Immunopurification and insertion into liposomes of native and mutant H-2Kb: quantification by solid phase radioimmunoassay

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IMMUNOPURIFICATION AND INSERTION INTO LIPOSOMES OF NATIVE AND MUTANT H-2Kb: QUANTIFICATION BY SOLID PHASE RADIOIMMUNOASSAY FRANCOISE Centre

d’lmmunologic

ALBERT, CLAUDE BOYER, LEE D. LESERMAN ANNE-MARIE SCHMITT-VERHULST INSERM-CNRS

de Marseille-Luminy.

Case 906. 13288 Marseille

and Cedex 9. France

Abstract--To study the interaction between T cells and isolated H-2Kb. we developed protocols for the immunopurification of the molecule from monoclonal anti-H-2Kh immunoadsorbent columns and for its insertion in lipid vesicles. Patterns of reactivity of two anti-H-2Kb monoclonal antibodies (mAb) (20-8-4 and Y3) on H-2 recombinant and H-2Kh mutant mice indicated that mAb Y3 reacted with all six mutant forms of H-2K” tested. Binding competition studies indicated that Y3 and 20-8-4 recognized distinct epitopes of H-2Kb. A solid phase radioimmunoassay was established using these two mAb to monitor H-2Kh activity in detergent containing cell lysates, after immunopurification. and after insertion in the into liposomes. About 70”,, of H-2Kb activity could be eluted from anti-H-2Kb-immunoadsorbents presence of 3 izI NH,SCN and octyl glucoside (pH 7.41. A procedure of liposome formation combining gel dilution and dialysis yielded liposomes bearing H-2Kb molecules which could inhibit antibody plus complement qtolysis and could stimulate in ~‘it’o primed T cells to generate cytotoxic T lymphocytes irl ~?tro. The present protocol can be extended to immunopurify and obtain H-2Kbm’ bearing liposomes.

INTRODUCTION

H-2 f TNP (Guimezanes et al., 1982). However, neither in these experiments nor in the recent cloning of the gene for H-2Kh and its expression in mouse L cells (H-2k) (Mellor et ul., 1982), is the molecule removed from other structures present on mouse cells. Thus, these experiments do not permit distinction between hypotheses suggesting that the molecule is recognized prr .W or in association with nonpolymorphic cell surface components (Matzinger & Bevan, 1977). Furthermore. if recognition by T cells were to be proven to involve allogeneic H-2 components alone, the question could be raised as to whether this recognition would be sufficient to stimulate a T cell function, or whether the activation step would require the involvement of other cell surface components (as an example. see Golstein et al., 1982). An approach to these questions thus requires a study of the interaction between T cells and isolated H-2 products. Since these amphipatic molecules must be purified in detergent solutions, their reconstitution into lipid vesicles (liposomes) is necessary when studying their interactions with cells (Engelhard et [II., 1978; Herrmann & Mescher, 1981; Hale et ul., 1981). Initially, insertion of MHC molecules in liposomes was done with glycoproteins isolated by Lens culinuris lectin

The major histocompatibility complex (H-2) of the mouse codes for, among other proteins, the class I transplantation antigens which are expressed on most cells of the organism (Klein, 1975). These are glycoproteins of 45,000 mol. wt non-covalently associated with a 12,000 mol. wt protein, 82-microglobulin. The heavy chain consists of hydrophilic extracellular and cytoplasmic domains separated by a hydrophobic transmembrane portion (Nathenson et al.. 1981). Class I products are implicated in immune recognition and are target antigens for alloreactive (Alter et al., 1973) and antigenrestricted cytotoxic T cells (Zinkernagel & Doherty, 1974; Shearer, 1974). Techniques of purification (Freed rt ul., 1979) have yielded data on primary structure of H-2K, H-2D, and H-2L molecules (Coligan et al., 1980; Nathenson ct al., 1981: Maloy & Coligan, 1982). The primary structures of the H-2Kh molecule (Nathenson et ml., 1981), as well as those of various mutant forms of the molecule are known (Nairn et al., 1980). In order to study those features of H-2Kh required in T cell interactions we derived T cell clones specific for different antigenic sites of the molecule in allogeneic recognition (Albert et al., 1982). and in recognition of self 655

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656

affinity chromatography (Turner & Sanderson, 1978: Litman et al., 1979) or with class I MHC antigens purified on anti-/?2-microglobulin antibody immunoadsorbent columns (Engelhard et al., 1978). The availability of anti-H-2 mAb (Oi et al., 1978: Lemke et cl/., 1979: Ozato & Sachs. 1980) permitted further purification of the H-2 proteins using specific immunoadsorbent columns. as has been described for H-2Kk (Herrmann & Mescher. 1979). We here describe protocols for the immunoadsorbent purification of native or mutant forms of the molecule H-2Kb which allow quantitative recovery, as verified by a solid phase radioimmunoassay. Protocols for the insertion of the H-2 molecules in liposomes arc also presented. together with data indicating that these liposomes are capable of stimulating T cells.

