FUNCTIONAL SITES ON THE A,-CHAIN

0022-1767/89/1435-1472$02.00/0

Copyright 0 1989 by The Amerlcan Assoclatlon of lmmunologists THE JOURNAL OF IMMUNOLOGY VOl. 143. 1472-1481. No. 5. September 1 . 1989

Printed In U. S. A.

FUNCTIONAL SITES ON THE A,-CHAIN

Polymorphic Residues Involved in Antigen Presentation to Insulin-Specific, A,b:A,k- Restricted T Cells‘

ANGELIKA B. RESKE-KUNZ2* DIDIER LANDAISt JEAN PECCOUD,+ CHRISTOPHE BENOIST,+ A~~

DIANE MATHISt From the *Institut fur Irnrnunologie. Johannes Gutenberg-Universitat, 0.6500 MAINZ, FRG; and the ‘Laboratoire d e

Genetique Moleculaire des Eucaryotes du CNRS. Unite I85 d e Biologie Moleculaire et de Genie Genetique de I’INSERM, Institut d e Chirnie Biologique, Faculte de Medecine. 1 1 , rue Humann, 67085 Strasbourg Cedex, France

The interaction between the clonally selected TCR, the processed Ag peptide and the Ia molecule is not fully understood in molecular terms. Our study intended to delineate the residues of AB molecules that function as contact sites for Ag and for the TCR of a panel of T cells specific for the A chain of insulin in combination with mixed haplotype AB:A: molecules. Multiple L cell transfectants expressing a,@-heterodimers composed of wild-type AB- and chimeric or mutant A,-chains served as antigen presenting cells. The recombinant &-chains had been generated by an exchange of allelically hypervariable regions (ahv) or amino acids. The results point out a broad spectrum of b sequence requirements for the bovine insulin-specific activation of the various T cell populations. Activation of some T cells seemed quite permissive, requiring b-haplotype amino acids in any one of the three ahv, while others had strict requirements, demanding b-haplotype sequence in all three ahv. Our data stress the role of ahvII and especially ahvII1 in T cell activation. Interestingly, single amino-acid substitutions in ahvII or ahvIII of A,k were sufficient to bring up full stimulation potential for two T cell hybridomas. We also found that some ahv permutations influenced the Ag preference (beef insulin versus pig insulin) of some T cells. These data suggest a critical role for the three-dimensional structure of the complex formed by la and the processed Ag peptide. The stability of the trimolecular complex essential for T cell activation is envisioned as being the sum of the interactions between A@-A, TCR/Ag, and TCR/I-A, each variable in strength and compensated for by the others.

The I region of the MHC codes for class I1 (or Ia) molecules that are crucially involved in cell: cell communica-

Received for publication September 26. 1989. Accepted for publication June 5. 1989. The costs of publication of this article were defrayed in part by the

advertlsernent in accordance with 18 U.S.C. Section 1734 solely to indi- payment of page charges. This article must therefore be hereby marked

cate this fact.

SFB 31 1, the Centre National de la Recherche Scientifique (CNRS) and ‘ This work was supported by the Deutsche Forschungsgemeinschaft.

the Instltut National de la Santi et de la Recherche Midicale. J.P. is supported by a fellowship from the CNRS and RousseljUCLAF.

dressed. Author to whom correspondence and reprlnt requests should be ad-

tion during the immune response (1). The Ia molecules are transmembrane glycoprotein complexes consisting of two noncovalently linked polymorphic chains, a and p. In the mouse, two isotypic complexes exist: I-A composed of A, and Ap. and I-E comprised of E,, and Eo (2. 3). Subunits a and p have similar structures: each consists of an amino-terminal giobular domain (al, PI). a globular domain adjacent to the plasma membrane ( ( ~ 2 , p2), a transmembrane segment and an intracellular carboxy- terminal tail. Although the Ea-chain is relatively invariant, extensive allelic polymorphism is characteristic of A,, A,, and E,. Sequence analysis has revealed that virtually all allelic variation resides in the amino-terminal a1 and domains (4-8). Within these domains, polymorphic residues are clustered in distinct regions, termed “allelically hypervariable regions” (ahv3). Not surpris- ingly, then, most epitopes for alloreactive mAb have been mapped to the a1 and p1 domains (9-15). Polymorphism in the amino-terminal half of the A,] domain was shown to influence a,D-chain interaction and thus the efficiency of dimer expression (1 5). This result is consistent with a hypothetical model of the three-dimensional structure of the class I1 foreign-Ag-binding site (1 6). extrapolated from the crystal structure of class I molecules (17). In this model, the Ag-binding groove is flanked by the C-terminal a-helices of the a1 and domains, while the bottom of the cleft is formed by the N-terminal 0-strands of each domain.

T cells of the helper/inducer phenotype (Th cells) recognize processed Ag in association with Ia molecules on the surface of APC. These encompass diverse cell types like spleen dendritic cells (18). B cells (19), activated T cells (20, 21), and macrophages treated with IFN--y or granulocyte-macrophage-CSF(22-24). The formation of a ternary complex involving nominal antigen, the appropriate Ia molecule, and the TCR is considered to be pre- requisite for T cell activation (see Ref. 1 for discussion). I-A-restricted recognition of Ag was reported to be dependent on polymorphic residues situated in both the a- and P-chain (25). Multiple sites in the A,] domain appear to contribute to I-A-restricted Th cell activation (1 1, 12, 15, 25). while the Agz domain apparently is not involved.

