medium). Lymphoblasts were prepared following culture at a density of 5 × 10s cells/ml in medium containing 5 p.g/ml of concanavalin A (Sigma). Cell suspensions for subsequent culture or binding assays were centrifuged over Ficoll-Hypaque, s.g. 1.077, to remove dead cells al~d were washed three times in HBSS containing 25 mM aMM before use and resuspended in complete medium containing 12.5 mM aMM (Sigma). Cells for analysis by flow cytometry were subjected to hy-
potonic shock to lyse remaining erythrocytes and were then washed and resuspended in complete medium.
T cell growth factor Pig T cell growth factor (TCGF) was prepared according to the method of Charley et al. (personal communication). Freshly isolated peripheral blood lymphoeytes were rested in culture for 24 h before adding ConA to a final concentration of 5 /~g/ml. After a further 24 h in culture the supernatant was harvested and excess ConA removed by two incubations with Sephadex G-50 (Pharmacia) pre-swollen in RPMI 1640 (Rich and Pierce, 1974). TCGF was dialysed overnight against RPMI 1640, sterilised by filtration (0.2 /~m, Sarstedt, Leicester) and stored in aliquots at - 20oc. RhIL-2 (specific activity 697 Ci/mmol) was obtained from Amersham International (Aylesbury) and stored in aliquots at -20°C. The ability of TCGF/IL-2 to maintain proliferation was assayed in culture using 3 or 4 day ConA blasts. 100 /~1 volumes of cells at 10~ cells/ml were cultured in medium containing 12.5 mM aMM (Sigma) in 96 well tissue culture plates (Falcon, Becton-Dickinson, Oxford) with 100/~1 of dilutions of TCGF/IL-2 and/or monoclonal antibody. After 24 h, 0.25/~Ci of tritiated thymidine (Amersham) per culture was added and 24 h later the cells were harvested onto glass fibre filter disks using a Titertek cell harvester (Flow Laboratories). Incorporated radiation was measured in a 1219 Rackbeta scintillation counter (LKB-Wallac, Milton Keynes).
Monoclonal antibody production Lymphocytes were prepared from the peripheral blood of six pigs and were pooled and cultured with 5 /zg/ml of ConA. After 24 h cells were reisolated on Ficoll-Hypaque gradients, washed and 0.1 mi (approximately 5 × 10a) of packed cells injected i.v. into each mouse. 2 weeks later a second injection was given and 5 days later the mice were killed. Spleen cells from the immunised mice were fused with cells from the mouse myeloma line P3-X63-Ag.8.653, using a modification of the method of Oi and Herzenberg (1980) Briefly,
myeloma cells were fused with spleen cells in selective H A T RPMI 1640 medium containing 15% FCS and supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 50 I U / m l penicillin, 50 /.Lg/ml streptomycin and 2.5 # g / m l amphotericin B. Growth promoting factors were added as mouse conditioned medium prepared by incubating spleen cells from a normal mouse in 50 ml of H A T medium for 2 days. 50% of spent supernatant was replaced every 3 - 4 days by fresh unconditioned H A T medium. After 10 days HAT medium was replaced by HT medium for the next two feeds. The first test for antibody production was performed 10 days post-fusion. Hybridoma supernatants were screened by their ability to inhibit proliferation of 3 day pig ConA blasts in the presence of pig TCGF. Pig lymphoblasts were prepared and cultured in 96 well plates as described with 50/zl of hybridoma supernatant and a concentration of pig T C G F giving 50% of maximum proliferation. Incorporation of tritiated thymidine was determined as described and wells with less than 50% of the expected incorporation were regarded as positive. Positive wells were cloned twice by limiting dilution until a stable hybrid cell line was obtained. The immunoglobulin isotype of the hybridoma 231.3B2 was determined to be lgG1 using a commercial isotyping kit (MMT RC1, Serotec).
",9
91%
4%
Low High FALS Fig. I. Stylised two parameter histogram used for analysis of activated cells by flow cytometry, High and low quadrants were defined using control, non-activated cells in order to maintain as close as possible to 95% low. FALS; forward angle light scatter. GFL; green fluorescence.
tured in the absence of ConA) were analysed first and the resulting two parameter histogram used to set 'high' and "low' FALS and G F L / F A L S ratio gates to maintain less than 5% unstimulated cells in the 'high' quadrants (Fig. I). Activated populations were subsequently analysed using identical gates to determine the percentage of the population with 'high' FALS a n d / o r G F L / F A L S .
