monoclonal antibodies detect polymorphic determinants shared by i-a and i-e antigens

Molecular Immunology, Vol. 21. No. 4, pp. 293-299, 1984 Printed in Great Britain

0161-5890/84 $3.00 + 0.00 IC 1984 Pergamon Press Ltd

MONOCLONAL ANTIBODIES DETECT POLYMORPHIC DETERMINANTS

SHARED BY I-A AND I-E ANTIGENS

NORBERT KOCH,* MAURO S. SANDRIN.~ G~NTER J. H~MMERLINC.*

NABOHIKO TADA.: SHOJI KIMURA~ and ULRICH H~MMERLING~

*Institute of Immunology and Genetics, German Cancer Research Center, Heidelberg, F.R.G.; TResearch Centre for Cancer and Transplantation Department of Pathology. University of Melbourne,

Parkville. Victoria 3052. Australia; and $Memorial Sloan-Kettering Cancer Center. 1275 York Avenue, New York, NY 10021, U.S.A.

(First recehed 30 June 1983; accepted in recked form 31 October 1983)

Abstract-Binding data on inbred mouse strains and immunochemical isolation of Ia antigens with subsequent separation on non-reduced/reduced two-dimensional gels provide evidence for the crossreactivity of monoclonal antibodies with I-A and I-E products. Thus two monoclonal antibodies were found to react with A,A, as well as E,E, dimers. One of these mAbs, K22-42. reacts with the precursor form of E, chain of BIO.GD mice which is associated with the invariant chain (Ii). This indicates that the respective determinant on E,, is formed prior to association of E,, with E,.

INTRODUCTION

Class I and Class II antigens are products of the major histocompatibility complex (MHC).$ They consist of a number of highly polymorphic glycoproteins which are expressed on the cell surface (Klein, 1975), where they are recognized by activated T cells (Klein, 1979). The I region of the MHC encodes four polypeptide chains A,, A,, E, and E, (Cullen et al., 1978; Cook et al.. 1978). A,A, and E,E,{ form dimers, and in the cytoplasmic reticulum these dimers are associated with a nonpolymorphic polypeptide chain, the invariant chain (Ii). During trans- port to the cell surface this complex is broken such that on the cell surface A,A, dimers and E,E, dimers are not associated with the invariant chain (Jones et

al., 1978~; Koch et al., 19826). Similarly class I H-2 antigens also form dimers consisting of a highly polymorphic heavy chain and a less polymorphic light chain, &microglobulin (Nakamuro et al., 1973; Grey et al., 1973). Many monoclonal antibodies (mAbs) and antisera generated against H-2 antigens exhibit a high level of crossreactivity on the polymorphic K, D and L heavy chains (David et al., 1973; Murphy and Shreffler, 1975; Lemke et al., 1979). However, for Ia antigens less crossreactivity between I-A and I-E products has been described (Bhattacha-

§Abbreviations: Ii, Ia associated invariant chain; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; PBS, phosphate-buffered saline. pH 7.4; PMSF, phenyl- methylsulfonylfluoride; NP40, Nonidet P-40; PPO, 2,5-diphenyloxazol; LPS, lipopolysaccharide; mAb. monoclonal antibody; SDS-PAGE. sodium dodecyl- sulfate-polyacrylamide gel electrophoresis;.MHC, major histocompatibility complex.

rya et al.. 1981; Birnbaum et al., 1982: Pierres et ul.,

1982; Symington and Sprent, 1981). The demonstration of such crossreactivities is im-

portant and could be useful for studying evolutionary relationship between genes of common origin. The observation may also answer whether gene duplication and gene conversion could lead to the large polymorphism observed with Ia antigens.

In this report we present biochemical data for shared determinants between I-Ad and I-Ed antigens by using mAbs which crossreact with I-A and I-E products.

MATERIALS AND METHODS

Hybridomas and monoclonal antibodies

Hybridomas 171227 and K24-64 have been

described elsewhere (Lemke et al.. 1979; Koch et al.,

19826). 17/227 was obtained from an immunization of ATL mice with ATH spleen cells and K24-64 by immunization of A/J mice with 129 tumor. Hybrid- oma K22-42 was derived from a fusion of C57BL/6 mice, immunized with the tumor 129 (H-2’), and fused with NS-1 lymphoma cells. All three mAbs were of the yZar K class and bind protein A. Hybridomas were cultured in RPM1 medium containing 10% FCS from which protein A binding components had been removed by passage over a protein A column.