Although a number of studies have focused on the role of the A,-chain in T cell stimulation, information about

gions: BI, PI, SI . HI, beef, pig, sheep, and horse insulin. respectively. Abbreviations used in this paper: ahv. allelically hypervariable re-

1472

POLYMORPHIC A, RESIDUES INVOLVED IN INSULIN PRESENTATION 1473 TABLE I

Amino acid substitutions in species variant i n s u h s and insulin fragments as compared with mouse insullns"

Amlno Acld Posltlons

that presentation to these hybridomas occurs essentially only in the context of A::Ai molecules, and then attempt to define what distinguishes AI: from A$ in this system.

Insulin Fragments Insulln and A chain B chaln

4 8 9 10 3 30

Mouse insulin Asp Thr Ser lle Lys Ser BI PI SI HI

Glu Ala Ser Val Asn Ala Glu Thr Ser Ile Asn Ala Glu Ala Gly Val Asn Ala Glu Thr Gly Ile Asn Ala

BI-DesB1-3 Glu Ala Ser Val Ala BI-DesB22-30 Glu Ala Ser Val Asn BI-DesA1-4,18-2 1 Ala Ser Val

Bl-A(SS)s BI-BfSSl

Glu Ala Ser Val Asn Ala

acid sequence of both forms of mouse insulin (30). "Sequence differences are represented in comparison with the amino

Aa! first domain I I

L Cell ahvl ahvll ahvlll transfectants n

KKK

BKK KBK KKB

KBB BKB BBK BBB

B53 856 857 K53 K56 K57 K59 K69 K70 K75

K76

derived from A," and are shown as black or white rectangles, respec- Figure 1. Structure of the chimeric and mutant A, cDNA. Regions

tively. For the chimeric L cell transfectants. the three letter nomenclature indicate the haplotype derivations of the first, second and third allelic hypervarlable regions. L cell transfectants expressing single-site A, mutants are referred to by a letter signifying the A,-chain haplotype, and a number indicating the position of the mutated residue.

the role of A, is rather scarce. In this report, we describe experiments aimed at dissecting in molecular detail the role of A, in Ag presentation. A s APC we employ a panel of I-A+ L cell transfectants expressing a&heterodimers composed of wild-type Ai-chains and chimeric or mutant A,-chains. The Ag is insulin, presented by the APC to a set of T cell hybridomas reactive to determinants on the A chain of bovine insulin presented by APC from H-2bxk F, mice, but not from parental animals (26). We confirm

MATERIALS AND METHODS

Antmals. (B10 x BIO.BR)Fl mice were bred in the animal facilities of the Institut fur Immunologie, Mainz.

Ag. Monocomponent insulins of cow (BI), pig (PI). and sheep (SI) origin were generously donated to us by Dr. G. Seipke. Hoechst AG, Frankfurt. They were purified to homogeneity by ion-exchange chromatography. Horse insulin (HI) was purchased from Sigma (Tauf- kirchen). The isolated A and B chains of BI. as well as BI-DesB1-3 and BI-DesB22-30. were a generous gift of Drs. H. Brandenburg and H. G. Gattner. Deutsches Wollforschungsinstitut, Aachen. BI-A(SS)* and BI-B(SS) were obtained by reduction of the isolated S-sulfonated chains followed by reoxidation and purification by gel filtration (27). BI-DesB1-3 represents a modified BI. lacking amino acids 1 to 3 of the B chain (BI-Des(Phe-Val-Asn) B1-3(Pyr)B4) (28). BI-DesB22-30 is a modified BI which lacks amino acids 22-30 of the B-chain (BI-Des- nonapeptide. B22-30) (29). V 8 protease cleavage of the isolated A- chain of BI yielded BI-DesA1-4,18-21, which lacks amino acids 1-4 and 18-21. This derivative was kindly given to us by Dr. E. Rude, Institut fur Immunologie. Mainz. The purity of the insulins and their derivatives was assessed by reversed-phase HPLC. Table I depicts the amino acid substitutions of the various antigens as compared with mouse insulins.

Reagents. The mAb 10-2.16 (31), originally obtained from the Salk Institute. San Diego. CA, reacts with the (3-chain of I-Ak molecules (32). This antibody was employed as ascites fluid prepared as described elsewhere (33).

dium (GiBCO-BRL, Eggenstein) supplemented with 2 mM L-gluta- The basic culture medium was Iscove's modified Dulbecco's me-

mine, 100 IU penicillin. 100 pglml streptomycin, 5 X M 2-ME and 5% heat-inactivated FCS (Boehringer Mannheim). L cell transfectants bearlng wlld-type, chlmerlc and mutant

cDNA. L cell transfectants expressing wild-type A, cDNA have been reported previously (34). The transfectants bearing chimeric A,- chains have also been described (9). They are referred to as BBB, KBB. BKB, BBK. BKK, KBK, KKB, and KKK. The three letters denote the A, allele (k orb) from which ahvI. ahvII, and ahvIII. respectively. were derived. A schematic representation of these chimaeric A, cDNA is shown in Figure 1.

substitutions at positions 53, 56, and 57 (referred to as B53(A). Three L cell transfectants carry At-chains with single amino acid

B56(R), and B57(R)). These changes introduce the amino acid nor- mally found at that position in the A,-chain of the k allele. Con- versely. 8 L cell transfectants bear A$chains in which single amino acid residues were substituted by residues occurring at these positions in At (K53(G), K56(A), K57(S). K59(D), K69(V). K70(V), K75(G). and K76(V)). These changes were introduced by oligonucleotide- directed mutagenesis. as described elsewhere (9).

All transfectants express the A:-chain (34) in conjunction with the A, chimeric or mutant A, molecules.