IL-2 binding Flow cytometry Cells were incubated with hybridoma supernatant (1/1000) for 2 h, on ice in the presence of 0.2% sodium azide, washed and incubated for l h in the presence of FITC-conjugated goat antimouse Ig (Sigma). FITC stained cells were quantitated using an EPICS CS flow cytometer (Coulter Electronics). In initial experiments log green fluorescence (LGFL) and forward light scatter (FLS) were collected. For time course studies, FALS, green fluorescence (GFL) and the ratio of G F L to FLS were collected. A two parameter histogram of FALS on the x axis and the ratio of G F L to FALS on the y axis was used for analysis. The ratio was used to reduce the incidence of false positive increases in G F L since activated cells show increased FALS associated with an increase in size. The control population (lymphocytes cul-
Binding of 1~-51-conjugated rhlL-2 was measured following incubation for 60 rain at room temperature in 200/.d volumes containing 5 × 106 cells in RPMI 1640 plus 19% FCS. Control tubes also contained 250 U / m l of unlabelled rhIL-2. Cells were then directly centrifuged at 10,000 × g for 5 rain through cushions of 20% paraffin oil and 80% silicon oil. The tubes were frozen and the tips cut off and cell-associated radiation counted in a 1282 Compugamma (LKB-Wallac). Specific binding was calculated after subtraction of uninhibitable binding detected in control tubes in the presence of excess unlabelled rhlL-2. Inhibition experiments were performed in the presence of 250 U / m l of unlabelled rhIL-2 or of serial dilutions of the monoclonal antibody 231.3B2 or a second, isotype-matched monoclonal 230.3G4 which also binds to lymphocyte cell surfaces.
lmmunoprecipitation Surface molecules on 3 day activated pig lymphoblasts were iodinated (~251, Dupont, Hertfordshire) using the lactoperoxidase method. Iodinated cells were lysed in 10 mM Tris-buffered saline (pH 7.4) containing 1% deoxycholate, 1% Triton X-100 and 0.1% SDS. Nuclei were removed by centrifugation and the supernatant stored at -70°C. Thawed supernatant was precleared with protein G coated beads (Affigel, Bio-Rad, Hemel Hempsted) and subsequently incubated overnight with an equal volume of hybridoma supernatant. Bound antigen was precipitated with protein G beads, released by boiling in Laemmli sample buffer and run on 8.5% polyacrylamide gel under reducing conditions. Dried gels were autoradiographed using Kodak X-Omat film at - 7 0 ° C for 3 - 5 days.
Results
Appearance of antigen on cell surfaces Control and test histograms of fresh and 48 h activated cells are shown in Fig. 2. Numbers of freshly isolated cells expressing the antigen recognised by the monoclonal antibody 231.3B2 was low and expression was of low intensity. In con-
(b)
TABLE 1 PERCENT OF CELLS APPEARING IN 'LOW' AND 'HIGH' FLOW CYTOMETRY QUADRANTS (see Fig. I) FOLLOWING ACTIVATION WITH CONCANAVALIN A. Quadrants were defined using unstimulated cells to achieve as close as possible to 5% in the 'low' window for each parameter. GFL green fluorescence;FALS, forward angle light scatter GFL/FALS FALS Time (h) 3 6 12 24 48
Low Low
High
High Low
High
02.65 86.18 57.77 34.39 25.35
4.44 8.69 4.23 2.00 5.15
2.41 4.86 34.47 41.78 31.18
0.50 1.27 3.53 21.83 38.82
trast, the majority of cells recovered after 48 h expressed high levels of the antigen. Table I shows the percent of cells appearing in flow cytometry quadrants at time intervals following activation compared with non-activated cells. Between 6 and 12 h there was an increase in cells appearing in the high fluorescence quadrants. Initially these cells appeared in the low FALS quadrant, i.e., there was an increase in expression of the antigen without an increase in size. Subsequently, between, 12 and 48 h, cells accumulated in the high fluorescence, high FALS quadrant. These cells exhibited a much increased expression of surface antigen and increased size. In contrast there was no increase in the number of cells with increased size but not expressing increased surface antigen. The results suggest that the antigen was expressed following activation, prior to any increase in size, but that all ~arge blast cells expressed the antigen.
Effect on TCGF / IL-2 maintained blast cell proliferation
1
10
100
1000
Green Fluorescence Fig. 2. Expression of 231.3B2 antigen by (a) fresh cells; (b) following 48 h activation with ConA. Solid lines, cells incubated without monoclonal antibody; dotted line, cells incubated with monoclonalantibody 231.3B2.
Fig. 3 shows the effect of hybridoma supernatant on proliferation of 3 day ConA blasts in the presence of increasing concentrations of pig T cell growth factor or of rhlL-2. The addition of low concentrations of monoclona! antibody 231.3B2 increased the concentration of rhlL-2 required to maintain blast cell proliferation. However, this inhibition was not apparently due
100 ~" o
/-.:~ -.-f
~ s
(b)
TABLE II INHIBITION OF SPECIFIC BINDING OF =~l-rhlL-2 TO PIG LYMPHOBLASTS BY MONOCLONAL ANTIBODY 231.3B2.
,'1=
.'
Inhibitor °
0
0.01 0.1
1
10 100
1
10
100
rh 1[.-2(ulmi) % TCGF Fig. 3. Effect of monoclonalantibody231.3B2on proliferation of concanavalinA-activated pig lymphocytesmaintainedwith (a) recombinant human IL-2; (b) pig TCGF. Hyhridoma supernatant was added to a final concentrationof: a, 1/3125; b, 0; c, I/6Z'~; d, !/125; e, 1/25; f, I/5.
to simple competition, since higher concentrations o f 231.3B2 did not f u r t h e r increase the concentration o f rhIL-2 required.