MATERI.-\LS

AND

BlO.BR. C57BL:lO (BlO), C57BLj6 (B6) and recombinant mice were obtained from OLAC (Shaw Farms, Blackthorn, England) or CSEAL (Orleans, France) or maintained in our animal B6.C-H-2bm1, facilities. Mutant mice B6.C-H-2bm4, B6-H-2bm5, B6-H-2bm3, B6-H-2bmh, B6-H-2bmX were kindly provided by Dr. B. Kindred (Max Planck Institute, Tiibingen, F.R.G.). These were originally derived from C57BL/6 (D. W. Bailey. Bar Harbor. Maine. U.S.A.) for bml and bm4. C57BL;‘6 YEg (1. Egorov, Moscow. U.S.S.R.) for bm3, and from C57BL;‘6Kh (H. I. Kohn, Boston. Massachusetts. U.S.A.) for bm5. bm6 and bm8. Sex-matched male or female mice, 2-5 months old. were used. antibodies

et (11

anti-CKB immunization (Oi ~‘t (I[.. 1978). of which the two former antibodies were kind11 given respectively by D. H. Sachs (National Institute of Health. Bethesda. MD, U.S.A.) and S. Nathenson (Albert Einstein College of Medicine, Bronx. NY. USA). The Salk Institute Distribution Center (La Jolla. California. U.S.A.) provided the latter antibody. The ;,2a,r<-producing cells were maintained in culture in 5”,, heat inactivated fetal calf serum (FCS. Gibco-Europe. U.K.) RPM1 1640 medium containing lOm,Z4 HEPES. 5 x 10 ’ 2-mercaptoethanol. and antibiotics (100 U ml penicillin. 100 &ml streptomycin). MAbs from culture supernatants were purified by passing over a Protein A Sepharose 4B column (Pharmacia. Uppsala. Sweden). The clution was with phosphate buffered saline (PBS) pH 7.4 containing 2 12/1NH,SCN. Eluted Ah wcrc dialyzed against PBS.

METHODS

Mice

Monoclona1

ALBERT

(mAh)*

The mAb used were 20-8-4 (anti-H-2Kb, anti-H-2Db) ;‘2a,ri Ab obtained from C3H antiC3H.SW immunizations (Ozato & Sachs, 1980); Y3 (anti-H-2Kb), a ;‘2b,k- Ab obtained from a BALBic anti-BALB.B immunization (Jones & Janeway. 1981) and 11-4-1 (antiH-2Kk) 72a.ti Ab obtained from a BALB/c * Abbreviations used: Ab. antibody: mAb. monoclonal antibody : BSA. hovinc w~m albumm : C. uomplcmcnt : Con A, concana\alin A: CTL. cqtotoxic T lymphocyte: DMPC. dimyristoyl pho~phatidqlcholine: DPPE. dipalmltoy1 L-r-phosphatidylethanolamine: FCS. fetal calf serum; LPS, E. coli lipopolysaccharide: NP-40, Nonidet P-40; PBS. phosphate buffered saline: PS, phosphatidylserme: RIA, radioimmunoassay : SDS. sodium dodecyl sulfate.

The mAb (100 /lg) were iodinated with 500 IlCi 1’ 5l-sodium iodide (New England Nuclear, Boston, MA. U.S.A.) using chloramine T (Hunter & Greenwood, 1962). diluted to 5 x 10” cpm:ml in PBS containing 0.2”,, BSA and 0.02”,, NaN,, and stored at 4 C. For binding studies 100 ltl of spleen cells (5 x lo5 cells) in RPM1 1640 containing I”,, FCS were distributed in round bottomed microtiter plates (Greiner, F.R.G.) with 50 ~(1 of “‘1 mAb. After incubation for 30 min at 4 C‘ the plates were washed three times in PBS 0.2”,, BSA 0.02”,, NaN,. Cells were then resuspended in 100 /ll and bound radioactikit! counted in a ;’ counter.

Blast cells were obtained by culturing spleen cells for 48 hr in the presence of E. c,oli lipopolysaccharide (LPS) (Sigma, St Louis. MO. U.S.A.) in plastic flasks (NUNC. Denmark) (500 /lg LPS,‘SO mlj2.5 x lo8 spleen cells) in RPM1 1640 containing 5”,, FCS, 10 m!\f and HEPES, 5 x 1O-5 n/l 2-mercaptoethanol antibiotics (penicillin 100 U,:ml, streptomycin 100 jcg:‘ml). TtrJJJOr-

CC//a

The EL4 tumors originated from a B6 mouse leukemia and were obtained either from the Sloan-Kettering Institute for Cancer Research. New York. U.S.A. (EL4;SK) or from a ctonc selected for its production of Interleukin 2

Native and Mutant

(EL4-C16) by Farrar et a/. (1980). The tumors were maintained by passage in ascitic form in B6 mice.

When radiolabeled cells were used, the radioiodination procedure of Cone & Marchalonis (1974) was modified as follows. LPSinduced blast cells were washed 3 times with PBS. To 5 x 10’ cells in 0.15 ml PBS were added 50 ~1 of lactoperoxidase solution (Sigma) (2 mgjml in PBS) and 10 ~1 of 0.5 M phosphate buffer pH 7.0, followed by 1 mCi of “‘I-sodium iodide (New England Nuclear) and 60~1 of 0.03’? H202 in PBS. The mixture was incubated for 4 min at 30°C and for another 10 min at room temp after addition of more H202 (60 ~1 of 0.03% H,O,), after which 5 ml of cold PBS containing 0.02”/, NaN, and 2 mM KI (washing buffer) were added. Cells were washed twice in this same buffer, then spun once in 1 ml over 2 ml of FCS, washed two more times in washing buffer and transferred to a new tube.

NP-40 (0.52, w/v) in 15 mM phosphate buffer, pH 7.4, containing 1 mM dithiothreitol (Sigma), 200 U/ml Zymofren (Specia, Paris, France). and 2 x 10e5 M phenylmethylsulfonyl fluoride as protease inhibitors, was used for cell lysis. This was performed by a modification of the protocol of Schwartz et al. (1973). Briefly, to a pellet of IO* LPS blast cells, washed once with PBS. was added 1 ml of lysate buffer. After 20 min of incubation on ice insoluble material was removed by centrifugation at 1,100 8. then the recovered supernatant was ultracentrifuged at 140,OOOy for 45 min and material remaining in solution passed over the immunoadsorbents.

Sepharose 4B CNBR (Pharmacia) beads were coupled to mAb 20-g-4, Y3 or 11-4-1 at a concentration of 5 mg to 10 mg of mAb/ml of gel. These immunoadsorbents were kept at 4’C and used less than 10 times.