Fluorescence analysis. Fluorescence analysis was performed as reported elsewhere (35). with minor modifications. Cells (5 X lo5) in staining medium (PBS. supplemented with 2% FCS and 10 mM NaN,) were sedimented, resuspended in 50 pl mAb 10-2.16 at a predeter-

4°C for 30 min. After two wash steps, the cells were stained for 30 mined saturating concentration (ascites, diluted 1/500), and kept at

min with 50 pl FITC-conjugated goat anti-mouse IgG, F(ab'), f r a g ment, diluted 1/30 (Jackson Immuno Research, West Grove, PA). After two washes. the cells were fixed with 0.5 ml of 0.7% paraform- aldehyde in PBS. Samples were analyzed with a n Epics C cell sorter (Coulter Electronics, Hialeah, FL) equipped with logarithmic ampli- fiers. with a scale of 1 to 1024: an increase of 136 channels represents a doubling of fluorescence intensity. For each histogram IO4 cells were analyzed.

T cells. T cell hybridomas were established as described previously (26) from lymph node T cells of (B10 X BlO.BR)F, mice immunized with BI. They were cloned two to four times at 0.2 to 0.25 cells/well. Hybridomas 42.1.84.169.175 (42/175), 42.1.83.113.141 (42/141). 33.11.1 and 51.144.2 are stained by mAbKJ16-133, which binds to TCR @-chains expressing Vp8.1 or Vp8.2 (36) (a kind gift of

Hybridomas lG10.85.bl.15.42 (1/42). 14.37.15,56.12.1, and54.21.1 Drs. J. Kappler and P. Marrack, National Jewish Hospital. Denver).

were not stained by this antibody. Insulin recognition on splenic accessory cells was demonstrated to be restricted by I-A,b:A,k molecules (26). T hybridoma cells were grown in basic medium supplemented with 1 mM sodium pyruvate and were subcultured 1 :20 to 50 twice per week. The IL-2 dependent T cell clone ST2/K.9 has been described previously (37). It originates from (B10 x BIO.BR)Fl

1474 POLYMORPHIC A, RESIDUES INVOLVED IN INSULIN PRESENTATION

14.37.15 56.12.1

60 60

10 40 160 640 10 40 160 640

Antigen ( p g / m l ) Ffgure 2. T cell responses to BI and PI presented on splenic accessory cells. T hybridomas (lo5) or ST2/K.9 T cells (lo4) were cocultured with 2 X

supernatants (100 p l ) were tested for IL-2 content with 5 X 103/100 p l CTLL in the case of hybridoma cultures. and for IL-3 content with 2 X 103/100 10' syngeneic spleen cells and BI (0) or PI (0) at the concentrations indicated, or with medium as a control, in 0.2 ml volume for 24 h. Cellfree

pl SN1-B3 in the case of ST2/K.9. Compared with the proliferation of the indicator cells (CTLL or SNl-B3) induced by a lymphokine standard preparation. the proliferatlon of the indicator cells in the presence of the T cell hybridoma-supernatants. generated by stimulation with BI at optimal concentrations, amounted to 100% for 42/175 cells, 100% for 33.11.1, 100% for ST2/K.9, 67% for 54.21.1. 82% for 51.144.2. 78% for 42/141, 58% for 14.37.15, 59% for 56.12.1, and 55% for 1/42.

mice primed with PI and is propagated in basic medium containing 250 U/ml of rIL-2 (a kind gift of Dr. U. Schwulera, Biotest, Offen- bach). This clone recognizes BI and PI in the context of AP:Ai molecules, as well as BI with A2:A; elements.

sessed by culturing lo5 hybridoma cells with 3 x lo4 transfected L T cell assays. Production of IL-2 by T cell hybridomas was as-

cells or 2 X lo5 spleen cells and varying concentrations of Ag (or medium as a control) for 24 h in 0.2 ml of basic medium containing sodium pyruvate. An aliquot of 50 or 100 pI of cellfree supernatant from triplicate cultures was frozen, thawed, and added to 2.5 x lo3/ 50 pl or 5 X 103/100 pl, respectively, of IL-2-dependent CTLL cells (38). Forty hours later, 0.5 pCi = 18.5 kBq [3H]TdR was added, the cultures were terminated 8 h later and 13H]TdR incorporation determined by liquid scintillation counting. To measure production of IL- 3 by STZ/K.9 cells. lo4 T cells were cocultured with 3 x lo4 L cell transfectants or 2 x lo5 spleen cells and varying doses of Ag for 24 h. Cellfree supernatants were tested for IL-3 content by using 2 X lo3 SNl-B3 indicator cells (39). kindly given to us by Dr. E. Schmitt (Mainz). f'H]TdR (0.2 pCi = 7.4 kBq) was present during the last 18

cpm with Ag minus cprn without Ag. h of a 48-h incubation. Results are expressed as A cpm referring to

RESULTS

Characteristics of the Insulin-Specific T Cells

To study the role of the A,-chain in antigen presentation, we have used a panel of T cell hybridomas and a T cell clone, ST2/K.9. All derive from lymph node T cells of (B10 X BIO.BR)FI mice immunized with BI (hybridomas) or PI (ST2/K.9) and have been described earlier (26. 37). The response of these T cells to BI/PI, when presented by spleen cells, is restricted by I-At:Ai F1 hybrid molecules (26, 37) [our unpublished results). In addition, cells of the ST2/K.9 T clone are stimulated to proliferate when they recognize BI in the context of A::A,b determi-

nants (37). As shown in Figure 2, the T cell hybridomas 42/175 and 33.11.1 as well as the T clone ST2/K.9 are reactive towards PI as well as BI when presented on splenic accessory cells, although significantly higher concentrations of PI than BI are required to induce a similar level of lymphokine production. In contrast, all the other hybridomas used in this study react to BI, but not to PI, at the concentration range of antigen used. Doses of insulin beyond 1.5 mg/ml proved inhibitory for these T hybridoma cells (our unpublished observations).