Metabolic labeling qf‘ cells and immunoprecipitation

10’ LPS activated spleen cells obtained after 3 days of culture were washed intensively with methionine-free medium containing 10 mM HEPES and incubated for 5 min in 200~1 prewarmed medium containing 200 pCi 35S-methionine (Amersham). the incorporation of methionine being stopped by adding

293

194 NOKBEKT Koci-r et Eli.

Table I. Rractlwtv of mAb K22- 42 with soleen cells of 13 mouse strams”

K?? 47 1~100 _

dilution of’ K A,; A, E, E, D ascites Raid No Ah

f57BL, 10 h b h h -1, h 529 386 BIO D? d d d d d d 4544 30-I BI0.M f 1 f f /I f 1277 ?I:! BlO.BR k h b h I\ 6 3462 314 B1O.P P P P P P P 7323 266 Bl0.Q q q Li q iI q .wo 304 BIO RIfI(7INS) r A.TL s 1: ; ; ; ;

37cfl ihi I IO? 13’1

A.TH ji 5 i \ s d l2c?h 171 BIO.HTT

: ;, ; ; h d 997 35Y

BIO.A(SR) k d 1957 7% BIO A 14R) k k b k ’ h II91 216 BlO.GD d d d d:b’ -’ h 30’) 56X

“The reactivity of mAb K??--42 was determined in a cellular radioimmunoassay with brndmg of ‘%iabeled protein A. The Iwed values represent mean cpm of triplicates. The reactwztq partcrn OF l7/227 has already been described by Lemke et al. (1979) using a “‘1 protein A bindmg test.

*The defect to generate E, chains is due to: deletion m the E, gene (haplctypes b and s). mRNA of aberrant sire (haplotype f) and a defect in RNA processing or stability (haplotype q) as desonhed by Mathis CI al. (1983).

‘The E.. chain of H-2” mice IS different to the E,, cham nf the parental mioc of b md d haplotyes as &scribed by Jones ( I9801

of cold phosphate-buttered saline {PBS) (4°C). Alter- natively, labeling was performed for I hr with 10’ cells in 1 ml medium containing 109, dialyzed FCS. Labeled cells were resuspended in 500~1 Tris- buffered PBS pH 7.4 containing 10mM PMSF and 1: 1000 dilution of Trasylol and lysed with 50 ~1 of 100, NP40 (final c~)ncentration 1:;) (~obberstein et nl., 1979). DNA and debris were removed by centri- fugation and glycoproteins were isolated from the supernatant with Lens culinaris lectin affinity chro- matography (Hayman and Crumpton, 1972). Immu- noprecipitation was performed by using lo6 cpm of Lens cu~~nar~~ purified glycoproteins incubated with 50 11 of hybridoma supernatant (20-fold concen- trated) and mixed with 5 ~1 protein A Sepharose and rotated for 12 hr at 4 C. Immunoprecipitates were washed three times with 0.2.5”,, NP40 containing PMSF and Trdsylol and denatured prior to electrophoresis by boiling for 5 min in nonreducing sample buffer containing 3”+, SDS.

Two-dimensional nonreducedjreduced SDS-poly- acrJ*lamide gel electrophoresis (SDS-PAGE)

Samples were loaded on tube gels for the first dimension (19 cm running, 1 cm stacking gel) and electrophoresed at 1 mA!gel. The gels were then

incubated for 1 hr at 60°C in sample buffer containing 5’$, 2-mercaptoethanol and fixed on the top of a slab gel with hot agarose (0.4”,) and electrophoresed in the second dimension with 100 mA/gel (Koch and Haustein. 1980). Gels were treated with PPO as described (Bonner and Laskey, 1974). dried and exposed at - 70 C for 3-5 days with Cronex 4 X-ray films (DuPont).