A lysate derived from lo9 cells in 10 ml was passed over an anti-H-2Kk 11-4-l immunoadsorbent to remove non-specifically binding material. The pass-through was applied to an anti-H-2Kb (20-8-4) immunoadsorbent. The

657

H-2Kb

column was washed with 15 to 20 column volumes of the lysate buffer then 5 ml of a 1 M Tris-HCl pH 7.8 buffer were passed to eliminate the remaining non-specifically bound material. The column was then equilibrated with elution buffer lacking the NH,SCN. The elution was achieved with elution buffer:Tris buffer (50 mM) containing 0.1 M NaCI, 30 mM n-octyl glucoside (Sigma), the aforementioned protease inhibitors and 2 M NH$CN (pH 7.4). The eluate (approximately 2.5 ml) was immediately passed over a G25 column (PDlO Pharmacia) to eliminate the NH$CN and tested in a double mAb RIA. Solid-phase of H-2Kb

radioimmunoassa~~ for

the detection

We used two mAb, Y3 and 20-g-4, that recognize different epitopes on the H-2Kb molecules, in a solid-phase radioimmunoassay for monitoring H-2Kb purification. MAb Y3 (50 kltl of a 50 pg/ml solution in PBS) was distributed in the wells of flat bottomed microtiter plates (Greiner). After 1 hr of incubation at room temp the plates were washed in PBS containing 1% BSA and air dried. Then 20 ~1 of PBS 2”,‘, BSA were added per well followed by 20 ~1 of a cell lysate equivalent to IO8 cells/ml, 20 ~1 of the pass-through, or 20~1 of the recovered eluate. After incubation for 1 hr at 37’C and overnight at 4-C the plates were washed three times with PBS l”,, BSA and dried. Fifty ,nl of ‘251-labeled mAb 20-8-4 (lo6 cpm/ml) were then added per well in PBS 0.2”,, BSA 0.029, NaN,. After incubation for I hr at 37 ‘C and 1 hr at 4 C the plates were washed three times in PBS. Bound radioactivity was eluted from the plates with 200 it1 of 1 N HCl and counted in a :’ counter. Prepaution

of lipid vesicles

Dimyristoylphosphatidylcholine (DMPC; synthetic) and phosphatidylserine from beef brain (PS) were purchased from Sigma. Dipalmitoyl L-sc-phosphatidylethanolamine (DPPE; synthetic) was obtained from CalbiochemBehring A.G. Cholesterol was from Applied Science Laboratories. These were used in a lipid to cholesterol ratio of 70:30 (w/w). The composition in weight of the lipids was as follows: DMPC: 5601~; PS: 14”:); DPPE: 300/;. The 1ipid:cholesterol mixture was dried from chloroform: methanol (9~: 1v) in N, and kept at -20°C. “‘1-DPPE prepared by modification of DPPE with the Bolton-Hunter reagent

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65X

(Bolton & Hunter, 1973). according to a technique similar to that recently described (Schroit, 1982). was added in trace amounts to the mixture of lipids for liposome analysis by flotation. One mg of the lipid preparation was mixed in a total 2.5 ml with a preparation of H-2K” obtained from lo9 cells [in Tris buffer (50 mM) containing 0.1 M NaCl. 30 mM octyl glucoside and the protease inhibitors]. This mixture was incubated for 15 min at room temp, and then passed over a G25 column (PDlO. Pharmacia) to eliminate the majority of detergent. Further elimination of detergent was achieved during a 12 hr dialysis against PBS at 10 c. The liposomes were tested in the solid-phase radioimmunoassay, then filtered on 0.45 11 filters (Gelmann) and kept sterile at 4 C.

Pol~wrylumide

gel ~1ectrophowsi.s

in SDS rrnd

Ul(to~rl~fioy~crph!’

Electrophoresis was performed in 15”,, (w,;v) polyacrylamide slab gels using the Tris discontinuous buffer system of Laemmli (1970). Samples precipitated with trichloracetic acid (TCA) were dissolved by heating at 100 C for 5 min in sample buffer. Molecular weights were calculated by reference to the mobilities of Coomassie blue stained standard proteins. Autoradiography of dried gels was done using Kodak X R5 film.

PrOfeilI

d~tC’J-JJIiJl~~tiOJZ

Protein determinations of octyl glucoside containing samples were carried out using the Folin-Lowry method (Lowry ef (I[., 1951) on 12.5”,, TCA precipitated samples which were washed with acetone and dissolved in 1 N NaOH. Bovine serum albumin in octyl glucoside. precipitated in the same way. was used as a standard. Sucros~~ dcnsit!~ yr-adknt centr@gation Centrifugation was in a Beckman SW41 swinging bucket rotor at 140.000 9 overnight. Samples were placed at the bottom of the centrifuge tubes. diluted in 60’!,, sucrose to give a final concentration of 45”,, sucrose in 1 ml, and overlaid successively with 1 ml of 35?,,, 1 ml of 25”,,, 1 ml of 15”,, sucrose and finally with PBS. Fractions (0.4 ml) were collected from the bottom by aspiration.