To localize the epitope on the insulin molecule recognized by the various T cell populations, the cells were stimulated with several insulin derivatives whose structure is given in Table I. As shown in Figure 3, all hybridomas react against A chain determinants. The isolated A chain of BI, BI-A(SS)2, exhibits a stimulatory capacity comparable to BI, whereas the isolated B-chain, BI-B(SS), has no activity. The lack of influence of the B-chain is further substantiated by the good responses induced by BI-DesB1-3 and BI-DesB22-30, which lack amino acids 1-3 or 22-30 of the B chain, respectively. In contrast, all responses are abolished by the deletion of A chain amino acids 1-4 and 18-21 (BI-DesA1-4,18-21).

Although all hybrids show a similar reactivity pattern towards the various derivatives of BI, a significant heterogeneity exists between them. The importance of A loop residues (positions 8- 10) varies considerably, as evidenced by the responses to pig or sheep insulin (Figs. 2 and 3). The two PI-crossreactive hybridomas [42/175 and 33.11.1) are also efficiently reactive to SI. Within the

33J.l 42/175

80

Antigen ( p M )

Flgure 3. Ag fine specificity of T hybridomas. T hybridomas ( 10’) were cultivated in 0.2 ml volume with 2 x lo5 syngeneic spleen cells and BI (01, SI (A], BI-DesB1-3 (0). BI-DesB22-30 (A], BI-DesA1-4. 18-21 (0). BI- A(SS)n (@, BI-B(SSJ (01. or medium as a control. Twenty-four hours later.

CTLL cells. 50 p1 of cellfree supernatant was tested for IL-2 activity on 2.5 X lo3

group of PI non-reactive hybridomas, responses to S I range from substantial (54.21 .I) to virtually absent (1 / 42).

A further indication of the heterogeneity of the hybrids comes from a preliminary analysis of the V, usage of their TCR. Hybridomas 42/175, 33.11.1, 51.144.2, and 42/141 stain with KJ16-133, and thus express Vg8.l or Vp8.2 (36). The other hybridomas do not (data not shown).

with spleen cell APC also apply to L cell transfectants. We utilized a set of four transfectant lines: they display at their surface the single-haplotype A,k:A$ or A::A,b complexes or the mixed-haplotype At:A,b or A::A,” molecules. The four lines were tested for their capacity to present BI or PI to the T hybridomas; the results are shown in Table 11. All of the hybrids are restricted by the A?A$ molecule. This finding is in good agreement with our ObseNatiOn that such hybridomas preferentially employ F,-unique A,b:A$ restriction elements on splenocytes (26). The weak presentation of BI to 33.11.1 by At:Ag transfectants was unexpected, as this hybridoma is not activated at all by BI on H-2b splenocytes. This finding might be due to an augmented level of I-A molecules on the transfectants as compared with splenocytes, rather than a cell-specific property, because 33.11.1 cells are also activated by I-Ab- positive LB-27.4 €3 hybridoma cells (40) (data not shown).

In general, the antigen specificities noted with splenocytes as APC are maintained when L cell transfectants are used. Reactivity to PI is found with 42/175 and 33.11.1, but not with the other hybrids. There is one notable exception: 42/141. which is activated by PI presented on A%:A;-bearing L cells, but not by spleen cells. This finding was verified in several experiments using splenocytes and L cell transfectants in parallel. The basis for this discrepancy is not clear. It is conceivable that an elevated expression of I-A on the transfected cells or a processing difference is involved. At any rate, and with these two exceptions, insulin presentation by the transfectants is as expected.

Activation of Insulin-Specific T Cells by L Cells Expressing Chimeric or Mutant A, Chains

The results described above confirm the importance of both A, and A4 for presentation of insulin to T cells. They also provide us with a clean experimental system with which to define functional regions of A,. Can one pinpoint regions or isolated amino acids that are responsible for the major differences in presentation capacity evinced by A,k:A,k and A,b:A,k?

Sequence analysis of 8 allelic cDNA had revealed three

Insulin Presentation by L Cell Transfectants Expressing Single-Haplotype or Mixed-Haplotype A

Molecules

allelically hypervariable regions in a1 : ahvI (residues 1 1 - 15). ahvII (residues 53-59) and ahvIII (residues 69-76) (4, 7). There are two amino acid differences between the A: and A,k in ahvI, four in ahvII, and four in ahvIII (Fig.

Before embarking on an extensive analysis with chi- 1). We employed as APC L cell transfectants expressing meric and mutant &:A, molecules, we deemed it neces- wild-type A i chains along with chimeric A, chains that sary to verify that restrictions on presentation observed were a composite of ahv sequences from b and k haplo-

TABLE I1 Ag presentatlon by L cells bearing wild-type A, and A, chains of dtfferent haplotypes

Prollferatlon of CTLL in Presence of Hybrldoma Supernatants [ A cpm x

APC 421175 33.11.1 54.21.1 51.144.2 421141 14.37.15 1/42 56.12.1

BI PI BI PI BI PI BI PI 8 1 PI BI PI BI PI 81 PI

0.3 0.6 1.3 0.8 0.2 0.1 1 .0 0.7 1.9 0.7 0.4 0.3 0.6 0.3 0.4 0.4 46.2 69.4 89.1 95.7 9.3 1.5 38.3 0.5 66.9 42.4 81.2 4.0 31.1 0.4 29.4 0.4

0.8 0.6 0.4 0.4 0.4 0.2 0.4 0.5 0.4 0.3 1.2 0.5 0.8 0.4 0.4 0.4 ””-

1.0 0.4 10.9 0.6 0.2 0 . 1 0.6 0.3 0.6 0.9 0.6 0.5 0.3 0.3 0.5 0.4 0.3 0.6 0.4 0.5 0.2 0.4 1 . 0 0.6 0.6 0.3 0.3 0.2 0.3 0.4 0.6 0.3

supernatants (50 pl ] were tested for IL-2 content using 2.5 X lo3 CTLL cells. Optimal proliferation of CTLL with the IL-2 standard amounted to “ T hybridoma cells (10’) were cocultivated with 2 X lo5 splenocytes or 3 X lo4 L cell transfectants and antigen [640 p&lml) for 24 h. Cellfree

115,973 cpm. L cells transfected only with the thymidine kinase gene.