Competitive inhibition of binding Specific binding o f rhlL-2 to pig lymphoblasts u n d e r the range o f conditions used indicated a single affinity r e c e p t o r (Fig. 4; K o approximately 65 pM; 720 receptors per cell). In subsequent assays a final concentration o f 60 p M o f rhlL-2 was used. Table 11 sbows the counts added as 1251labelled rhlL-2 and counts recovered in the cell pellet in the absence of any inhibitor and in the presence of 250 U / m l of cold rhlL-2 or of dilutions of the monoclonal 231.3B2 or 230.3G4. Specific binding was calculated after subtraction of
None rhlL-2 231.3B2 230.3G4
Concentration
CPM Added
Bound
% specific binding
13,693 13,¢,32 !3,755 13,9,!8 14,070 13,9~,'5 13,423
2,435 360 473 436 i,536 2,579 2,437
100.0 0.0 5.5 3.7 56.7 106.9 100.1
250 U/ml I / 10 1/ 100 1/1000 I / 10 I / I0(1
non-specific counts b o u n d in the presence o f 250 U / m l o f cold rhlL-2.
Molecular weight T h e monoclonai antibody 231.3132 precipitated a single b a n d on P A G E with a molecular weight o f 6 7 - 6 8 kDa (Fig. 5).
66 ~ Kd = 65 pM 720 molecules/cell
45
~
~:"
,==IP~
36 29 24 ,--I=~ 20 ==1=~ 500 0 Bound (molecules/cell) Fig. 4. Scatchard plot of specific (uninhibitable) binding of l~Sl-labelled.rhIL-2to 3 day pig lymphoblasts.
Fig. 5. Autoradiograph of polyacrylamide gel loaded with IZ~l-labelled surface proteins immunoprecipitated from pig ConA blasts with the hybridoma 231 3B2. The position and size (kDa) of molecular weight markers run concurrently are shown.
Discussion The monoclonal antibody 231.3B2 recognises an antigen which is expressed early on cell surfaces following activation by concanavalin A. The antigen is expressed on cells before increases in size can be detected by flow cytometry; however, cells which do demonstrate increased forward angle light scatter all express increased levels of the antigen. The surface antigen recognised is thus closely associated with cell activation and the time course of expression and heterogeneous distribution of staining intensity is consistent with the expression of IL-2 receptors in populations of activated human lymphocytes (Cantrell and Smith, 1983, 1984). The ability of the monoclonal antibody to inhibit specific binding of rhlL-2; to pig blasts suggest that it is recognising an epitope on or close to a binding site for rhiL-2, taken together with its ability to inhibit rhlL-2 maintained proliferation this demonstrates that the rhlL-2-binding site is functionally active, i.e., that it is acting as a receptor. The non-competitive nature of the inhibition is consistent with :'~erferenee with the formation of high affinity receptors rather than with competetive occupancy of a single IL-2 binding site. Monoclonal anti-Tac (IL-2Ra, CD25) behaves similarly (Leonard et ai., 1982, Wang and Smith, 1987). The molecular weight of the antigen recognised by the monoclonal 231.3B2 (65-70 kDa) is greater than that reported for the human a chain. It is, however, consistent with the finding only of relatively high affinity receptors and a single 6570 kDa molecular weight band from activated pig lymphocytes following covalent linking to t251rhlL-2 (Takamatsu et ai., 1989). In comparison, on activated ruminant iymphocytes the same authors found both low and high affinity receptors and 50-55 kDa and 65-80 kDa IL-2-crosslinking molecules, and a 55 kDa homologue of the bovine a chain has been sequenced (Weinberg et al., 1988). Thus, a phyiogenetic modification may have occurred resulting in a and /3 chains of similar weight in the pig, or, alternatively the porcine /3 chain or a novel 70 kDa component may be heavily upregulated following activation. The determined affinity of the specific binding
of rhlL-2 to pig lymphocytes (K d approximately 65 pM) is intermediate between the human medium (K d = 1 riM) and high (K d = 20 pM) affinity receptors (Tsudo et al., 1987). Since no evidence was obtained in this study for more than a single receptor this difference is likely to be attributable to a redueed affinity of a pig high affinity receptor complex for rhlL-2 compared to native pig IL-2. Low affinity of an a chain equivalent would also account for the failure to detect low affinity receptors on activated cells. The estimated number of receptors per cell (less than 1000) is also lower than estimates for human high affinity receptors (Robb et al., 1984) but agrees with previous estimates of number and affinity of pig IL-2 receptors (Takamatsu et al., 1989). In summary, we have demonstrated that a monoclonal antibody 231.3B2 recognises an epitope on a 65-70 kDa molecule associated with a receptor for interleukin-2 on pig lymphocytes. The molecule recognised is not present on resting cells but is upregulated following nonspeeific activation in vitro activation. Binding of the antibody to activated cells inhibits IL-2 driven proliferation.
Acknowledgements This work was supported by a Link Grant from the Agriculture and Food Research Council and by the Institute for Animal Health Compton.