ALBERT

<‘I
Inhibition of’~ntihd~~ p/us calm/ l3i.s

coJ~Jp/~~JJJcwf

~nrrfialcd

The liposomes made as described above were tested as inhibitors of the complement (C) dependent lysis by 20-8-4 or Y3 of (“‘Crl (sodium chromate, New England Nuclear) labeled BlO.A(SR) LPS blast cells. as previously described (Albert cat rrl., 1982). MAb Y3 and 20-8-4 were used at 40ng,;ml. which normally gives 60”,, “Cr release from H-2h cells with a preselected rabbit C used at 1 20. F-o1 the inhibition experiments serial dilutions of liposomes and liposomes containing H-2Kh were incubated with 20-8-4 or Y3 at 4 C fol 20 min in round bottomed microtiter plates. then 50 1’1 of a suspension containing 2 x 10’ BlO.A(SR) LPS blast cells,ml were added and incubated 20 min more. After centrifugation at 650 9 for 5 min. 50 /Al of a 1 :20 dilution of rabbit C were added to the cell pellets which were incubated for 45 min at 37 C after resuspcnsion. At the end of the incubation 150 /II of RPM I I”,, FCS were added per well. the plates were centrifuged for 5 min at 650 8. and 100 itl of supernatant was counted in a ;’ counter. Generatior? of‘ c!~/ol~.tic T CY//.\irl c~~r/tuw Spleen cells from CBA/J mice primed bk intraperitoneal injection of 3 x IO” allogeneic tumor cells [EL4/SK (H-2h)] from 3 wcehs to 3 months earlier, were harvested and put in culture as previously described (Schmitt-Vcrhulst et ~1.. 1978) in RPM1 1640 medium supplcmented with 2 mM glutamine, 100 mM pyruvate, 5 x IO- ’ !If 2-mercaptocthanol, 10 mM HEPES buffer. 100 U;ml penicillin, 100 &ml streptomycin, and 5”,, dccomplementcd FCS (Gibco-Europe. U.K.). Six million responding cells were cultured in 2 ml in Costar cluster wella in the presence of \,arious numbers of 2000 lad irradiated stimulating cells or liposomes, as indicated in the results. For stimulation with liposomes responding cells and liposomcs were first incubated for I hr at 37 (‘ in 0.5 ml in the culture wells before addition of another 1.5 ml of medium. Incubation was fog 4 days in a humidified 5”,, CO, in air incubator at 37 C. Tc,,st$N ~.~~of!.fi~.L/ctiritj After 4 days of culture, cells harvested from triplicate wells were pooled and resuspended in RPM]-5”,, FCS medium. The cytotoxic assay was done in triplicate in V-shaped microtiter

659

Native and Mutant H-2Kh

with both H-2Kb [BlO.A(SR)] and H-2Db [BlO.A(4R)J and weakly with H-2Kd but not with H-2Kk (Ozato & Sachs, 1980). Reactivity on H-2K” mutant cells could be tested directiy for mAb Y3 (no reactivity with H-2Db) and was found positive for all the tested mutant cells as indicated by Ab pius C lysis of 51Cr labeled blast cells. Analysis of the reactivity of mAb 20-8-4 on the mutant cells required the use of the solid-phase radioimmunoassay (see later). allowing for the selective detection of H-2K but not of the H-2Db present on the mutant cells. Results indicated that mAb 20-8-4 was reactive with all mutants tested except for bm3. In order to determine their potential use as paired mAb in the solid-phase RIA we determined that mAb Y3 and 20-S-4 recognized distinct epitopes on the H-2Kb molecule by their capacity to inhibit each other’s binding to BlO.A(SR) cells. Results in Fig. 1 indicate that the presence of mAb 20-8-4 did not affect the binding of lz51 mAb Y3, which was inhibited by unlabeled mAb Y3 (Fig. ZA), and that mAb Y3 did not affect binding of “‘1 mAb 20-8-4 (Fig. 1B). A solid-phase RIA was set up using

plates with 2 x lo4 51Cr-labeled tumor target cells and various effector to target cell ratios in 0.2 ml, as indicated in the results. After an initial 2 min 400g centrifugation the microplates were incubated for 4 hr at 37”C, then centrifuged for 5 min at 6509 and 0.1 ml of supernatant was collected from each well for determination of radioactivity in a y counter. Per cent specific “Cr release is expressed as: cpm experimental - cpm medium cpm for 1 N-HCl incubated target cells RESULTS

Selection of mAb .for the detection of solubilized

H-2Kb in a solid phase radioimmunoassay A summary of the reactivities of the mAb Y3 and 20-8-4 which were used in this study is presented in Table 1. Results of direct binding studies of 1251 mAb Y3 and 20-8-4 indicated that mAb Y3 was specific for H-2Kb, since it reacted with BlO.A(SR) (KbDd) but not with D2.GD (KdDb); it was, however, strongly cross-reactive with H-2Kk, as indicated on BlO.BR and BlO.A(4R). MAb 20-S-4 reacted

Table I. Reactivity patterns of the two anti-H-2Kb mAb on H-24 H-Zk, H-td H-2 recombinant and mutant H-2 spleen cells

Mouse strain

K

I-A

B10 BIO.BR BALB/c B lO.A(4R) BIO.A(SR) DZ.GD

h

;t b d

b k d k b d

bml bm3 bm4 hm5 bm6 bm8

b b b b b b

B6.C-H-Zbm’ B6.H-2hm3

k

H-2” I-E

D

L

20-g-4

Y3

(b) k

c+~ -b

++” ++b

++b

++”

(:)

b k d b d b

(t)

ndc

-b

(b) (b) (b) (b) (b) (b)

b h b b b b

(b) (b) (b) (b) (h) (b)

(b) k (:)

++i-i< +ie ++c ++p

++d ++d ++d ++d ++d ++“

“Alleles expressed at the K, I-A, I-E. D and L regions of the H-2 complex are given according to Klein ef ui. (1978). Parentheses indicate that no gene product has been identified in that atlehc form. ‘Binding of “‘1 mAb Y3 and 20-8-4 on 1 x lo6 spleen cells: + + represent 100% binding; + represent 65% binding; + represent 20:; binding; - represent less than 5% binding. ‘nd = not done. ‘Tested by mAb + C mediated cytotoxicity: t + represent >50”/;, of specific “Cr released from target cells when incubating 50~11of Y3 at 40 ng/ml in the presence of 2 x 104 5’Cr ConA blast cells + C; maximum release in presence of HCI I N was 2087 + 140 and medium release was 45 + 16. ‘Binding of “‘1 mAb 20-W in the RIA after incubation of lysate on Y3 coated plates. Since Y3 does not react with H-2Db. only H-2K is detected; + + represent > lO.OOOcpm; - represent 12OOcpm; background binding for control BtO.A(4R) cells was 500 cpm.