Figure 4. Responses of BI/PI-reactive T cells to insulin presentation by L cell transfectants carrying chimeric A, and wild-type A, genes. T hybridoma cells ( 1 05) and ST2/

tants listed (3 X lo4] and BI (0) or PI (0) at the concentra- K.9 cells (lo4) were cocultivated with the L cell transfec-

tlons indicated in 0.2 ml volume. IL-2 (42/175. 33.11.1) and IL 3 (ST2/K.9) content was measured in the supernatants (50 pl) of 24-h cocultures by using 2.5 x 103/50 pl CTLL and 103/50 p1 SNl-B3. respectively. Control cpm without Ag were less than 1000 cpm. Mean fluorescence intensity channel numbers were for BBB: 486, KKK: 547, KBB: 717. BKB: 622, BBK: 683. BKK: 444. KBK: 738. KKB: 564.

421175 33.1.1 r ST2/K.9 8

4

8

4

8

4

h 8

U

'2

-1 4 1 V - 4 - 2 -

e ?

a

4

10 40 160 640 10 40 SO 640 2.5 10 40 160 640

Antigen (,ug/ml)

BBB

KBB

BKB

BBK

BKK

KBK

KKB

KKK

type molecules. These chimeric A, molecules are tran- scribed and translated from in vitro-permutated cDNAs inserted into a eukaryotic expression vector and transfected into L fibroblasts. The L cell lines are denoted BBB, KBB, BKB, BBK, KKK, BKK, KBK, and KKB. The three letters indicate the haplotypes of the A,-chain from which ahvI, ahvII, and ahvIII, respectively, were derived. A schematic representation of the wild type and chimeric A,-chains is shown in Figure 1.

To evaluate the contribution of single amino acid residues within ahvI1 and ahvIII to antigen presentation, we developed a set of L cell lines carrying wild-type AB chains together with A, polypeptides modified by oligonucleotide-directed mutagenesis to yield single-site substitutions (9). Single amino-acid residues in the ahvII region of the A2-chain were substituted with residues which occur at the same position in A:: alanine at 53 (B53(A)), arginine at 56 (B56(R)), arginine at 57 (B57(R)). The re- ciprocal substitutions were made on the Af: chain: glycine at 53 (K53(G)), alanine at 56 (K56(A)), serine at 57 (K57(S)), aspartic acid at 59 (K59(D)). In addition, single amino acid residues within ahvIII of Af: were replaced by the corresponding amino acids of the b-type: valine at 69 (K69(V)), valine at 70 (K70(V)), glycine at 75 (K75(G)) and valine at 76 (K76(V)).

The Ia density on transfected L cells was assessed by flow cytometry at the same time as assays for APC function were conducted. This was necessary, because the

capacity to activate T cells has been shown to be dependent on the level of Ia expression (41). The cell surface density of Ia molecules on L cells bearing chimeric or mutant molecules was similar to or higher than on the BBB line used as the wild-type standard for APC potential.

I. Bl/Pl-reactive T cells. The results obtained by stimulating the three BI- and PI-reactive T cell populations with APC displaying chimeric A,-chains are depicted in Figure 4. The hybridomas 421175 and 33.11.1 exhibit a congruent pattern of reactivity towards the L cell transfectants. All L cells are able to present BI, as if the presence of b-haplotype residues in any one region suf- fices for efficient presentation. One notes that the influence of ahvI does seem weaker than that of ahvII or ahvIII, since there is a strong shift in the dose/response curves for BBB vs BKK. The T cells are not, however, effectively stimulated when PI is presented on KKB cells (one of the few findings applicable to all hybridomas).

We wondered whether the presence of a b-haplotype residue at just one position of the Af: chain might be sufficient for stimulation of these two T hybridomas. Interestingly, from the panel of transfectants carrying single-site substituted A,-chains, K56(A) cells were per- fectly able to present BI and PI to these T cells (Fig. 5). whereas K53(G), K57(S), and K59(D) cells did not act as APC (not shown). Moreover, K70(V) transfectants, but not K69(V), K75(G), and K76(V), induced lymphokine

POLYMORPHIC A, RESIDUES INVOLVED IN INSULIN PRESENTATION 1477

BI 61 BI 42 1175 33.II.1 6 -

42/t41 856

8 51

%

$ " p [ I @ B 5 6 E 53

4 - BBB

BBB 2 - -

PI PI PI

P z -

X v

E P V 4

10 40 160 660 10 40 160 Si0 l'0 60 160 640

Antigen ( p g / m l )

FZgure 5, APC function of L cell transfectants carrying single-slte substituted A, chains. T hybrldoma cells (lo') were cultured In the presence of

pl of 24 h culture supernatants. Background cpm In the absence of Ag amounted to less than 800 cpm. Mean fluorescence lntenslty channel numbers 3 x lo' L cells carrying mutated A, chains, and BI (0) or PI (0) at the concentrations Indicated. CTLL (2.5 X 103/50 al) were used to detect IL-2 In 50

were for BBB: 455, B53(A]: 548, B56(R): 603, B57(R): 61 1 , K56(A): 745, K70IV): 515.

production by the T cell hybridomas when challenged with BI. However, K70(V) transfectants did not permit a response to PI, which is in accordance with our results with KKB L cells (Fig. 4). K56(A) and K70(V) transfectants did not activate 42/141 T hybridoma cells [data not shown].