References Aasted, B., Blixenkrone-Moller,M., Larsen, E.B., Ohmann, H.B., Simesen, R.B. and Uttenthal, A. (1988) Reactivityof eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals. Vet. lmmunol. Immunopathol. 19, 31-38. Bich-Thuy, L.T., Dukovich, M., Peffer, N.J., Fauci, A.S., Kehrl, J.H. and Greene, W.C. (1987) Direct activationof human resting T cells by IL 2; the role of an IL-2 receptor distinct from the Tac protein. J. Immunol. 139,1550-1556. CantrelL D.A. and Smith, K.A. (1983) Transient expression of interleukin 2 receptors. J. Exp. Med. 158, 1895-1911. Cantrell, D.A. and Smith, K.A. (1984)The interleukin 2 T cell system: a new cell growth model. Science224, 1312-1316. Dukovich, M., Wano, Y., Bich-Thuy, L., Katz, P., Cullen, B.R., Kehrl, J.H. and Greene, W.C. (1987) A second
human interleukin-2 binding protein that may be a component of high affinity interleukin-2 receptors. Nature 327, 518-522. English, L.S., Binns, R.M. and Licenee, S.T. (19851 Characterisation of pig T ceil growth factor and its species-restricted activity on human, mouse and sheep cells. Vel. Immunol. Immunopathol. 9, 59-69. Fong, S. and Doyle, M.V. (19861 Response of bovine and porcine peripheral blood mononuclear cells to human recombinant interleukin 2 (1251 Vet. Immunol. lmmunopathol. 11, 91-100. Hahn, H.J., Kuttler, B., Volk, H.D., Lucke, S. and Diamantstein, T. (1990) Intedeukin 2 receptor-targeted therapy in islet transplantation research. Herin. Metab. Res. 25, 177-180. Leonard, W.J., Depper, J.M., Uchiyama, T., Smith, K.A., Waldmann, T.A. and Greene, W.C. (19821 A monoclonal antibody that appears to recognise the receptor for human T-cell growth factor; partial eharacterisation of the receptor. Nature 300, 267-269. Oi, V.T. and Herzenberg, LA. (19801 Immunoglobulin-producing hybrid cell lines. In: B.B. Mishell and S.M. Shiigi (Eds.), Sdected methods in cellular immunology. Freeman, San Francisco, CA. Osawa, H. and Diamantstein, T. (19841 A ral monoelonal antibody that binds specifically to mouse T lymphoblasts and inhibits IL-2 receptor functions; a putative anti-lL-2 receptor antibody. J. Immunol. 132, 2445-2450. Ricordi, C., Socci, C., Davalli, A.M., Staudacher, C., Vertova, A., Bare, P., Freschi, M., Gavazzi, F., Bertuzzi, F., Pozza, G. aud Di Carlo, V. (1990) Swine islet isolation and transplm~tation. Herin. Metab. Res. 25, 26-30. Robb, R.J. and Greene, W.C. (19831 Direct demonstration of the identity of T cell growth factor binding protein and the Tac antigen. J. Exp. Med. 158, 1332-1337. Robb, R.J., Munck, A. and Smith, K.A. (19811 T cell growth factor receptors. Quantitation, specificity and biological relevence. J. Exp. Med. 154, 1455. Robb, R.J., Greene, W.C. and Rusk, C.M. (19841 Low and high affinity cellular receptors for interleukin 2. Implications for the level of Tac antigen. J. Exp. Med. 16(I, 1126-1146. Robb, R.J., Rusk, C.M., Yodel, J. and Greene, W.C. (19871 lnterleukin 2 binding molecule distinct from the Tac protein: analysis of its role in formation of high affinity receptors. Prec. Natl. Acad. SCi. USA 84, 2002-2006. Saragovi, H. and Malek, T.R. (19901 Evidence for additional
subunits associated to the mou~ interleukin-2 receptor p55/p75 complex. Proc. Natl. Acad. Sci. USA 87, 11-15. Sharon, M., Siegal, J.P,, Tosato, G,, Todoi, J., Gerrard, T.L. and Leonard, WJ. (1988)The human interleukin 2 receptor a chain (p70) Direct identification, partial purification. and patterns of expression on peripheral blood mononuclear cells. J. Exp. Mcd. 167, 1265-1270. Sharon, M., Gnarra, J.R., Leonard, W.J. (19911)A 100-kilodalton protein is associated with the routine interleukin-2 receptor: Biochemical evidence that pl00 is distinct from the alpha and beta chains. Proc. Natl. Acad. Sci. USA 87, 4869-4873. SiegaL J.P., Sharon, M., Smith, E L ' a n d I..¢onard, WJ. (19871 The IL-2 receptor ot chain (p70) : role in mediating signals for LAIL NK and proliferative activities. Science 238, 75-78. Stott, J.L., Fenwick, B.W. and Osboro, B.I. (1986) Human recombinant interleukin 2 augments in vitro blastogenisis of bovine and porcine lymphocytes. Vet. Immunol. Immunopathol. 13, 31-38. Sugamura, K., Takeshita, T., Asao, H., Kumaki, S., Ohbo, K., Ohtani, K. and Nakamura, M. (19901 IL-2-indueed signal transduction: Involvement of tyrosine kinase and IL-2 receptor gamma chain. Lympbokine Res. 9, 539-542. Takamatsu, H., Denycr, M.S., Collen, T., Stokes, A. and Tucker, D.L. (19891 Characterisation and comparison of putative interleukin-2 receptors of cattle, sheep, pigs and horses. Immunobiology 4 (suppl.), 72. Takeshita, T., Goto, Y.. Tada, K., Nagata, K., Asao, H. and Sugamura, K. (1989) Monoclonal antibody defining a molecule possibly identical to the p75 sobunit of interleukin 2 receptor. J. Exp. Med. 169, 1323-1332. Tanaka, T., Saiki, O., Satoro, D., Shigcru. N. and Kishimoto, S. (1988) lnterleukin-2 functions through novel intcrleukin-2 binding molecules on T cells. J. lmmunol. 140, 470-473. Tsudo, M., Kozak, R.W., Goldman, C.K. and Waldmann, T.A. (1987) Contribution of a p75 interleukin-2 binding peptide to a high affinity interleukin-2 receptor complex, Prec. Natl. Acad. Sci. USA 84, 4215-4218. Wang, H.-M. and Smith, K.A, (19871 The interleukin 2 receptor. Functional consequences of its bimolecular structure, J. Exp. Med. 166, 1055-1069. Weinberg, A.D., Shaw, L, Paetkau, V., Bleackley, R.C., Magn uson, N.S., Reeves, R. and Magnuson, LA. (19881 Cloning of eDNA for the bovine IL-2 receptor (bovine Tac antigen). Immunology 63, 603-610.