FRANCOISE

660

ALBERT

rt trl.

spleen cells and EL4SK

cells were respectively 1, 0.8, 0.5 and 0.23. BlO.A(SR) LPS blast cells were thus used routinely as a source of H-2Kb.

0125 025

05

,

pg

mAb

2

4

Fig. 1. Inhibition of binding of anti-H-2Kb mAb Y3 and 20-8-4 to BlO.A(SR) cells. Binding of “‘1 mAb Y3 (A) and 20-8-4 (B) in the presence of unlabeled mAb Y3 (a) and 20-8-4 (0) is expressed as per cent of binding in the absence of unlabeled mAb. as a function of the concentration of unlabeled mAb. IOO”, binding was 3550cpm for Y3 and 16.000 cpm for 20-8-4.

We used BlO.A(SR) LPS blasts as a source of H-2Kh and a 20-8-4 coupled immunoadsorbent for immunopurification. As described in detail in Materials and Methods. BlO.A(SR) lysates adsorbed on a 1 ml 20-8-4 immunoadsorbent column were washed and eluted with a buffer containing octyl glucoside and 2 M NH,SCN. Dilutions of an aliquot of the initial lysate in the RIA allowed quantification of soluble H-2Kb and thus an estimation of the percentage of recovery of H-2Kb activity in the eluate could be made. RIA data in Fig. 3 compare the H-2Kb activity in the initial lysate, in the passthrough from the 20-8-4 immunoadsorbent. and in the material eluted from that immunoadsorbent. In the experiment shown in Fig. 3 no H-2Kb activity was detected in the passthrough, but about 65’!,, of the activity of the lysate was recovered in the eluate. From such RIA curves cell equivalents of H-2Kb will be defined for each H-2Kh preparation in comparison to the lysate: as an example. in the linear part of the RIA, the eluate point corresponding to 0.26 x lo6 cells has an RIA value equal to 0.17 x lo6 cells initially present in the

mAb Y3 to coat plastic microtiter plates and “‘1 mAb 20-8-4 to reveal bound H-2Kh. This technique reveals only H-2Kh, even from such H-2Db bearing cells as the EL4 tumors and the H-2Kbm mutant cells, since only the H-2Kb molecule is bound to the Y3 mAb. Selection

of the source of H-2Kb expressing

cells

The cells used for purification of H-2Kb were selected by comparing the relative amounts of H-2Kb detected by the solid-phase RIA from lysates of different cells expressing H-2Kb. The data in Fig. 2 show a comparison of the H-2Kb NP-40 solubilization of detected after BlO.A(SR) (H-2Kb,Dd) normal spleen cells or 48 hr LPS stimulated splenic blast cells and the two H-2b tumor cells EL4/SK and EL4.Cl6. BlO.BR (H-2k) LPS induced splenic blast cells were used as control. Relative amounts of H-2Kb detected in lysates from 4 x lo5 cells for BlO.A(SR) LPS blasts, EL4.Cl6, BlO.A(SR)

IUMO~R

Of CELLS

[x 1o-6 I

Fig. 2. Estimation of the relative amounts of H-2Kh solubiked in NP-40 extracts from different cell sources using a solid phase I-adioimmunoa\sn~. Amounts of H-2K” detected bv “‘1 mAb 20-X-4 in NP-40 I\batcs from BlO.A(SR) spleen cells (0) or LPS-induced h&t cells IO). EL4,SK (D) or EL4.C.16 (A) tumor cella and BlO.BR LPSinduced blast cells (tl) hound to mAh Y3 coated microtitcr ~~11s in a solid phase radioimmllnoa~~nv

Native

and Mutant

661

H-2K”

30-

0

2

I NUMBER

OF CELLS

77 I x 10-G

87

I

Fig, 3. Detection of H-2Kb activity by solid phase RIA after immunopurification and insertion in liposomes. H-2Kb activity as detected by iZ51 mAb 20-8-4 on Y3 coated plates as a function of the number of cells initially present in the NP-40 lysates (0). the pass-through from the 20-8-4 mAb immunoadsorbent (---). the material eluted from that immunoabsorbant before (A) and after (m) insertion in liposomes. H-2K” activity measured on the H-2Kb-liposomes in the presence of excess detergent is also shown (0). A control sample of H-2Kk bearing liposomes (see legend to Fig. 8) was also tested (0). The abscissa indicates the number of cells initially present in a given sample. corrected for the different volumes, i.e. I x IO9 cells were present in 20 ml of the lysate or of the pass-through, and in 2.5 ml of the eluate and of the liposome preparation. Bars indicate standard deviations only when they are larger than the size of the represented point.

lysate. This indicates a 65% yield of H-2Kb activity from lysate to eluate and this value will be defined as corresponding to 0.17 x 1Oh cell equivalents. Folin-Lowry protein determination indicated that the immunopurification yielded an eluate containing 20,~g of protein and 3 x IO* cell equivalents of H-2Kb activity from a lysate of 4 x 10’ cells containing 4 mg of protein: this corresponds to a 150-fold increase in specific activity of H-2Kb. The high salt wash (see Materials and Methods) contained 300 pg of protein, but no detectable H-2Kb activity. Liposome

H-2Kb was compared to that of the lysate and of the eluate (Fig. 3). Dilutions of the liposomes in the RIA indicated that about 50”,, of the H-2Kb activity from the lysate became incorporated in liposomes. Addition of detergent to the liposomes only slightly increased the level of detection of H-2Kb. indicating the preferential exposure of H-2Kb on the outer surface of the liposomes. When more diluted H-2 preparations were used the loss of H-2 during the process of insertion in liposomes was more important (see Fig. 7).