If processed PI was capable of binding to the I-A molecules of K70(V) transfectants, then PI should competitively inhibit the stimulation of 42/175 T cells by BI presented on K70[V) L cells. As shown in Figure 6, the presence of PI in addition to BI indeed reduced the level of activation of the hybridoma cells. These data suggest that processed PI fragments can bind to mutated I-Ak molecules containing a valine at position 70 of the A,- chain, although in a manner which does not allow stimulation of 42/175 and 33.11.1.

The third BI/PI-reactive line, ST2/K.9, has very different requirements for activation (Fig. 4). BBB and KBB are efficient APC for BI, while BKB and BBK transfectants are less active in this respect, suggesting that a good T cell response to BI depends on the presence of b-haplotype residues in both ahvII and ahvIII. A b-haplotype stretch in ahvIII alone seems to engender only a very weak response (KKB). This interpretation is substantiated by the results obtained with L cells displaying At- chains with point mutations in ahvfI. B56(R) L cells, which express A:-chains bearing the k-haplotype amino acid residue at position 56, exhibit an about 13-fold reduced presentation capacity, though an amino acid substitution at position 53 and 57 has no such adverse effect (data not shown).

2. Bl-specifc, PI-nonreactive T cells. A representative experiment in which the group of BI-specific, PI-nonreactive hybridomas was challenged with insulin-presenting chimeric transfectants is presented in Table 111. Hybrid- omas 54.21 .I and 51.144.2 exhibit a similar pattern of reactivity to the L cell APC. Every chimeric L cell transfectant except BKK can present BI, indicating that the presence of a b-haplotype segment in either ahvII or ahvIII is sufficient for potent T cell stimulation (Table 111). The L cell transfectants that carry A:-chains with point mutations-B53(A) and B56(R)-stimulate these T cells to a similar degree as do BBB L cells (shown representatively for 51.144.2 cells in Fig. 5). As B53(A) and B56(R) cells express more I-A molecules on their cell surface than BBB cells, this might indicate that k haplotype amino acids, when present at positions 53 and 56, exert a negative influence. In contrast, B57(R) transfectants proved more efficient in T hybridoma activation than BBB L cells which might be due to the higher density of class I1 molecules.

Interestingly, these two hybridomas show a response to PI in the presence of of KBB and KBK, but not BBB APC. Also, the transfectants carrying A:- or A:-chains with single-site substitutions in ahvII could not serve as APC (not shown). It may be that the combination of a b- haplotype stretch in ahvII and a k-haplotype segment in ahvI permits the formation of a particularly stable I-A/ PI/TCR complex. The relatively high density of surface Ia molecules on these two transfectants could also influence presentation ability (although BBK has virtually the same amount of surface Ia as does KBK, with which it

1478

3

2

1

f - t - I 3

2

1

POLYMORPHIC A, RESIDUES INVOLVED IN INSULIN PRESENTATION

10 61 (Id glml)

102

oma cells by PI. T hybridoma cells (lo5) were cocultured with 3 X lo4 L FLgure 6. Competition of 81-mediated stimulation of 42/175 T hybrid-

cell transfectants. BI at the concentrations indicated and PI at 640 (O),

pl of cellfree supernatant were tested for 1L-2 activity on 2.5 X lo3 CTLL 320 (A) and 160 (0) pg/ml or wlthout PI [O). Twenty-four hours later, 50

cells.

shares ahvII and ahvIII; BBK does not present PI). Activation of the hybridoma 14.37.15 [Table 111) shows

requirements similar to those of ST2/K.9: effective presentation of insulin requires that both ahvII and ahvIII are b-haplotype; ahvI is of little importance (KBB). Only very weak stimulation occurs in the presence of BI, when b- haplotype sequence is confined to ahvIII. Lymphokine production by 14.37.15 in the presence of B56(R) and B53(A) presenters is slightly lower than in the presence of BBB L cells. Considering the high density of I-A on the transfectants with mutated A:-chains, single point mutations in ahvII seem to have a negative influence on presentation of BI to 14.37.15 cells (Fig. 4).

Hybridomas 1/42 and 56.12.1 represent a separate class, whose activation requires b-haplotype residues in all three ahv. This requirement is absolute for 56.12.1; for 1/42 one still sees a low level of presentation with b- haplotype sequence in ahvIII plus either ahvI or I1 (KBB, BKB). The B53 (A) transfectant cells are highly efficient antigen presenters relative to the other ahvII mutants, which might point to a negative influence of the b-haplotype amino acid at position 53 [shown representatively for 56.12 I in Fig. 5).

3. 42/141, a hybridoma with reversible aflinity for BI and PI. A s mentioned above, the hybridoma 42/141 is responsive to both BI and PI presented on L cells, but not on splenocytes. The response of 42/141 to B1 focuses mainly on ahvIII: efficient activation by KKB and BKB, weak stimulation by BBK, and no stimulation by KBK

(Table 111). In line with these data, mutants B53(A), B56[R), and B57[R) are potent APC (Fig. 5). Interestingly, stimulation of 42/141 by PI has different requirements: it depends primarily on ahvII with some participation by either ahvI or ahvIII. Substitution of a single b-haplotype amino-acid residue in ahvII by a k-haplotype residue does not abrogate the T cell response (Fig. 5).