85
~) 1992 Elsevier SciencePublishers B.V. All rights reserved 0022-1759/92/$05.00
J1M06390
A monoclonal antibody recognising an epitope associated • 41,~ ~ ~. i°cccp~ors with pig m,er.,,,.k...-,, M i c h a e l Bailey, Karin Stevens, Paul W. Bland and C h r i s t o p h e r R. Stokes Department of Veterinary Medicine. Unirersity of Bris.~ol,Langford Hovw. Lang[ord. Bristol. UK
(Received I May 1991,revised received 13 March 1992.accepted 31 March 1992
A monoclonal antibody is described which recognises an epitope associated with a receptor for interleukin-2 (IL-2) on pig lymphocytes. The monoclonal antibody inhibits high affinity binding of radiolabelled recombinant human IL-2 (rhlL-2) by pig lymphoblasts and also non-competitively inhibits both pig-TCGF and rhlL-2 maintained proliferation. By flow cytometry the antigen is apparently not present on freshly isolated blood lymphoeytes but is detectable on small cells between 6 and 12 h after activation and on large cells by 24-h. These findings are comparable with those obtained using monoclonal antibodies recognising the 55 kDa a chain of the human and mouse IL-2 receptor (p55, TAC) expressed on activated cells in vivo and in vitro. However, the molecular weight of the porcine antigen is between 65 and 70 kDa. Key words: lnterleukin-2receptor; Monoclonalantibody;(Pig)
Introduction
In both mouse and man binding studies have demonstrated three classes of cell surface IL-2 receptor with dissociation constants of approximately 30 nM, 1 nM and 20 pM (Robb et al., 1981, 1984; Dukovich et al., 1987; Sharon et al., 1987). Recent studies in both species suggest that the high affinity receptor represents a complex of, at least, the two lower affinity receptors (Robb
Correspondence to: M. Bailey, Department of Veterinary Medicine, Langford House, Langford, Bristol BSI8 7DU, UK. Abbreriations: rhlL-2, recombinant human interleukin-2; PBL, peripheral blood lymphocytes;aMM, a-methyl mannoside; TCGF, T cell growth factor, ConA supernatant; FCS, foetal calf serum; FALS, forward angle light scatter; GFL, green fluorescence.
et al., 1987; Tsudo et al., 1987) now termed the a (p55, CD25, TAC) and /3 (p70, p75) chains. Signal transduction and internalisation is apparently dependent upon the medium affinity /3 chain (Kd = 1 nM), which may be expressed by a number of resting cell types (Bich-Thuy et al., 1987; Siegal et al., 1987; Tanaka et al., 1988; Sharon et al., 1988), and a recently identified y chain (Sugamura et al., 1990). Several other possible components of an 1L-2 receptor complex have been described (Saragovi et al., 1990; Sharon et al., 1990). The a chain is expressed following activation and co-operates in the formation of high affinity receptors on activated cells. Monoclonal antibodies have been characterised which recognise, and interfere with activation through, mouse and primate receptors (Robb et ai., 198Z: Osawa et al., 1984; Takeshita et al., 1989) and have been used in vivo for suppression of immune
responses following transplantation (Hahn et al., 1990). Pig iymphoblasts are responsive to both native and recombinant human IL-2 (Eqglish et al., 1985; Fong and Doyle, 1986; Stott et al., 1986). It appears, therefore, that pig iymphoblasts can express a receptor capable of IL-2-binding and signal transduction. However, covalent cross-linking using IZSl-labelled recombinant human IL-2 (rhlL-2) has identified only a single 70 kDa surface molecule (Takamatsu et al., 1989). Despite the use of pigs in transplantation studies (Ricordi et al., 1990), no monocionals specific for the components of the pig IL-2 receptor have been described although one report found no cross-reactivity between pig and human IL-2 receptors (Aasted et ai., 1988). This report describes a monoclonal antibody which recognises a 65-70 kDa molecule expressed on activated, but not resting pig iymphocytes, and which inhibits the functional binding of IL-2.