RESULTS AND DISCUSSION

Binding studies were performed on H-2 congenic and recombinant strains of mice in order to charac-

terize the monoclon~~l antibodies (Table I ). The genes coding for the determinant detected by K22-42

were mapped to the H-2 complex by the reactions observed on the H-2 congenic strains: C57BL, 10 (N-2h) cells were negative, whereas cells of the other independent H-2 haplotypes were positive. The genes were further mapped by typing H-2 recombinaI~t strains. The negative reaction of BlO.GD excludes the H-2K molecule and AtA”, dimer and suggests that the antibody reacts with either the H-2D molecule or the E,E,, molecule which this strain does not express. Both H-2K and H-2D could be excluded as K22-42 does not ~mmunoprecipit~te any class I molecule. but immunoprecipit~tes class II molecules only (Figs 2, ? and 4). The binding of K22-42 with BlO.A(SR) cells

(Table 1) suggests that the antibody reacts with EiEt dimers; as H-_ qh strains are negative this excludes

AtA$ dimers of BlO.A(5R) and also H-2K. This binding is of particular interest because this antibody was derived from C57BLi6 (H-lh) itnmunjzed mice which do synthesize EF chains but do not express them on the cell surface (Jones L’I ul.. 19786) due to a deletion in the I$ gene to code for the E, chain

(Mathis PI ul., 1983). However, K22---42 also binds to BlO.A(4R) spleen

cells (Table 1). The reaction on this strain excludes H-2D and must be due to binding to AtA; dimers 3s this strain also does not express an E,,E, dimer due to a deletion in the Et gene (Mathis et al.. 1983). The

crossreactivity of K22. 42 to I-A products is strongly supported by binding data on spleen cells of Bl0.M. BIO.Q and A.TH. These strains lack the h-7 determinant, which is a marker for the EYE,< dimer. indicating that no E, chain is expressed (Jones ef crl.. 1981). Thus, K22-42 probably reacts with AJAI,.

A&IA”, and A:A$. It is obvious that no clear assignment of K22-42 to either I-A or I-E products is possible and it is best explained as crossreactivity with both

Polymorphic determinant detection 295

Two dimensional SDS-PAGE of la antigens

10 SDS-PAGE unreduced

80K 4OK

Fig. I. Illustration of I-A and I-E antigens separated in a two-dimensional nonreduced/reduced gel system. At the top of the first nonreduced dimension is demonstrated, where S-S linked complexes and monomers are separated. The second dimension, where the previous S-S linked subunits are separated correspond to the 2D gel pattern of Fig. 2, except that in Fig. 2 the 15K component runs off the front

of the gel.

products. Altogether the binding data suggests that

K22-42 binds to AiAi. A,kAi, A,9A& E,bEt and E$Ei dimers.

Monoclonal antibody 171227 has been previously mapped to the I-A subregion (Lemke et al., 1979). 17/227 binds to C57BL/lO. BlO.D2 and BlO.BR spleen cells but not to cells of the other independent H-2 haplotypes strains; this maps the genes to the H-2 complex. The results obtained with A.TL (posi-

tive) and A.TH (negative) map the genes to the I region. The reactivity can be mapped to the I-A subregion: reaction with C57BL/lO implies that the specificity is on the AkAi as no EtEj dimer is present: reaction with BlO.A(4R) implies reaction with AbAi as no EiE); dimer is made, and similarly reactivity with BlO.GD implies reaction with AiAi as no E;Ei dimer exists.

In order to obtain more detailed information about the crossreactivity of K22-42 with A,A,) and E,E,, dimers. a nonreducedireduced two-dimensional gel system was used for biochemical analysis of the Ia antigens. Ia polypeptide chains bind different amounts of SDS in a nom-educed compared to a reduced state due to intramolecular S-S bonds (Koch and Hlmmerling. 198 1). therefore in the nonreduced/ reduced two-dimensional gel the different Ia poly- peptides can be separated from each other even when their molecular weights are very similar. Further- more, I-A z and b chains but not I-E a and B chains artificially form interchain S-S linkages during solu- bilization of cells (Cook et ul., 1978), and in the nonreducedireduced gels such SX linked subunits

are situated in the same vertical line. An illustration

of I-A and I-E antigens separated in a nonreduced/reduced two-dimensional gel is demonstrated in Fig. 1.