prepurution

Liposomes were made after mixing immunopurified H-2Kb (from lo9 cells) and 1 mg of lipids. The elimination of detergent on a G25 column (PDlO) was almost total, as could be tested when using trace amounts of “C-octyl glucoside (F. Albert, unpublished data). The liposome-containing fractions were pooled and used after 12 hr of dialysis. Liposomes were sterilized on a 0.45 pm Gelmann filter without any loss of material (unpublished data). The activity of the liposome-associated

SDSPAGE analysis was performed on ‘251-labeled cell surface protein present in the initial NP-40 lysate (Fig. 4A), on material eluted from the 20-8-4 immunoadsorbent after washes with 1 M Tris pH 7.8 (Fig. 4B). and on the material eluted with octyl glucoside and 2 M NH,SCN (Fig. 4C). Washing with Tris appeared to remove two contaminants of low mol. wt and some material at the position of 45,000 mol. wt. In the eluted material two major bands can be detected corresponding to

662

FRANColSE

ALBERT

<‘I trl.

Fig. 4. SDS-polyxxylamidc gel electrophoresls of NP-40 Iysates and itnmtlnopllrllied H-2K”. Prom “‘I-labeled BIO.A(SR) LPS induced blast cells a NP-40 lysate was pl-cpuxi uhich wah loaded on ;I 20-X-4 mAb immunoadsorbcnt as described in Materials and Method\. A I j”,, (M A) SDS lrel was run on IO ~tl of lysate (equivalent to 3 x IO‘ cells) (lane A); on the material clutcd f~rom the immunondsorhcr,t with I &I Tris pH 7.8. after prwpiution with l2.5”,, TC‘A acid and dissolution m the gel buffer (lane B): and on the material clutcd from the immunoadaorhent with the 2 !If NH,SC‘N containing clution hull& after precipitation and dissolution as for the previous sample ~lanc C). Marker prokina detec~cd by Coomassie hluc stain were respecti\cl>: phosphorylase b (I). albumin (2). owlbumin (3). carbonic anhydrase (4). trypsin inhibitor (5) and x-lactalbumin (6). corresponding to mol. wtb ( x IO ‘) of 01. 67. 33. 30. ?O and 14 respectively.

mol. wts of 12,000 and 45,000, as expected for class I proteins. The purification is not total: as seen in Fig. 4C, small amounts of contaminating high molecular weight material are still present.

associated with lipids the H-2Kh protein was detected in fraction 5 together with ““I-DPPE (Fig. 5B).

Idlihitiorl

of’Ah plusC

c~~totoricit~~ by liposomt~-

H-2K"

Samples of liposomes containing trace amounts of “‘I-DPPE without protein (Fig. 5A) or with H-2Kb protein (Fig. 5B). or ‘251-H-2Kb alone. were analyzed for flotation on discontinuous sucrose gradients. After 20 hr of centrifugation at 140.000 g samples were collected from the bottom and radioactivity and H-2Kb activity in the RIA was measured in the fractions. In the absence of lipids the H-2Kb protein remained at the bottom of the gradient (fractions I and 2) (Fig. 50 whereas when

Liposome preparations were tested for thei! capacity to inhibit the Ab plus C mediated lysis of “‘Cr-labeled BlO.A(SR) target cells (Fig. 6). The anti-H-2Kh mAb Y3 and 20-8-4 were tested at a concentration giving around 60”,, (51Cr) release in the absence of inhibitor (40 ngjml). Dilutions of liposomes were incubated first with the mAb and further with the target cells before the addition of C as described in Materials and Methods. In the presence of liposomes lacking H-2Kb some inhibition of cytolysis was observed (30”;, for Y3 and X”,, for X-X-4). Adding increasing

Native

and Mutant

663

H-2Kh

H-2Db expressed on the mutant cells. We therefore tested whether the same technique of immunopurification using Y3 immunoadsorbents and the RIA to monitor H-2K purification could be applied to a mutant such as bml. which is generally distinguished from H-2Kb by T cells (Melief rt al., 1980; Albert et al., 1982). Figure 7 describes the recovery of H-2Kbm1 activity from 5 x 10’ B6. C-H-2bm’ LPS blast cells as tested by the RIA: about 70”,, of the activity of the lysate was recovered in the eluted material, of which about 501’,, was incorporated in liposomes.

%

oi 0

2

6

f RACTIOU

10

1’4

IllJM6ER

Fig. 5. Flotation analysis of H-2Kb-liposomes. Centrifugation on a discontinuous sucrose gradient was performed on: (A) sham-reconstituted phospholipid vesicles containing cholesterol and trace amounts of “‘1-DPPE; (B) immunopurified H-2K”-reconstituted phospholipid vesicles containing cholesterol and trace amounts of “‘1-DPPE at a lipid to protein ratio of 1O:l (w/w); (C) H-2Kh alone immunopurified from “51-labe1ed BIO.A(SR) LPS-induced blast cells. The position of the lipids in the gradient is detected by cpm of ‘“51-DPPE (O--O) (A and B): the position of the H-2K” is detected by cpm of “‘1 mAb 20-X-4 in the RIA when unlabeled H-2Kb was used (0 - 0) (B) and bq the cpm of the “‘1-H-2Kh (A- -A) in (C).