Materials
and methods
Lymphocytes PBL were prepared by centrifugation (700 × g, 40 min)of peripheral blood diluted 1/2 in Hanks' balanced salt solution (HBSS, Flow Laboratories, Rickmansworth) on a cushion of Ficoll-Hypaque s.g. 1.077 (Ficoll-Paque, Pharmaeia, Milton Keynes). Lymphocytes were recovered from the interface, washed three times in HBSS and resuspended in RPMI 1640 medium (Dutch modification, Flow Laboratories) supplemented with 10% foetal calf serum (FCS, Gibco, Uxbridge), 10 -5 M 2-mercaptocthanol, 200 IU penicillin and 100 #g streptomycin/ml (complete medium). Lymphoblasts were prepared following culture at a density of 5 × 10s cells/ml in medium containing 5 p.g/ml of concanavalin A (Sigma). Cell suspensions for subsequent culture or binding assays were centrifuged over Ficoll-Hypaque, s.g. 1.077, to remove dead cells al~d were washed three times in HBSS containing 25 mM aMM before use and resuspended in complete medium containing 12.5 mM aMM (Sigma). Cells for analysis by flow cytometry were subjected to hy-
potonic shock to lyse remaining erythrocytes and were then washed and resuspended in complete medium.
T cell growth factor Pig T cell growth factor (TCGF) was prepared according to the method of Charley et al. (personal communication). Freshly isolated peripheral blood lymphoeytes were rested in culture for 24 h before adding ConA to a final concentration of 5 /~g/ml. After a further 24 h in culture the supernatant was harvested and excess ConA removed by two incubations with Sephadex G-50 (Pharmacia) pre-swollen in RPMI 1640 (Rich and Pierce, 1974). TCGF was dialysed overnight against RPMI 1640, sterilised by filtration (0.2 /~m, Sarstedt, Leicester) and stored in aliquots at - 20oc. RhIL-2 (specific activity 697 Ci/mmol) was obtained from Amersham International (Aylesbury) and stored in aliquots at -20°C. The ability of TCGF/IL-2 to maintain proliferation was assayed in culture using 3 or 4 day ConA blasts. 100 /~1 volumes of cells at 10~ cells/ml were cultured in medium containing 12.5 mM aMM (Sigma) in 96 well tissue culture plates (Falcon, Becton-Dickinson, Oxford) with 100/~1 of dilutions of TCGF/IL-2 and/or monoclonal antibody. After 24 h, 0.25/~Ci of tritiated thymidine (Amersham) per culture was added and 24 h later the cells were harvested onto glass fibre filter disks using a Titertek cell harvester (Flow Laboratories). Incorporated radiation was measured in a 1219 Rackbeta scintillation counter (LKB-Wallac, Milton Keynes).
Monoclonal antibody production Lymphocytes were prepared from the peripheral blood of six pigs and were pooled and cultured with 5 /zg/ml of ConA. After 24 h cells were reisolated on Ficoll-Hypaque gradients, washed and 0.1 mi (approximately 5 × 10a) of packed cells injected i.v. into each mouse. 2 weeks later a second injection was given and 5 days later the mice were killed. Spleen cells from the immunised mice were fused with cells from the mouse myeloma line P3-X63-Ag.8.653, using a modification of the method of Oi and Herzenberg (1980) Briefly,
myeloma cells were fused with spleen cells in selective H A T RPMI 1640 medium containing 15% FCS and supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 50 I U / m l penicillin, 50 /.Lg/ml streptomycin and 2.5 # g / m l amphotericin B. Growth promoting factors were added as mouse conditioned medium prepared by incubating spleen cells from a normal mouse in 50 ml of H A T medium for 2 days. 50% of spent supernatant was replaced every 3 - 4 days by fresh unconditioned H A T medium. After 10 days HAT medium was replaced by HT medium for the next two feeds. The first test for antibody production was performed 10 days post-fusion. Hybridoma supernatants were screened by their ability to inhibit proliferation of 3 day pig ConA blasts in the presence of pig TCGF. Pig lymphoblasts were prepared and cultured in 96 well plates as described with 50/zl of hybridoma supernatant and a concentration of pig T C G F giving 50% of maximum proliferation. Incorporation of tritiated thymidine was determined as described and wells with less than 50% of the expected incorporation were regarded as positive. Positive wells were cloned twice by limiting dilution until a stable hybrid cell line was obtained. The immunoglobulin isotype of the hybridoma 231.3B2 was determined to be lgG1 using a commercial isotyping kit (MMT RC1, Serotec).
",9
91%
4%
Low High FALS Fig. I. Stylised two parameter histogram used for analysis of activated cells by flow cytometry, High and low quadrants were defined using control, non-activated cells in order to maintain as close as possible to 95% low. FALS; forward angle light scatter. GFL; green fluorescence.
tured in the absence of ConA) were analysed first and the resulting two parameter histogram used to set 'high' and "low' FALS and G F L / F A L S ratio gates to maintain less than 5% unstimulated cells in the 'high' quadrants (Fig. I). Activated populations were subsequently analysed using identical gates to determine the percentage of the population with 'high' FALS a n d / o r G F L / F A L S .