The reversal of this hybridoma's Ag preference with BBB and BBK APC appears to be due to the predominance of different a,-domain sites in BI- vs PI-induced activation. This change in antigen fine-specificity was studied more extensively in antigen titration experiments em- ploying BBB, BBK, and BKB APC for presenting BI, PI, SI , and HI. These insulins differ only in the loop region of the A chain (positions 8- 10, see Table I). When presented by BBB cells, PI, SI, and HI stimulated 42/141 to a significant degree, although they were inferior to BI in this respect (Fig. 7). In contrast, with BBK APC, PI proved superior to BI, S I , and HI in its capacity to activate 42/ 14 1 cells. This reversal of the BI/PI stimulatory potential appears to be due to a failure of BI to stimulate the T cells efficiently when presented by BBK cells, because the response of 42/141 cells to PI is only slightly reduced with BBK as compared with BBB transfectants. This result reminds one of the finding obtained with 42/175 cells: using KKB or K70(V) presenters the response to PI is lost, while the response to the species variant BI is not changed. BKB APC, on the other hand, activate 42/141 BI-specifically with reduced efficiency as compared with BBB, although they expose more I-A. PI is inefficient at activating the T cells, and S I and HI are only weakly stimulatory. Since PI competitively inhibited the response of 42/141 cells to BI in the presence of BKB cells (data not shown), binding of processed fragments of PI to the chimeric I-A molecules can apparently take place.

A summary of the entire set of reactivity patterns is presented in Table IV.

DISCUSSION

Starting with At:A,k [no presentation) and A::A,k (efficient presentation) we hoped to pinpoint which of the ten amino-acid differences between the two a chains dictate k-ness and b-ness, in terms of antigen presentation to a set of insulin-specific, At:A$-restricted T cell hybridomas. We also anticipated that antigen- or TCR-contact sites on A, could be distinguished: a b k change in an antigen- Ia contact site should influence presentation to all hybridomas, while an altered Ia-TCR contact site should only affect some of them.

Before discussing the results, let us consider a few points concerning the validity of the experimental system. First, the data should not be greatly influenced by AJAa pairing problems (1 5). since the starting combination for an efficient response to insulin represents the most unfavorable pairing combination, A::A,k. Any mod- ification of A, should (and does) improve its ability to pair with cotransfected Ai. Thus, any loss of presentation potential as a result of b+k changes cannot be attributed to less favorable pairing. Second, the chimeric and mutant &-chains are not monstrously deformed molecules: they assemble with the A,-chain, are readily expressed at the cell surface, and the complexes are recognized by a number of A,- and A,-specific mAbs (9). Further con- firmation of this point derives from the fact that all of

POLYMORPHIC A, RESIDUES INVOLVED IN INSULIN PRESENTATION

TABLE Ill

1479

Antigen presentatfon by L cells bearfng chimeric A, and wfld-type AkB chalns to a panel of BI-specific T cell hybridomas

Prollferatlon of CTLL ( A cpm) In Supernatants"

Hybrldomas Ag (rg/ml) BBB

(486)' (547) KKK KBB

(717) BKB (622)

BBK (683)

BKK (444)

KBK (7381

KKB 1564)

54.21.1

51.144.2

42/141

14.37.15

1/42

56.12.1

BI (640) BI (1 60) PI (640) PI (1 60)

BI (640) BI (1 60) PI (640) PI ( 160)

BI (640)

PI (640) BI ( 160)

PI ( 1 60)

BI (640) BI (160) PI (640) PI ( 1 60)

BI (640) BI ( 160) PI (640) PI (1 60)

BI (640)

PI 1640) BI [ 160)

PI i 16oi

26.261 27.367

2.857 1.250

28.716 30.044

21 1 0

73.849 68.106 37.472 25.984

56.558 20.171

1.224 286

29.424 20.647

0 0

31.511 27.952

0 0

226 45

140 0

85 0 0 0

1.685 1.510

472 207

0 0 0 0

242 4 0 0

342 97

100

63.924 19.096

22.931 21.143

50.066 66.718 9.464 7.405

78.757 71.150 45.450 30.027

66.460 72.535 12.760 1.759

8.964 4.083

384 533

0

0 0

0

39.296 16.452

405 275

15.881 18.592

227 4

46.246 58.272 4.04 1

819

12.216 6.667

477 718

3.813 1.701

0 0

340 84

282 7.16

20.75 1 20.124

1.099 25 1

11.324 12.638

0 0

7.136 6.486

3 1.804 20.450

248

173 191

43

563 192 342 194

0

0 0

n

682 424

0 29

0 38 0

99

2.540 818 233

10

36 0

0 0

0 0 0 0 0

0 0

0

20.840 60.925 24.312 24.567

52.107 53.988

7.276 6.854

516 1.105 2.724

406

62 0

168 78

0 0

0 0

0 0

41 0

22.504 16.754

0 0

12.307 8.662

228 55

17.997 18.892

338 69

5.061 4.080

222 123

269 228

510 0

0

0 0

0 I , 155 ." -

Cellfree supernatants (100 PI) were tested for IL-2 content uslng 5 X lo3 CTLL. Optimal proliferation of CTLL obtained "T hybridoma cells ( lo5) were cocultivated with 3 X lo4 L cell transfectants and Ag a s indicated for 24 h in parallel.

with the IL-2 standard preparation amounted to 84.582 cpm. The cpm in the absence of Ag were below 1,400 cprn. Mean fluorescence intensity channel number.

3-

2-

1-

3-

2-

1-

b

3-

2-

1-

d

-\

BKB

P

5 20 80 320 Antigen ( p g l m l )

Ffgure 7. Reversal of the antigen preference of 42/141 T hybridoma cells. 42/141 T cells (lo5) were cocultivated with 3 X IO4 L cells and varlous doses of species variant insulins for 24 h. IL-2 content was determined in cellfree supernatants.