Ia antigens were immunoprecipitated with K22-42 and 17/227 from glycoproteins prepared from LPS- activated BALB/c spleen cells which had been pulse labeled for 5 min with -“S-methionine (Figs 2a and b). In this short labeling period the polypeptide chains contain only high mannose oligosaccharides and thus show no complex protein-linked oligosaccharides (Hubbard and Robbins. 1979). K22Z42 immuno- preclpltates E, strongly and A,{ more weakly (Fig. 2a). The A, chain can be distinguished from E, by its lower apparent mol. wt and also because A,$ occurs in two distinct forms, probably due to different intramolecular SSS bonds (Koch and Hgm- merling, 1981). The immunoprecipitation of AzAi/ dimers by K22-42 was unexpected because this mAb does not bind to BlO.GD cells which express only A&1A$ dimers. However, it is possible that the respective determinant is more readily available in solubilized material than on whole cells. The invariant chain (Ii) which is always coprecipitated with Ia antigens, and which forms various dimers with other components (Koch and Hgmmerling, 1982) (see leg-

end to Fig. 2) is seen as a large spot which super- imposes A, and E, chains in Fig. 2a.

17/227 immunoprecipitated predominantly the A,A,] dimer and additionally EXE, chains (Fig. 2b). In this case A, and E, chains can be better resolved from the Ii spot. A, and A, chains are also present in a

296 Nonrmr KOCH et ul.

Fig. 2. Nonreduced:reduced two-dimensional SDS-gel of 5 min pulse-labeled Ia antigens. LPS stimu- lated blast cells of BALB,‘c spleen cells after 3 days of culture were labeled for 5 min with “S-methionine. Cells were lysed with NP40 and glycoproteins isolated by using Lens cuiinaris Sepharose. Immuno- precipitation was performed by using mAbs (a) K22-42 (because mAb K22-42 binds on BlO.D2 cells but does not bind to BIO.GD it was designed as a-1-E) and (b) 173227 was designed a-1-A. by Lemke et ul. (1979). Additionally to Ia antigens the invariant chain (Ii) is coprecipitated. Ii chains form several dimers as described by Koch and Hgmmerling (1982). From left to the right Ii is S-S linked to a 41K component. Ii forms a homodimer and Ii is linked to a 15K component which in this gel runs off the front of the gel. Additionally Ii occurs as a monomer. Further a 25K component is present which is noncovalently

associated to Ii.

position where they previously were S-S linked (left side of Fig. 2b). These previously S-S bonded A, and A, chains correspond from their mobility in the second dimension exactly to the positions of A, and A,$ chains in the monomeric state (right part of Fig. 2b). The A, chain is always less intensively labeled compared to A,. One explanation for different labeling is that A, chain contains less methionine compared to A,,, as in the dimeric state r and b chains are in a stoicheometry of 1: 1. Above the two A, chains at the right in Fig. 2b two faint spots appear, which possibly reflect A,j chains with different high mannose content. Superimposition of Figs 2a and b reveals that these spots do not correspond to E,j. The precipitation of EXE,, dimers by 17:‘227 was unexpected as this antibody was shown to react only with the I-A products (Lemke et al.. 1979). However. the biochemical data shows a crossreaction with

EXE,,.

After labeling for 1 hr additionally processed forms

of Ia antigens become visible (Fig. 3). The pattern obtained with K22-42 shows three strongly labeled E,, spots (Fig. 3a). In addition two very faint A,, spots appear. A, and E, chains cannot be resolved from each other because the glycosylated forms of these polypeptide chains become very diffuse bands.

Figure 3b demonstrates Ia antigens immunoprecipitated by mAb 17,i227. In addition to E,,. A, spots are also present. Processed forms of the two A,) chains and their respective precursors can be seen. In contrast the I-A subregion specific mAb K24464 immunoprecipitates only A,A,, dimers and no E,, chain is detectable (Fig. 3~).

In order to investigate the location of the allo- determinants. LPS activated spleen cells of BlO.GD mice were labeled for 5 min with “‘S-methionine. BIO.GD mice lack E, chains and thus form no EXE,! dimers; however, the E,; chain is present in the


L-i-E K22-42 BALB/c

T-l -A I 71227 BALB/c

4lk

A j ap i A/lP

3-I-A K24-64 BALB/c

0 a 69” )

46 ,

.