IL-2 binding Flow cytometry Cells were incubated with hybridoma supernatant (1/1000) for 2 h, on ice in the presence of 0.2% sodium azide, washed and incubated for l h in the presence of FITC-conjugated goat antimouse Ig (Sigma). FITC stained cells were quantitated using an EPICS CS flow cytometer (Coulter Electronics). In initial experiments log green fluorescence (LGFL) and forward light scatter (FLS) were collected. For time course studies, FALS, green fluorescence (GFL) and the ratio of G F L to FLS were collected. A two parameter histogram of FALS on the x axis and the ratio of G F L to FALS on the y axis was used for analysis. The ratio was used to reduce the incidence of false positive increases in G F L since activated cells show increased FALS associated with an increase in size. The control population (lymphocytes cul-
Binding of 1~-51-conjugated rhlL-2 was measured following incubation for 60 rain at room temperature in 200/.d volumes containing 5 × 106 cells in RPMI 1640 plus 19% FCS. Control tubes also contained 250 U / m l of unlabelled rhIL-2. Cells were then directly centrifuged at 10,000 × g for 5 rain through cushions of 20% paraffin oil and 80% silicon oil. The tubes were frozen and the tips cut off and cell-associated radiation counted in a 1282 Compugamma (LKB-Wallac). Specific binding was calculated after subtraction of uninhibitable binding detected in control tubes in the presence of excess unlabelled rhlL-2. Inhibition experiments were performed in the presence of 250 U / m l of unlabelled rhIL-2 or of serial dilutions of the monoclonal antibody 231.3B2 or a second, isotype-matched monoclonal 230.3G4 which also binds to lymphocyte cell surfaces.
lmmunoprecipitation Surface molecules on 3 day activated pig lymphoblasts were iodinated (~251, Dupont, Hertfordshire) using the lactoperoxidase method. Iodinated cells were lysed in 10 mM Tris-buffered saline (pH 7.4) containing 1% deoxycholate, 1% Triton X-100 and 0.1% SDS. Nuclei were removed by centrifugation and the supernatant stored at -70°C. Thawed supernatant was precleared with protein G coated beads (Affigel, Bio-Rad, Hemel Hempsted) and subsequently incubated overnight with an equal volume of hybridoma supernatant. Bound antigen was precipitated with protein G beads, released by boiling in Laemmli sample buffer and run on 8.5% polyacrylamide gel under reducing conditions. Dried gels were autoradiographed using Kodak X-Omat film at - 7 0 ° C for 3 - 5 days.
Results
Appearance of antigen on cell surfaces Control and test histograms of fresh and 48 h activated cells are shown in Fig. 2. Numbers of freshly isolated cells expressing the antigen recognised by the monoclonal antibody 231.3B2 was low and expression was of low intensity. In con-
(b)
TABLE 1 PERCENT OF CELLS APPEARING IN 'LOW' AND 'HIGH' FLOW CYTOMETRY QUADRANTS (see Fig. I) FOLLOWING ACTIVATION WITH CONCANAVALIN A. Quadrants were defined using unstimulated cells to achieve as close as possible to 5% in the 'low' window for each parameter. GFL green fluorescence;FALS, forward angle light scatter GFL/FALS FALS Time (h) 3 6 12 24 48
Low Low
High
High Low
High
02.65 86.18 57.77 34.39 25.35
4.44 8.69 4.23 2.00 5.15
2.41 4.86 34.47 41.78 31.18
0.50 1.27 3.53 21.83 38.82
trast, the majority of cells recovered after 48 h expressed high levels of the antigen. Table I shows the percent of cells appearing in flow cytometry quadrants at time intervals following activation compared with non-activated cells. Between 6 and 12 h there was an increase in cells appearing in the high fluorescence quadrants. Initially these cells appeared in the low FALS quadrant, i.e., there was an increase in expression of the antigen without an increase in size. Subsequently, between, 12 and 48 h, cells accumulated in the high fluorescence, high FALS quadrant. These cells exhibited a much increased expression of surface antigen and increased size. In contrast there was no increase in the number of cells with increased size but not expressing increased surface antigen. The results suggest that the antigen was expressed following activation, prior to any increase in size, but that all ~arge blast cells expressed the antigen.
Effect on TCGF / IL-2 maintained blast cell proliferation
1
10
100
1000
Green Fluorescence Fig. 2. Expression of 231.3B2 antigen by (a) fresh cells; (b) following 48 h activation with ConA. Solid lines, cells incubated without monoclonal antibody; dotted line, cells incubated with monoclonalantibody 231.3B2.
Fig. 3 shows the effect of hybridoma supernatant on proliferation of 3 day ConA blasts in the presence of increasing concentrations of pig T cell growth factor or of rhlL-2. The addition of low concentrations of monoclona! antibody 231.3B2 increased the concentration of rhlL-2 required to maintain blast cell proliferation. However, this inhibition was not apparently due
100 ~" o
/-.:~ -.-f
~ s
(b)
TABLE II INHIBITION OF SPECIFIC BINDING OF =~l-rhlL-2 TO PIG LYMPHOBLASTS BY MONOCLONAL ANTIBODY 231.3B2.
,'1=
.'
Inhibitor °
0
0.01 0.1
1
10 100
1
10
100
rh 1[.-2(ulmi) % TCGF Fig. 3. Effect of monoclonalantibody231.3B2on proliferation of concanavalinA-activated pig lymphocytesmaintainedwith (a) recombinant human IL-2; (b) pig TCGF. Hyhridoma supernatant was added to a final concentrationof: a, 1/3125; b, 0; c, I/6Z'~; d, !/125; e, 1/25; f, I/5.
to simple competition, since higher concentrations o f 231.3B2 did not f u r t h e r increase the concentration o f rhIL-2 required.