TABLE IV A gradient of b-haplotype ahv requirements for BI-dependent

activation

Requirements

Either ahvI. I1 or 111 T Cell

42/175 33.11.1

51.144.2 54.21.1 Either ahvll or 111

ahvllI 42/14 1

Both ahvll and I11 ST2lK.9 14.37.15

ahvI. I1 and 111 1/42 56.12.1

the transfectants are capable of activating at least some T hybridomas [in this study or in parallel studies mentioned below, in which KKK is the wild-type presenter).

All chimeric L cell transfectants were able to present BI to at least two T hybridomas, 42/175 and 33.11.1 (although with BKK presenters a higher concentration of the antigen was required). Therefore processed fragments of BI must be capable of binding to the antigen groove of the respective I-A molecules. Very striking is the finding that the presence of just one b-haplotype polymorphic residue in ahvII (at position 56) or ahvIII [at position 70) of the At-chain permits binding. Polymorphic determining residues for Ia/BI interactions may thus exist in, or be influenced by, each of the ahvs. Similar to BI, PI was presented to at least two hybridomas by the various chimeric L cell transfectants except by KKB cells. Accordingly K70(V) transfectants, carrying a valine at position 70 of the At-chain, did not permit a PI-mediated T cell response. However, by antigen competition experiments PI was found to bind to this mutated I-A molecule.

The conclusion that Ia/Ag interactions are dispersed


over the Ia complex, is quite different from that drawn from some other, simpler models of allelically restricted antigen presentation. Ronchese et al. (42) have shown, for example, that a single amino-acid difference (EP position 29) accounts for the differential presentation of pigeon cytochrome c by Ep” and E:, I t is true that the I-A/ insulin system has more potential complexity than the I- Elpigeon cytochrome C system: ten amino-acid differences between A2 and A2 (and a variable A,-chain) vs four differences between E: and Ep” (and a constant E,- chain) and, in addition, a larger and less well-defined peptide in the case of insulin. One notes that the immune system makes full use of this potential complexity: not only is both A, and AB polymorphism perceived (Table 11) (26), but all three ahv regions of A, are seen in Ab:Ak distinctions during BI presentation.

An alternative explanation for the binding of BI and PI to I-A molecules of all the L cell transfectants would be that both insulins interact with nonpolymorphic residues shared by A: and At. Certainly, H-2” mice are low re- sponders for BI and PI (43). but previous results obtained with distinct Ir-gene-controlled systems had indicated that APC from low responder animals were capable of presenting the nonpermissive antigen to selected T cell clones (44, 45). Thus their Ia molecules could form an appropriate Ia/Ag-complex recognizable by the T cells. This notion is supported by binding studies using purified Ia molecules and peptides derived from protein antigens (46, 47). Although, in general, a strong correlation between MHC restriction and peptide binding was observed (46, 47), the mere binding of an antigen fragment to Ia was not always sufficient to generate a T cell response (47). Thus it is possible that all the APC used here can bind the antigenic BI and PI peptide.

Our finding that some T hybridomas were nonreactive to BI and/or PI in the presence of distinct chimeric L cell transfectants might thus be due to an inability of their TCR to interact with the I-A/Ag complex. The results have turned out significantly more complex than expected revealing a gradient of b-haplotype sequence requirements for effective BI-mediated activation of the different A::Ap”-restricted T hybridomas (see Table IV). This gradient ranges from the very permissive (b-haplotype sequence in any one of the three A,-ahv; hybridoma 42/175) to the very strict (b-haplotype amino acids in all three A,-ahv; hybridoma 56.12.1). Such a gradient of b- haplotype sequence requirements probably reflects the fact that Ag presentation depends on the formation of a stable trimolecular complex of A@-A/TCR. Thus, a particularly favourable TCR/Ag combination could compen- sate for weak TCR/I-A interactions.

Overall, though, the three ahvs prove to be of variable importance for T cell stimulation. ahvI clearly has the least influence: KBB presents BI efficiently to all but two of the hybridomas; BKK activates only a few of the T cells, and never presents as well as BBB. This finding could well reflect the relatively minor differences between A,k and At in ahvl: Ser‘’+Thr, and Thr’’43er. ahvII and especially ahvIII have more influence: KBB can activate almost all of the hybrids (Table 111). Quite similar results have been obtained in the analysis of A”-directed alloreactivity and Ak-restricted lysozyme peptide presen-

tation using the same panel of transfectant APC (48, 49). The dominance of ahvIII was found to be even more pronounced in the latter study (49).

How can one integrate our data and the discussion made above with current hypotheses on the three-dimensional structure of class I1 molecules? The most widely accepted model is clearly that of Brown et al. (1 6), extrapolated from the crystal structure of class 1 molecules (17). According to this model, k/b variability in ahvI lies in one of the @-strands which form the base of the Ag-binding groove, ahvII is at the very beginning of the long helical structure which flanks the groove, and ahvIII is at the other end. One can easily picture how each of these locations could be capable of controlling I-A/TCR interactions. Our observations on BI/PI presentation argue that no single stretch of the A,-chain is uniquely responsible, but that spatially distant regions can make impor- tant contributions. With different T cells, or with slightly different antigens, a particular region can be seen to exert a variable influence.

Acknowledgments. We are grateful to U. Arndts, U. Bonifas and P. Gerber for excellent technical assistance, to U. Klutentreter and C. Waltzinger for help with the flow cytometry, and to A. Staub and F. Ruffenach who synthesized the oligonucleotides.

1 .

2.

3.

4.

5.

6.

7.

8.

9.

10.

1 1 .

12.

13.

14.

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FUNCTIONAL SITES ON THE A,-CHAIN

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