amounts of liposomes containing from 0.625 to 10 x lo6 cell equivalents of H-2Kb gave increasing inhibition of cytolysis. Fifty per cent specific inhibition (as compared to inhibition by liposomes alone) was obtained for about 2.5 x lo6 and 5 x lo6 cell equivalents of H-2Kb-liposomes for mAb 20-8-4 and Y3 respectively. Under similar assay conditions about 2.5 x 10’ B10 spleen cells were required to obtain 5O”i;; inhibition of 20-8-4 induced lysis. C’sr of Y 3 irnmunoudsorbents $kation cf H-2Kh””

for the iwnunopw-

The summary of reactivities of mAb Y3 (Table 1) on H-2Kb mutant spleen blast cells indicated that this Ab reacted with each of the tested mutant forms of H-2Kb and not with the

Spleen cells from in cico primed CBA mice (with H-2b EL4/SK tumor cells) were restimulated in vitro with BlO.MBR (KbIkDq) spleen cells, with H-2Kb or H-2Kk containing liposomes, or with liposomes lacking H-2 as controls. Results in Fig. 8 indicate that about lOO-fold more cell equivalents of H-2Kb in liposomes were required to give a similar level of CTL activity as with the BlO.MBR cells. No stimulation was observed when non-protein bearing or H-2Kk-liposomes were used. The H2Kk-liposomes were prepared from immunoadsorbent purified H-2Kk as described in the legend to Fig. 8.

100

I

0

,625

1.25

I

8

I

2.5

5

10

cull equirllrnts

,x166

)

Fig. 6. Inhibition of mAb plus complement cytolysis by H-2Kb-liposomes. Per cent ‘ICr released from 1 x IO4 “Cr-labeled BIO.A(SR) Con A-induced blast cells was. after incubation with 40 ng.‘ml of mAb Y3 (0.0) or 20-8-4 (A. A) followed by rabbit complement. respectively 62 and 49”,,. The effects of preincubation of the mAb with H-2Kh liposomes (0. A). or sham-reconstituted liposomes (0. A) on the degree of cytolysis is expressed as the per cent of inhibition of “Cr-release as a function of the cell cquivalents of H-2Kh-liposomea or sham-reconstituted liposomes present.

664

FRANCOISE

ALBERT

et ol

Ix lo6

NUMBER OF CELLS

1

Fig. 7. lmmunopurification of H-2Kbm’ dnd its insertion in liposomes. The H-2Kbm’ activity was measured in the NP-40 lysate from 5 x IO* B6.C-H-2 bm’ LPS-induced blast cells (0). in the material eluted with 2 M NH,SCN containing buffer from a Y3 mAb immunoadsorbent column (0). and in reconstituted liposomes (I mg of lipids for 3 ml of eluate) (0); cpm of ‘*‘I mAb 20-8-4 bound in the RIA with mAb Y3 coated on the microwells is expressed as a function of the number of cells initially present in the different samples (as corrected for the differences in volume).

50

a\0

0

-.A .

I,

I

1

3X104

MO3 NUMBER

OF

STIMULATING

MO5 CELL

3x106

3x10’

ECIUIVALENTS

Fig. 8. Comparison of secondary cytotoxic T lymphocyte inductton by BlO.MBR spleen cell\ and H-2Kh-liposomes. Spleen cells from CBA mice (H-ZL) primed with 3 x 10” EL4,‘SK (H-2? tumor cells 3 weeks earlier. were cultured with various numbers of BlO.MBR (KhDq) stimulating cells (0) or various cell equivalents of H-2K”-1iposomes (A). H-2K”-liposomes (A) or sham-reconstituted liposomes (0). CTL activity was measured 4 days later at different effector to target cell ratios ( from 5O:l to 3.0:1) by the “0-release assay on BlO.A(SR) (K”Dd) LPS-induced blast cells or CBA LPS-induced blast cells as controls. The values of the per cent of specific “Cr released from the BIO.A(SR) target cells are cxpresscd for an elfector to target cell ratio of 12.5:l. whrch was in the linear part of the effector dilution curves. Specific “Cr released from CBA target cellc was i l”,, for all of the effecters. H-2Kk-liposomcs were prcpar-cd by immunopurifcation on an immunoadsorbent of H-2K”-spcc1hc mAb (100-30-33. Lemke ef (I/.. 1979) wtth washes and elution conditions as described for the H-2Kh ~mmunopuritic~~tiot~ in Materials and Methods except for the omission of initial passage on the 11-4-l immunoadsor-bent. I-ecogni/The H-2Kk activity was assessed by the solid phase RIA using “‘1 mAb 100-5-28 (anti-H-2K’ ing a different epitope of the H-2K’ molecule than 100-30-33. Lcmke “I ii/.. 1979) to re\cal the H-2Kh hound on microwcll\ coated with mAh 100-30-33.

Native

and Mutant

DISCUSSION

We developed protocols for the immunopurification of H-2Kb molecules and their insertion in phospholipid vesicles with the aim of studying their interaction with T-lymphocytes. H-2Kb was chosen because its primary structure is known (Nathenson et al., 1981) and because many mutant forms exist which present one or two amino acid substitutions (Nairn et ul., 1980). The analysis of the effects of such mutations on different interactions between T cell and H-2Kb expressing cells could be studied using monoclonal T cells (Albert et al., 1982). Further understanding of the role of H-2Kb as an entity isolated from the cell surface requires its puri~cation. Classical biochemical techniques (Freed et LII., 1979) or their combination with the use of alloantisera coupled to Sepharose (Rogers rt ul., 1979) gave low yields of recovery of purified H-2K products. Recently the use of immunoadsorbents obtained by coupling mAb to Sepharose has been developed for the immunopuri~cation of class I (Herrmann & Mescher, 1979; Stallcup rt al., 1981) as well as class 11 (Zecher & Reske, 1982) MHC antigens. While the immunopurification of H-2Kk on an I l-4-1 mAb immunoadsorbent was for us as successful as the results described by Herrmann & Mescher (1979), this technique could not be applied to the ilnmunopurification of H-2Kb (F. Albert, unpublished). Indeed, when elution was performed using sodium deoxycholate and NaCl no H-2Kb could be recovered from either 20-8-4 or Y3 immunoadsorbents* Thus the elution protocol used by Herrmann and Mescher appears applicable only with mAb such as 11.4.1, which seem to have low affinity for their antigen (F. Albert, unpublished). For this reason a different protocol was developed for the immunopurification of detergent solubilized H-2K b. A solid-phase radioimmunoassay was established as a simple assay to monitor H-2Kb activity during purification and formation of liposomes. This assay uses two mAb (Table 1) recognizing different epitopes of the H-2Kb molecule (Fig. 1). It could be used to estimate the relative recovery of H-2Kb in NP-40 lysates from different cell sources (Fig. 2), and to choose as starting material for the immLinopurification LPS blast cel’ts. Previous reports used the inhibition of antibody plus complement cytolysis (Turner & Sanderson. 1978; Hale et rrl., 1980) or competition in a classical RIA (Rogers et al.. 2979) as means of