Competitive inhibition of binding Specific binding o f rhlL-2 to pig lymphoblasts u n d e r the range o f conditions used indicated a single affinity r e c e p t o r (Fig. 4; K o approximately 65 pM; 720 receptors per cell). In subsequent assays a final concentration o f 60 p M o f rhlL-2 was used. Table 11 sbows the counts added as 1251labelled rhlL-2 and counts recovered in the cell pellet in the absence of any inhibitor and in the presence of 250 U / m l of cold rhlL-2 or of dilutions of the monoclonal 231.3B2 or 230.3G4. Specific binding was calculated after subtraction of
None rhlL-2 231.3B2 230.3G4
Concentration
CPM Added
Bound
% specific binding
13,693 13,¢,32 !3,755 13,9,!8 14,070 13,9~,'5 13,423
2,435 360 473 436 i,536 2,579 2,437
100.0 0.0 5.5 3.7 56.7 106.9 100.1
250 U/ml I / 10 1/ 100 1/1000 I / 10 I / I0(1
non-specific counts b o u n d in the presence o f 250 U / m l o f cold rhlL-2.
Molecular weight T h e monoclonai antibody 231.3132 precipitated a single b a n d on P A G E with a molecular weight o f 6 7 - 6 8 kDa (Fig. 5).
66 ~ Kd = 65 pM 720 molecules/cell
45
~
~:"
,==IP~
36 29 24 ,--I=~ 20 ==1=~ 500 0 Bound (molecules/cell) Fig. 4. Scatchard plot of specific (uninhibitable) binding of l~Sl-labelled.rhIL-2to 3 day pig lymphoblasts.
Fig. 5. Autoradiograph of polyacrylamide gel loaded with IZ~l-labelled surface proteins immunoprecipitated from pig ConA blasts with the hybridoma 231 3B2. The position and size (kDa) of molecular weight markers run concurrently are shown.
Discussion The monoclonal antibody 231.3B2 recognises an antigen which is expressed early on cell surfaces following activation by concanavalin A. The antigen is expressed on cells before increases in size can be detected by flow cytometry; however, cells which do demonstrate increased forward angle light scatter all express increased levels of the antigen. The surface antigen recognised is thus closely associated with cell activation and the time course of expression and heterogeneous distribution of staining intensity is consistent with the expression of IL-2 receptors in populations of activated human lymphocytes (Cantrell and Smith, 1983, 1984). The ability of the monoclonal antibody to inhibit specific binding of rhlL-2; to pig blasts suggest that it is recognising an epitope on or close to a binding site for rhiL-2, taken together with its ability to inhibit rhlL-2 maintained proliferation this demonstrates that the rhlL-2-binding site is functionally active, i.e., that it is acting as a receptor. The non-competitive nature of the inhibition is consistent with :'~erferenee with the formation of high affinity receptors rather than with competetive occupancy of a single IL-2 binding site. Monoclonal anti-Tac (IL-2Ra, CD25) behaves similarly (Leonard et ai., 1982, Wang and Smith, 1987). The molecular weight of the antigen recognised by the monoclonal 231.3B2 (65-70 kDa) is greater than that reported for the human a chain. It is, however, consistent with the finding only of relatively high affinity receptors and a single 6570 kDa molecular weight band from activated pig lymphocytes following covalent linking to t251rhlL-2 (Takamatsu et ai., 1989). In comparison, on activated ruminant iymphocytes the same authors found both low and high affinity receptors and 50-55 kDa and 65-80 kDa IL-2-crosslinking molecules, and a 55 kDa homologue of the bovine a chain has been sequenced (Weinberg et al., 1988). Thus, a phyiogenetic modification may have occurred resulting in a and /3 chains of similar weight in the pig, or, alternatively the porcine /3 chain or a novel 70 kDa component may be heavily upregulated following activation. The determined affinity of the specific binding
of rhlL-2 to pig lymphocytes (K d approximately 65 pM) is intermediate between the human medium (K d = 1 riM) and high (K d = 20 pM) affinity receptors (Tsudo et al., 1987). Since no evidence was obtained in this study for more than a single receptor this difference is likely to be attributable to a redueed affinity of a pig high affinity receptor complex for rhlL-2 compared to native pig IL-2. Low affinity of an a chain equivalent would also account for the failure to detect low affinity receptors on activated cells. The estimated number of receptors per cell (less than 1000) is also lower than estimates for human high affinity receptors (Robb et al., 1984) but agrees with previous estimates of number and affinity of pig IL-2 receptors (Takamatsu et al., 1989). In summary, we have demonstrated that a monoclonal antibody 231.3B2 recognises an epitope on a 65-70 kDa molecule associated with a receptor for interleukin-2 on pig lymphocytes. The molecule recognised is not present on resting cells but is upregulated following nonspeeific activation in vitro activation. Binding of the antibody to activated cells inhibits IL-2 driven proliferation.
Acknowledgements This work was supported by a Link Grant from the Agriculture and Food Research Council and by the Institute for Animal Health Compton.
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