Fig. 3. Nonreducedjreduced two-dimensional SDS-gel of 1 hr labeled la antigens. BALB/c spleen cells were activated as described in Fig. 1 and labeled for 1 hr with 35S-methionine. Other details as in Fig. 1.

Immunoprecipitation was performed by using mAbs (a) K22-42, (b) 17/227 and (c) K24-64.

cytoplasm (Jones ef al., 1981). la antigens immu- that these mAbs do not react with free E,! chains. The noprecipitated from this strain are shown in Fig. 4. 17/227 and K24-64 react with A,A,< dimers (Figs 4b

two Faint spots above the two A,j chains in Fig. 4b and c are, as mentioned before, not in the position of

and c). No E,, chain is visible on these gels. indicating E,,. In contrast to Figs 4b and c, mAb K22-42

298 NORBERT KOCH er aI.

3-l-E W-42

f3lO GD :,:

Fig. 4. Nonreducedlreduced two-dimensional SDS-gel of 5 min pulse-labeled la antigens. LPS blast cells of BlO.GD spleen cells after 3 days of culture were labeled for 5 min with “S-methioninc. Other details as in Fig. 1. lmmunoprecipitation was performed by using mAbs (a) K21-31. (b) 17’717 and (c) K34664.

immunoprecipitates E,, chain together with the invariant chain (Fig. 4a). This E,, spot can be super- imposed with E,! in Fig. 2a. The reduced form of the E,! chain has a mobility similar to the invariant chain and thus can be distinguished from the two A,, chains which run faster in the reduced second dimension. I-A products are not visible in this gel probably because the affinity of K22Z42 to I-A products is not very high (compare Figs 2a and 3a). Suggesting that the determinant for K23-42 is formed by assembly of E,{ chain with the invariant chain, this finding implies a functional role for the invariant chain in assembly of Ia antigens, which is supported by the fact that K2Z42 is not able to immunoprecipitate single E,,

chain synthesized in a cell-free translation system (unpublished).

The strain distribution pattern of mAb K22-42 and 11/221 exhibits differences in binding to 11. p, q. r, f and s haplotypes (compare Table 1 and Lemke et

ul., 1979), thus it can be concluded that these mAbs do not bind to the same determinant.

Crossblocking studies were performed on whole spleen cells to examine whether both mAbs bind to

labeled 17:‘227 to BALBic spleen cells was blocked by cold K22-42. However. binding of “‘I-labeled K22Z42 was not blocked by cold 17/227. This one way inhibition suggests a conformational change by binding of K23-42 to the rQ dimer. The binding of K22--42 should alter the determinant for 17,1X7. Conversely the binding of 17/X7 does not alter the determinant for K22 -42. This one way inhibition at least indicates that both mAbs bind to the same r- /j dimer: however, it does not prove whether the blocking takes place on I-A or I-E products.

The results described herein clearly demonstrate that mAbs 17,‘227 and K22-~42 crossreact with I-A

Fable 1 C’roasbloching between ant, I-A” tnAhs 011 BALB,c ,plren cell?

“‘I-labeled Inhibition wth cold InhIbitor mAh K??-42 I7 727

K?7 -III c I7 227 25 c

Bindlng of “51-labeled mAbs to spleen cells was inhibited by cold mAb as described by Koch e, al. (1981~).

c = complete inhibitlon was obtained. = no inhibition at 100 $g:ml of cold InhibItor

the same a-p dimers (Table 2). The binding of ‘?‘I 25 = 50”,, inhibitlon at 75 ~cg~ml 01 cold lnhlbltor


and I-E products both in direct binding studies and

also immunochemically. Whether the allo- determinant shared between the I-A and I-E products

is due to a common ancestral gene which has given rise to gene duplication as suggested by Klein and Figueroa (1981). or to gene conversion as suggested for H-2K antigens (Pease et al., 1983; Weiss er al., 1983) is unclear. Probably the sequence of Ia genes will give more information on homology between x and B chains of the two subregions.

Acknowledgement--We wish to thank Professor I. F. C. McKenzie for critical reading of the manuscript and valu- able comments.

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monoclonal antibodies detect polymorphic determinants shared by i-a and i-e antigens

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