H-2Kb

665

detecting H-2K activity. The first assay requires significant dilution of detergent containing samples to prevent lysis of the target cells (Schwartz & Nathenson, 1971), which greatly lowers the sensitivity of the assay: the second requires a stable radiolabeled purified product. With mAb Y3 coated on the microplates and “‘1 mAb 20-8-4 revealing bound H-2Kb the assay was quite reproducible and allowed estimation of H-2Kb activity in lysates. column effluents and eluates and H-2K”-liposomes (Figs 3 and 7). In order to obtain a satisfactory purification of H-2Kb, NP-40 lysates were first passed on an anti-H-2Kk immunoadsorbent then on the mAb 20-8-4 in~munoadsorbent which retained all H-2Kb activity (Fig. 3). Washing with lysate buffer and with 0.5 M NaCl and/or 1 A4 Tris. pH 7.8 consistently detached two major contaminants, as detected by SDS-PAGE analysis (Fig. 4). The H-2Kb activity was then &ted with 2 M NH,SCN containing 30 mM octyl glucoside (Fig. 3) as two major bands of mol. wt 45,000 and 12,000 according to SDS---PAGE analysis (Fig. 4). Such procedures yielded from 65% to lOOo/;,of the H-2Kh activity from the lysates in the eluates. as detected by the solidphase RIA (Fig. 3 and results not shown). The two main techniques for liposome formation from detergent solutions involve dialysis (Kagawa & Racker. 1971) or gel filtration (Brunner et al., 1978). We used a combination of these techniques by eliminating the detergent first on a G-25 Sephadex column and further by dialysis against PBS at 10-C. Simple dialysis would normally require 48 hr, but the choice of phospholipids with saturated fatty acid side chains of more than 14 carbons would in addition require that liposome formation be at a temperature above 23°C (the transition temperature of DMPC), during which time it is difficult to avoid growth of microorganisms and potential degradation of the purified products, Using a low molecular weight detergent such as octyl glucoside, which has a high critical micellar concentration and is thus easily dialysable (Helenius ef ai., 1977: Mimms et cd., 1981), it was possible to eliminate the majority of the detergent [as tested using “C-octyl glucoside (results not shown)] on the G-25 Sephadex column at room temp. and to further dialyse the formed liposomes to remove any residual detergent in the cold for 12 hr. Analysis by electron microscopy indicated that liposomes were obtained under these

666

FRANCOISE

conditions (data not shown). H-2Kh activity could be quantitatively recovered in the liposomes. The molecule appeared to be inserted with the H-2Kh molecule facing the exterior. since no major increase in activity could be detected after liposome Iysis by excess detergent (Fig. 3). Association of the H-2Kh activity and the liposomes was confirmed by co-fotation on discontinuous sucrose gradients using lz51-DPPE (Fig. 5). The H-2Kh-liposomcs could inhibit mAb plus complement cytolysis of BlO.A(SR) target cells (Fig. 6) and they were effective in stimulation of anti-H-2K h cytolytic T lymphocytes in culture of in r,iro primed lymphocytes (Fig. 8). HoLvever. as previously described for H-2Kk-bearing liposomes (Herrmann & Mescher, 1981). liposomes bearing H-2Kh antigens were much less efficient than whole cells in terms of cell-equivalents required to generate a given level of CTL response. Since the induction of CTL is a complex phenomenon requiring not only antigen stimulation of various clones of CTL precursor cells. but also the involvement of radio-resistant adherent cells (Pettinelli et trl., 1979) and of products secreted by helper T cells (Weinberger cr 01.. 1981). it is difficult to evaluate which step or steps are limiting (Herrmann c’t II/.. 1982). The comparison of stimulating properties of H-2Kh on cells and H-2Kh on liposomes suggests that qualitative differences might exist in terms of T cell stimulation between the two forms of H-2Kh presentation (Albert cjt ~1.. 1983). As anti-H-2Kh T cell clones with defined stimulating properties are now available, the importance of factors such as the lipid composition. fluidity and si;le of liposome, as well as density of H-2 molecules per liposome and the presence of other cell surface components can be studied for the stimulation of individual T cell clones (F. Albert et al.. in preparation). Thus. we here describe protocols to obtain serologically and biologically active H-2Kh bearing liposomes which can also be used for the preparation of mutant H-2Kb-liposomes (Table 1. Fig. 7).

,4~~no1~/~,r/llc,1,1c’nr\~WC wish to thank G. Iasa for the manufacture of the slab gel electrophoresis apparatus. J. Barbe! C. Goridig. A. Guimeranes and B. Rubin for critical reading of the manuscript and V. Bernay and M. Issa for efficient secretarial help. This uork waab supported by grants from DGRST (ACC 81.L.0711) and INSERM (CRL 82.1035). F. Albert v,as the recipient of a fellowship from DGRST.

ALBERT

<‘r trl REFERENCES

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Native

and Mutant

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